Patent Publication Number: US-2005142610-A1

Title: Variant polypeptides containing plekstrin homology domains and uses therefor

Description:
RELATED APPLICATIONS  
      This application claims priority to U.S. Provisional Application No. 60/509777, filed Oct. 7, 2003, the entire contents of which are incorporated herein by reference. 
    
    
     GOVERNMENT FUNDING  
      The work described herein was supported, at least in part, by funding from the National Institute of Health Grant DK 60564. 
    
    
     BACKGROUND  
      Lipid binding domains that target intracellular membranes play a crucial role in the assembly of signaling and trafficking complexes and in membrane remodeling events such as vesicle budding, phagocytosis, and cell motility. The biological significance of membrane targeting is underscored by the prevalence of lipid binding domains, which rank amongst the most common domains in the eukaryotic proteome, and by the discovery of major proto-oncogene proteins and tumor suppressors containing essential lipid binding domains and/or lipid metabolic activities that regulate membrane association (1-4), There are several major classes of lipid binding domains including pleckstrin homology (PH), FYVE (acronym of Fabl, YOTB, Vacl, and EEA1), plant homeodomain (PHD), phox homology (PX), and C2 (named for homology with protein kinase C, PKC) domains as well as variety of smaller domain families and peptide motifs. The variation in physical properties and recognition mechanisms between and within families is striking.  
      Pleckstrin homology domains are commonly found in eukaryotic signaling proteins. The family possesses multiple functions including the ability to bind inositol phosphates. PH domains have been found to possess inserted domains, e.g., such as syntorphins in PLC gamma, and to be inserted within other domains. Mutations in Burtons tyrosine kinase within its PH domain causes X-linked agammaglobulinaemia (XLA) in patients.  
      Multiple species of 3′-phosphorylated inositol lipids are thought to be involved in a number of cellular signaling and membrane trafficking pathways, including membrane ruffling (Parker, P. J.(1994)  Curr. Biol.  5:577; Wennstrom, S. et al. (1994)  Curr. Biol.  4:385), chemotaxis (Parker, P. J.(1994)  Curr. Biol.  5:577; Wennstrom, S. et al. (1994)  Curr. Biol.  4:385), secretory responses (Parker, P. J.(1994)  Curr. Biol.  5:577; Wennstrom, S. et al. (1994)  Curr. Biol.  4:385), membrane trafficking of growth factor receptors (Okada, T. et al. (1994)  J. Biol. Chem.  269:3568; Kanai, F. et al. (1993)  Biochem. Biophys. Res. Commun.  195:762), insulin secretion, cell regulated adhesion and insulin-mediated translocation of glucose transporters to the cell surface (reviewed in Czech (1995)  Annu. Rev. Nutri.  15:441-471). A relatively large, constitutive pool of PI 3-phosphate is present in resting cells, while very low levels of PI 3,4-biphosphate and PI 3,4,5-triphosphate are rapidly increased in response to a number of external cellular stimuli (reviewed in Cantley et al. (1991)  Cell  64:281-302 and Kapeller, R. and L. C. Cantley (1994)  Bioessays  16:565-578). The pool of PI 3-phosphate may be largely due to PI (Bonnema, J. D. et al. (1994)  J. Exp. Med.  180:1427; Yano, H. et al. (1993)  J. Biol. Chem.  268:25846)-kinases such as PtdIns 3-kinase (Bonnema, J. D. et al. (1994)  J. Exp. Med.  180:1427; Yano, H. et al. (1993)  J. Biol. Chem.  268:25846), a mammalian homolog of the yeast VPS34 protein (Herman, P. K. and S. D. Emir (1990)  Mol. Cell. Biol.  10:6742-6754), which can utilize only PI as substrate. In contrast, a second category of PI 3-kinases, isoforms of the p110 PI 3-kinase, are capable of phosphorylating PI 4-phosphate and PI 4,5-bisphosphate at the 3′ position (Hiles et al. (1992)  Cell  70:419-429; Hu et al. (1993)  Mol. Cell. Biol.  13:7677-7688; Kippel et al. (1994)  Mol. Cell. Biol.  14:2676-2685; Stoyanov et al. (1995)  Science  269:690-693). These enzymes apparently contribute to the regulated pools of PI 3,4-P 2  and PI-3,4,5-P 5  stimulated by receptor or non-receptor tyrosine kinase activation (in the case of isoforms p110 and p110β) or G protein activation (in the case of p110γ). The existence of multiple PI 3-kinase isoforms suggests the influence of multiple signaling pathways on these enzymes and, possibly, divergent reactions of the individual 3′-phosphoinositides.  
      The extensive literature on phosphoinositide metabolism by lipid kinases and phosphatases is covered in two recent reviews (5, 6). Given a high negative charge density, distributed over 2-4 phosphates in close proximity, it is not surprising that a strong positive electrostatic potential should be a common feature of the various domains that recognize phosphoinositides. What is more remarkable, in view of the pseudo-symmetry of the D-myo-inositol head group, is the high degree of stereochemical selectivity that lipid binding domains have evolved to distinguish even the most structurally similar phosphoinositides.  
      Several groups have reported a novel protein module of approximately 100 amino acids termed the pleckstrin homology (PH) domain located at the carboxy-terminal of several proteins involved in signal transduction processes (Haslam et al. (1993)  Nature  363:309-310; Mayer et al. (1993)  Cell  73:629-630; Musacchio et al. (1993)  Trends Biochem. Sci.  18:343-348). PH domains have been implicated in the binding to membranes containing PI 4,5-bisphosphate, as well as to the binding of several proteins βγ subunits (Gβγ) of heterotrimeric G proteins (Touhara et al. (1994)  J. Biol. Chem.  269:10217-10220; Satoshi et al. (1994)  Proc. Natl. Acad. Sci. USA  91:11256-11260; Lemmon et al. (1995)  Proc. Natl. Acad. Sci. USA  92:10472-10476), protein kinase C (Yao et al. (2994)  Proc. Natl. Acad. Sci. USA  91:9175-9179), WD motifs (Wang et al. (1994)  Biochem. Biophys. Res. Commun.  203:29-35  
      PH domains have been found in a number of proteins including protein kinase C α, phospholipase C-δ1, the serine/threonine kinase known variously as protein kinase B, Akt and Rac (Burgering, B. M. T. and P. J. Coffer (1995)  Nature  376:599-602; Franke et al. (1995)  Cell  81:727-736; Coffer, P. J. and J. R. Woodgett (1991)  Eur. J Biochem.  201:475-481) among others.  
      Phosphoinositides are second messengers that have been shown to play a critical role in cell survival, membrane trafficking, insulin regulation, adhesion, migration and cytoskeletal dynamics. Based on the prevalence of PH domains and the biological importance of phosphoinositide, a need exisists for understanding and controlling the selectively of various PH domain containing polypeptides for one or more given phosphoinositides.  
     SUMMARY OF THE INVENTION  
      The instant invention is based on the discovery that mutants of PH domains result in polypeptide that have significantly altered affinity (i.e., increased or decreased) for given phosphoinositides. Variants that differ by as little as one amino acid in the PH domain can have completely different ligand recognition and/or can have vastly different affinity for the natural ligand when compared to the wild type polypeptide.  
      Accordingly, in at least one embodiment, the invention provides polypeptides comprising a variant PH domain. In one specific embodiment, the polypeptide has increased binding specificity for a phosphatidylinositide molecule to which the PH domain naturally binds. Alternatively, the polypeptide has decreased binding specificity for a phosphatidylinositide molecule to which the PH domain naturally binds.  
      In another related embodiment, the polypeptide has increased binding specificity for a phosphatidylinositide molecule to which the PH domain naturally does not bind. In yet another related embodiment, the polypeptide has decreased binding specificity for a phosphatidylinositide molecule to which the PH domain naturally does not bind. In specific embodiments, the phosphatidylinositide molecule is phosphatidylinositol-3,4,5 (PI-3,4,5)P3 or phosphatidylinositol-4,5 (PI-4,5)P2.  
      In related embodiments, the variant PH domains have increased or decreased affinity for phosphatidylinositol-3,4,5 (PI-3,4,5)P3 or phosphatidylinositol-4,5 (PI-4,5)P2.  
      In one specific embodiment the variant PH domain has at least one, two, or three glycine residues inserted in the β1/β2 loop as compared to the wild-type sequence.  
      In another embodiment, the variant PH domain comprises an amino acid substitution in a residue that does not contact the head group of a given phosphatidylinositol.  
      In another specific embodiment, the PH domain is present within a Grp1/ARNO/ Cytohesin family polypeptide.  
      In another embodiment, the invention provides a method of using a PH domain variant to selectively detect the presence of a specific phosphatidylinositide. In related embodiments, the phosphatidylinositide molecule is phosphatidylinositol-3,4,5 (PI-3,4,5)P3 or phosphatidylinositol-4,5 (PI-4,5)P2.  
      In specific embodiments of the invention, the polypeptide comprising a variant PH domain has a 10, 100, or 1000 fold higher specificity for a given phosphatidylinositide molecule than the wild-type polypeptide.  
      In one specific embodiment, the polypeptide comprising a variant PH domain has lost the ability to bind and/or recognize the natural ligand (e.g., phosphatidylinositol-3,4,5 (PI-3,4,5)P3 or phosphatidylinositol-4,5 (PI-4,5)P2).  
      In another embodiment, the invention provides a polypeptide comprising a variant PH domain wherein the variant (i) increases the affinity of the PH domain for one ligand while not changing the affinity for a second ligand: (ii) increases the affinity of the PH domain for one ligand while decreasing the affinity for a second ligand; or (iii) increases the affinity of the PH domain for one ligand while increasing the affinity for a second ligand. In certain embodiments the second ligand is a natural ligand of the PH domain. In another embodiment, the second ligand is not a natural ligand of the PH domain.  
      In another embodiment, the invention provides a polypeptide comprising a variant PH domain wherein the variant (i) decreases the affinity of the PH domain for one ligand while not changing the affinity for a second ligand; (ii) decreases the affinity of the PH domain for one ligand while decreasing the affinity for a second ligand; or (iii) variant decreases the affinity of the PH domain for one ligand while increasing the affinity for a second ligand. In certain embodiments the second ligand is a natural ligand of the PH domain. In another embodiment, the second ligand is not a natural ligand of the PH domain.  
      In one embodiment the invention provides a variant GRP1 polyeptide with a substitution selected from the group consisting of K273A, K282A, R284A, Y295F, R277A, R277C, V278A, V278C, K279A, K279C, T280A, T280C, R305A, K343A, N354A, and H355A of SEQ ID NO:1. In a related embodiment the invention provides a variant GRP1 polyeptide having one or more of the following substitutions: of K273A, K282A, R284A, Y295F, R277A, R277G, V278A, V278C, K279A, K279G, T280A, T280G, R305A, K343A, N354A, and/or H355A of SEQ ID NO:1.  
      In one embodiment the invention provides a variant ARNO polyeptide with a substitution selected from the group consisting of K273A, K283A, R285A, Y296F, R278G, V279G, K280G, T281G, R306A, K344A, N355A, and H356A of SEQ ID NO:3. In a related embodiment the invention provides a variant ARNO polyeptide having one or more of the following substitutions: K273A, K283A, R285A, Y296F, R278G, V279G, K280G, T281G, R306A, K344A, N355A, and/or H356A of SEQ ID NO:3. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       FIG. 1  Depicts PI 3-Kinase Signaling  
      In response to extracellular signals, PI 3-kinases catalyze the formation of 3-phosphoinositides. These second messengers are critical to cellular functions such as cell survival, membrane trafficking, insulin regulation, adhesion, migration and cytoskeletal dynamics.  
       FIG. 2  Depicts PH Domain Structures  
      Space filling models of PH domains bound to the phosphoinositides IP3 and IP4. Grp1 Btk and Dapp1 all bind IP4 in a similar orientation but do so making contacts with different loops. PLCδ binds IP3 in a flipped orientation compared to the way Grp1, Btk and Dapp1 bind IP4. Grp1 possesses a hairpin insertion of the β6/δ7 loop that Btk and Dapp1 are missing. Btk and Dapp1 possess longer β1/β2 loops than Grp1. Differences in the loop regions may explain the wide range of ligand affinities.  
       FIG. 3  Depicts the Phosphoinositides Relevant to PH Domains  
      The major signaling phosphoinositides are represented as red and yellow stick models. Pleckstrin homology domains may bind to a diverse selection of phosphoinositides with varying degrees of specificity.  
       FIG. 4  Depicts the Comparison of β1/β2 Loops of PH Domains  
      These PH domains recognize phosphoinositides with a wide range of affinities and specificities. Despite almost 90% identity between the Grp1, ARNO and Cytohesin domains, there are drastic differences in their affinity and specificity for PIP3 and PIP2. The affinity for PIP2 over PIP3 can be affected by presence or absence of a third glycine in the β1/β2 loop.  
       FIG. 5  Depicts the Structure of the ARNO PH Domain Bound to IP3  
      A. A 1.8 Å resolution xray data set was collected on crystals of ARNO bound to IP3. In the first round of refinement, electron density for the IP3 head group is present.  
      B. A ribbon diagram depicts the structure of ARNO bound to IP3.  
      C. The PH domains of ARNO and Grp1 bound to IP3 and IP4 respectively is shown for comparison purposes. Many of the residues in Grp1 that contact the 3 and 4 phosphates of IP4 contact the 4 phosphate of IP3 in ARNO. Consequently, many of the residues that contact the 4 and 5 phosphate of IP4 in Grp1 make contact with the 5 phosphate of IP3 in ARNO.  
       FIG. 6  Depicts the Selection Against IP3 in Grp1  
      A. The β1/β2 loops of ARNO bound to IP3 and Grp1 bound to IP4 have been superimposed. The IP4 in the Grp1 structure has been removed to show the placement of the IP3 in relation to Grp1. The shorter β1/β2 loop of Grp1 (2G) brings a valine in close proximity to the 1 phosphate of IP3. The longer β1/β2 loop in ARNO keeps the valine at 279 away from the 1 phosphate in IP3.  
      B. The Grp1 PH domain bound to IP4 is shown with the IP3 of ARNO modeled in the phosphoinositide binding pocket. ARNO binds IP3 in a shifted or rotated manner compared to the way in which Grp1 and Btk bind IP4. The contacts made with the 3 phosphate in Grp1 are made with the 4 phosphate in ARNO, while those residues that contact the 4 and 5 phosphates in Grp make contact with the 5 phosphate in ARNO. Furthermore, ARNO does not bind IP3 like PLCδ, which binds the ligand in a flipped orientation compared to Grp1 and Btk. This suggests that ARNO binds IP3 in a somewhat novel manner.  
      C. The β1/β2 loops of ARNO bound to IP3 and Grp1 bound to IP4 are superimposed with the IP4 of Grp1 removed. IP3 and valine are rendered to show proximity of atomic radii. The valine of Grp1 clashes sterically with IP3 while the valine of ARNO is juxtaposed away from IP3. With its longer β1/β2 loop, ARNO can accommodate IP3 and IP4. Grp1 possesses a shorter β1/β2 loop that cannot accommodate IP3 as easily as IP4. This may explain the specificity Grp1 has for IP4 over IP3.  
       FIG. 7  Depicts the Structures of the ARNO and Grp1 PH Domains Bound to IP4  
      A. A ribbon diagram is shown of a 2.3 Å resolution crystal structure of the ARNO PH domain bound to IP4. Note the canonical beta barrel and variability loops that surround the opening of the barrel.  
      B. The ARNO and Grp1 structures bound to IP4 are superimposed. IP4 is bound in the same orientation and mode in Grp1 and ARNO. Note the shorter β1 /β2 loop of Grp1 is brought close to the 1 phosphate of IP4 while the longer β1/β2 loop of ARNO is farther away from IP4. The longer β1/β2 loop of ARNO can accommodate binding IP3 and IP4 with little specificity while the shorter loop of Grp1 helps enforce binding IP4 over IP3.  
       FIG. 8  depicts the Electron Density for Unbound Grp1 (3G) PH domain A sigma weighted map of the electron density for the phosphoinositide binding pocket of the Grp1 (3G) PH domain is shown. Despite the lack of an IP3 ligand, there are sulfate ions in the IP3 position, suggesting a preset position for recognizing phosphoinositides in a limited orientation.  
       FIG. 9  depicts an Isothermal Titration Calorimetry Experiments  
      Isothermal titration calorimetry (ITC) experiments were performed on wild type and mutant constructs of GST tagged Grp1 (2G) and Arno (3G). IP3 or IP4, was titrated into a sample cell containing appropriate Grp1 family construct.  
      A. Sample curve of IP4 being injected into a cell containing wild type ARNO. Each peak represents heat released upon IP4 binding.  
      B. Plotting integrated heats of binding for wild type ARNO and IP4 follow a single site binding model.  
       FIG. 10  Depicts the Results of ITC Experiments  
      A. The structure of the Grp1 PH domain is depicted in ribbon form with the specificity determining regions color coded. Contacts between the side chains and IP4 are represented as dotted lines.  
      B. Constructs of GST tagged Grp1(2G) were titrated with IP4. The dissociation constant (Kd) for each interaction was determined and compared to the wild type. The relative Kds are plotted for each Grp1 mutant.  
      C. &amp; D. Constructs of GST tagged ARNO (3G) were titrated with IP3 or IP4. The dissociation constant (Kd) for each interaction was determined and compared to the wild type. The relative Kds are plotted for each ARNO mutant. The V279G mutant bound IP3 and IP4 with higher affinity than wild type.  
       FIG. 11  Depicts an Alignment of Various Pleckstrin Homology Domains Against a Consensus PH Domain.  
       FIGS. 12A  and B depict the PH domains of GRP1 (SEQ ID NO:5) and ARNO (SEQ ID NO:6), respectively. 
    
    
     DETAILED DESCRIPTION  
      The instant invention is based on the discovery that mutants of PH domains result in polypeptide that have significantly altered affinity (i.e., increased or decreased) for given phosphoinositides. Variants that differ by as little as one amino acid in the PH domain can have completely different ligand recognition and/or can have vastly different affinity for the natural ligand when compared to the wild type polypeptide.  
      Accordingly, in at least one embodiment, the invention provides polypeptides comprising a variant PH domain. In one specific embodiment, the polypeptide has increased binding specificity for a phosphatidylinositide molecule to which the PH domain naturally binds. Alternatively, the polypeptide has decreased binding specificity for a phosphatidylinositide molecule to which the PH domain naturally binds.  
      In another related embodiment, the polypeptide has increased binding specificity for a phosphatidylinositide molecule to which the PH domain naturally does not bind. In yet another related embodiment, the polypeptide has decreased binding specificity for a phosphatidylinositide molecule to which the PH domain naturally does not bind. In specific embodiments, the phosphatidylinositide molecule is phosphatidylinositol-3,4,5 (PI-3,4,5)P3 or phosphatidylinositol-4,5 (PI-4,5)P2.  
      In related embodiments, the variant PH domains have increased or decreased affinity for phosphatidylinositol-3,4,5 (PI-3,4,5)P3 or phosphatidylinositol-4,5 (PI-4,5)P2.  
      In one specific embodiment the variant PH domain has at least one, two, or three glycine residues inserted in the β1/β2 loop as compared to the wild-type sequence.  
      In another embodiment, the variant PH domain comprises an amino acid substitution in a residue that does not contact the head group of a given phosphatidylinositol.  
      In another specific embodiment, the PH domain is present within a Grp1/ARNO/ Cytohesin family polypeptide.  
      In another embodiment, the invention provides a method of using a PH domain variant to selectively detect the presence of a specific phosphatidylinositide. In related embodiments, the phosphatidylinositide molecule is phosphatidylinositol-3,4,5 (PI-3,4,5)P3 or phosphatidylinositol-4,5 (PI-4,5)P2.  
      In specific embodiments of the invention, the polypeptide comprising a variant PH domain has a 10, 100, or 1000 fold higher specificity for a given phosphatidylinositide molecule than the wild-type polypeptide.  
      In one specific embodiment, the polypeptide comprising a variant PH domain has lost the ability to bind and/or recognize the natural ligand (e.g., phosphatidylinositol-3,4,5 (PI-3,4,5)P3 or phosphatidylinositol-4,5 (PI-4,5)P2).  
      In another embodiment, the invention provides a polypeptide comprising a variant PH domain wherein the variant (i) increases the affinity of the PH domain for one ligand while not changing the affinity for a second ligand: (ii) increases the affinity of the PH domain for one ligand while decreasing the affinity for a second ligand; or (iii) increases the affinity of the PH domain for one ligand while increasing the affinity for a second ligand. In certain embodiments the second ligand is a natural ligand of the PH domain. In another embodiment, the second ligand is not a natural ligand of the PH domain.  
      In another embodiment, the invention provides a polypeptide comprising a variant PH domain wherein the variant (i) decreases the affinity of the PH domain for one ligand while not changing the affinity for a second ligand; (ii) decreases the affinity of the PH domain for one ligand while decreasing the affinity for a second ligand; or (iii) variant decreases the affinity of the PH domain for one ligand while increasing the affinity for a second ligand. In certain embodiments the second ligand is a natural ligand of the PH domain. In another embodiment, the second ligand is not a natural ligand of the PH domain.  
      In one embodiment the invention provides a variant GRP1 polyeptide with a substitution selected from the group consisting of K273A, K282A, R284A, Y295F, R277A, R277C, V278A, V278C, K279A, K279C, T280A, T280C, R305A, K343A, N354A, and H355A of SEQ ID NO:1(Table 1).  
               TABLE 1                       GrP1 polypeptide sequence                                        LOCUS   NP_004218 399 aa linear PRI       DEFINITION   pleckstrin homology, Sec7 and coiled/coil domains 3; cytohesin 3;           ARF nucleotide-binding site opener 3; general receptor of           phosphoinositides 1 [ Homo sapiens ].       ACCESSION   NP_004218       VERSION   NP_004218.1 GI: 4758968       DBSOURCE   REFSEQ: accession NM_004227.3       KEYWORDS   .       SOURCE     Homo sapiens  (human)       ORGANISM     Homo sapiens             Eukaryota; Metazoa; Chordata; Craniata; Vertebrate; Euteleostomi;           Mammalia; Eutheria; Primates; Catarrhini; Hominidae; Homo.       REFERENCE   1 (residues 1 to 399)       AUTHORS   Ogasawara, M., Kim, S. C., Adamik, R., Togawa, A., Ferrans, V. J.,           Takeda, K., Kirby, M., Moss, J. and Vaughan, M.       TITLE   Similarities in function and gene structure of cytohesin-4 and           cytohesin-1, guanine nucleotide-exchange proteins for           ADP-ribosylation factors       JOURNAL   J. Biol. Chem. 275 (5), 3221-3230 (2000)       MEDLINE   20119275       PUBMED   10652308       REFERENCE   2 (residues 1 to 399)       AUTHORS   Venkateswarlu, K., Gunn-Moore, F., Oatey, P. B., Tavare, J. M. and           Cullen, P. J.       TITLE   Nerve growth factor- and epidermal growth factor-stimulated           translocation of the ADP-ribosylation factor-exchange factor GRP1           to the plasma membrane of PC12 cells requires activation of           phosphatidylinositol 3-kinase and the GRP1 pleckstrin homology           domain       JOURNAL   Biochem. J. 335 (Pt 1), 139-146 (1998)       MEDLINE   98416124       PUBMED   9742223       REFERENCE   3       AUTHORS   Franco, M., Boretto, J., Robineau, S., Monier, S., Goud, B., Chardin, P.           and Chavrier, P.       TITLE   ARNO3, a Sec7-domain guanine nucleotide exchange factor for ADP           ribosylation factor 1, is involved in the control of Golgi           structure and function       JOURNAL   Proc. Natl. Acad. Sci. U.S.A. 95 (17), 9926-9931 (1998)       MEDLINE   98374282       PUBMED   9707577       REFERENCE   4 (residues 1 to 399)       AUTHORS   Klarlund, J. K., Guilherme, A., Holik, J. J., Virbasius, J. V., Chawla, A.           and Czech, M. P.       TITLE   Signaling by phosphoinositide-3,4,5-trisphosphate through proteins           containing pleckstrin and Sec7 homology domains       JOURNAL   Science 275 (5308), 1927-1930 (1997)       MEDLINE   97228176       PUBMED   9072969       COMMENT   REVIEWED REFSEQ: This record has been curated by NCBI staff. The           reference sequence was derived from CB988199.1, AJ223957.1,           BC028717.1 and BG620295.1.           Summary: This gene encodes a member of the PSCD (pleckstrin           homology, Sec7 and coiled-coil domains) family. PSCD family members           have identical structural organization that consists of an           N-terminal coiled-coil motif, a central Sec7 domain, and a           C-terminal pleckstrin homology (PH) domain. The coiled-coil motif           is involved in homodimerization, the Sec7 domain contains           guanine-nucleotide exchange protein (GEP) activity, and the PH           domain interacts with phospholipids and is responsible for           association of PSCDs with membranes. Members of this family appear           to mediate the regulation of protein sorting and membrane           trafficking. This encoded protein is involved in the control of           Golgi structure and function, and it may have a physiological role           in regulating ADP-ribosylation factor protein 6 (ARF) functions, in           addition to acting on ARF1.       FEATURES   Location/Qualifiers       source   1..399           /organism=“ Homo sapiens ”           /db_xref=“taxon:9606”           /chromosome=“7”           /map=“7p22.2”       Protein   1..399           /product=“pleckstrin homology, Sec7 and coiled/coil           domains 3”           /note=“cytohesin 3; ARF nucleotide-binding site opener 3;           general receptor of phosphoinositides 1”       Region   65..248           /region_name=“Sec7 domain. The Sec7 domain is a           guanine-nucleotide-exchange-factor (GEF) for the pfam00025           family”           /note=“Sec7”           /db_xref=“CDD:pfam01369”       Region   66..248           /region_name=“Sec7 domain”           /note=“Sec7”           /db_xref=“CDD:14836”       Region   265..381           /region_name=“Pleckstrin homology domain”           /note=“PH”           /db_xref=“CDD:24224”       Region   265..381           /region_name=“Pleckstrin homology domain. Domain commonly           found in eukaryotic signalling proteins. The domain family           possesses multiple functions including the abilities to           bind inositol phosphates, and various proteins. PH domains           have been found to possess inserted domains (such as in           PLC gamma, syntrophins) and to be inserted within other           domains. Mutations in Brutons tyrosine kinase (Btk) within           its PH domain cause X-linked agammaglobulinaemia (XLA) in           patients. Point mutations cluster into the positively           charged end of the molecule around the predicted binding           site for phosphatidylinositol lipids”           /note=“PH”           /db_xref=“CDD:smart00233”       CDS   1..399           /gene=“PSCD3”           /coded_by=“NM_004227.3:105..1304”           /note=“go_component: membrane fraction [goid 0005624]           [evidence E] [pmid 9742223];           go_component: plasma membrane [goid 0005886] [evidence E]           [pmid 9742223];           go_function: phosphatidylinositol binding [goid 0005545]           [evidence E] [pmid 9742223];           go_function: ARF guanyl-nucleotide exchange factor           activity [goid 0005086] [evidence E] [pmid 9707577];           go_function: inositol-1,4,5-triphosphate receptor activity           [goid 0008095] [evidence P] [pmid 9742223];           go_process: vesicle-mediated transport [goid 0016192]           [evidence E] [pmid 9707577]”           /db_xref=“GeneID:9265”           /db_xref=“LocusID:9265”           /db_xref=“MIM:605081”                  
 
     
       
         
           
               
               
               
               
               
               
               
               
            
               
                   
               
               
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      In a related embodiment the invention provides a variant GRP 1 polyeptide having one or more of the following substitutions: of K273A, K282A, R284A, Y295F, R277A, R277G, V278A, V278C, K279A, K279G, T280A, T280G, R305A, K343A, N354A, and/or H355A of SEQ ID NO:1.  
      In one embodiment the invention provides a variant ARNO polyeptide with a substitution selected from the group consisting of K273A, K283A, R285A, Y296F, R278G, V279G, K280G, T281G, R306A, K344A, N355A, and H356A of SEQ ID NO:3 (Table II).  
               TABLE II                       ARNO polypeptide sequence                                        LOCUS   NP_004219 399 aa linear       DEFINITION   pleckstrin homology, Sec7 and coiled/coil domains 2 isoform 2;           pleckstrin homology, Sec7 and coiled/coil domains 2; cytohesin 2           [ Homo sapiens ].       ACCESSION   NP_004219       VERSION   NP_004219.1 GI: 4758966       DBSOURCE   REFSEQ: accession NM_004228.3       KEYWORDS   .       SOURCE     Homo sapiens  (human)       ORGANISM     Homo sapiens             Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi;           Mammalia; Eutheria; Primates; Catarrhini; Hominidae; Homo.       REFERENCE   1 (residues 1 to 399)       AUTHORS   Huh, M., Han, J. H., Lim, C. S., Lee, S. H., Kim, S., Kim, E. and           Kaang, B. K.       TITLE   Regulation of neuritogenesis and synaptic transmission by msec7-1,           a guanine nucleotide exchange factor, in cultured Aplysia neurons       JOURNAL   J. Neurochem. 85 (1), 282-285 (2003)       MEDLINE   22529431       PUBMED   12641750       REMARK   GeneRIF: The overexpression of ARNO, another mammalian GEF,           produces extensive neuritogenesis in Aplysia neurons       REFERENCE   2 (residues 1 to 399)       AUTHORS   Smith, J. S., Tachibana, I., Pohl, U., Lee, H. K., Thanarajasingam, U.,           Portier, B. P., Ueki, K., Ramaswamy, S., Billings, S. J.,           Mohrenweiser, H. W., Louis, D. N. and Jenkins, R. B.       TITLE   A transcript map of the chromosome 19q-arm glioma tumor suppressor           region       JOURNAL   Genomics 64 (1), 44-50 (2000)       MEDLINE   20175430       PUBMED   10708517       REFERENCE   3 (residues 1 to 399)       AUTHORS   Ogasawara, M., Kim, S. C., Adamik, R., Togawa, A., Ferrans, V. J.,           Takeda, K., Kirby, M., Moss, J. and Vaughan, M.       TITLE   Similarities in function and gene structure of cytohesin-4 and           cytohesin-1, guanine nucleotide-exchange proteins for           ADP-ribosylation factors       JOURNAL   J. Biol. Chem. 275 (5), 3221-3230 (2000)       MEDLINE   20119275       PUBMED   10652308       REFERENCE   4 (residues 1 to 399)       AUTHORS   Venkateswarlu, K., Oatey, P. B., Tavare, J. M. and Cullen, P. J.       TITLE   Insulin-dependent translocation of ARNO to the plasma membrane of           adipocytes requires phosphatidylinositol 3-kinase       JOURNAL   Curr. Biol. 8 (8), 463-466 (1998)       MEDLINE   98217355       PUBMED   9550703       REFERENCE   5 (residues 1 to 399)       AUTHORS   Cherfils, J., Menetrey, J., Mathieu, M., Le Bras, G., Robineau, S.,           Beraud-Dufour, S., Antonny, B. and Chardin, P.       TITLE   Structure of the Sec7 domain of the Arf exchange factor ARNO       JOURNAL   Nature 392 (6671), 101-105 (1998)       MEDLINE   98169075       PUBMED   9510256       REFERENCE   6 (residues 1 to 399)       AUTHORS   Mossessova, E., Gulbis, J. M. and Goldberg, J.       TITLE   Structure of the guanine nucleotide exchange factor Sec7 domain of           human arno and analysis of the interaction with ARF GTPase       JOURNAL   Cell 92 (3), 415-423 (1998)       MEDLINE   98135767       PUBMED   9476900       REFERENCE   7 (residues 1 to 399)       AUTHORS   Frank, S., Upender, S., Hansen, S. H. and Casanova, J. E.       TITLE   ARNO is a guanine nucleotide exchange factor for ADP-ribosylation           factor 6       JOURNAL   J. Biol. Chem. 273 (1), 23-27 (1998)       MEDLINE   98079021       PUBMED   9417041       REFERENCE   8 (residues 1 to 399)       AUTHORS   Chardin, P., Paris, S., Antonny, B., Robineau, S., Beraud-Dufour, S.,           Jackson, C. L. and Chabre, M.       TITLE   A human exchange factor for ARF contains Sec7- and           pleckstrin-homology domains       JOURNAL   Nature 384 (6608), 481-484 (1996)       MEDLINE   97100951       PUBMED   8945478       REFERENCE   9 (residues 1 to 399)       AUTHORS   Kolanus, W., Nagel, W., Schiller, B., Zeitlmann, L., Godar, S.,           Stockinger, H. and Seed, B.       TITLE   Alpha L beta 2 integrin/LFA-1 binding to ICAM-1 induced by           cytohesin-1, a cytoplasmic regulatory molecule       JOURNAL   Cell 86 (2), 233-242 (1996)       MEDLINE   96319726       PUBMED   8706128       COMMENT   REVIEWED REFSEQ: This record has been curated by NCBI staff. The           reference sequence was derived from X99753.1 and U70728.1.           Summary: Pleckstrin homology, Sec7 and coiled/coil domains 2           (PSCD2) is a member of the PSCD family. Members of this family have           identical structural organization that consists of an N-terminal           coiled-coil motif, a central Sec7 domain, and a C-terminal           pleckstrin homology (PH) domain. The coiled-coil motif is involved           in homodimerization, the Sec7 domain contains guanine-nucleotide           exchange protein (GEP) activity, and the PH domain interacts with           phospholipids and is responsible for association of PSCDs with           membranes. Members of this family appear to mediate the regulation           of protein sorting and membrane trafficking. PSCD2 exhibits GEP           activity in vitro with ARF1, ARF3, and ARF6. PSCD2 protein is 83%           homologous to PSCD1.           Transcript Variant: This transcript (2) is missing 3 bp in the PH           domain region, which results in a protein isoform missing a single           glycine residue.       FEATURES   Location/Qualifiers       source   1..399           /organism=“ Homo sapiens ”           /db_xref=“taxon:9606”           /chromosome=“19”           /map=“19q13.3”       Protein   1..399           /product=“pleckstrin homology, Sec7 and coiled/coil           domains 2 isoform 2”           /note=“pleckstrin homology, Sec7 and coiled/coil domains           2; cytohesin 2”       Region   13..54           /region_name=“Coiled-coil domain”       Region   60..243           /region_name=“Sec7 domain. The Sec7 domain is a           guanine-nucleotide-exchange-factor (GEF) for the           pfam00025           family”           /note=“Sec7”           /db_xref=“CDD:pfam01369”       Region   61..243           /region_name=“Sec7 domain”           /note=“Sec7”           /db_xref=“CDD:14836”       Region   72..252           /region_name=“Sec7 domain”       Region   260..375           /region_name=“Pleckstrin homology domain”           /note=“PH”           /db_xref=“CDD:24224”       Region   260..375           /region_name=“Pleckstrin homology domain. Domain commonly           found in eukaryotic signalling proteins. The domain           family           possesses multiple functions including the abilities to           bind inositol phosphates, and various proteins. PH           domains           have been found to possess inserted domains (such as in           PLC gamma, syntrophins) and to be inserted within other           domains. Mutations in Brutons tyrosine kinase (Btk) within           its PH domain cause X-linked agammaglobulinaemia (XLA) in           patients. Point mutations cluster into the positively           charged end of the molecule around the predicted binding           site for phosphatidylinositol lipids”           /note=“PH”           /db_xref=“CDD:smart00233”       Region   262..375           /region name=“PH domain”       CDS   1..399           /gene=“PSCD2”           /coded_by=“NM_004228.3:159..1358”           /note=“go_component: kinesin complex [goid 0005871]           [evidence IEA];           go_component: membrane fraction [goid 0005624] [evidence           TAS] [pmid 9417041];           go_component: plasma membrane [goid 0005886] [evidence           TAS] [pmid 9417041];           go_function: ARF guanyl-nucleotide exchange factor           activity [goid 0005086] [evidence TAS] [pmid 9417041];           go_function: guanyl-nucleotide release factor activity           [goid 0019839] [evidence IEA];           go_process: actin cytoskeleton reorganization [goid           0007012] [evidence TAS] [pmid 9417041];           go_process: endocytosis [goid 0006897] [evidence TAS]           [pmid 9417041]”           /db_xref=“GeneID:9266”           /db_xref=“LocusID:9266”           /db_xref=“MIM:602488”                  
 
     
       
         
           
               
               
               
               
               
               
               
               
            
               
                   
               
               
                 1 
                 medgvyeppd 
                 ltpeermele 
                 nirrrkqell 
                 veiqrlreel 
                 seamsevegl 
                 eanegsktlq 
                   
               
               
                   
               
               
                 61 
                 rnrkmamgrk 
                 kfnmdpkkgi 
                 qflvenellq 
                 ntpeeiarfl 
                 ykgeglnkta 
                 igdylgeree 
               
               
                   
               
               
                 121 
                 lnlavlhafv 
                 dlheftdlnl 
                 vqalrqflws 
                 frlpgeaqki 
                 drmmeafaqr 
                 yclcnpgvfq 
               
               
                   
               
               
                 181 
                 stdtcyvlsf 
                 avimlntslh 
                 npnvrdkpgl 
                 erfvamnrgi 
                 neggdlpeel 
                 lrnlydsirn 
               
               
                   
               
               
                 241 
                 epfkipeddg 
                 ndlthtffnp 
                 dregwllklg 
                 grvktwkrrw 
                 filtdnclyy 
                 feyttdkepr 
               
               
                   
               
               
                 301 
                 giiplenlsi 
                 revddprkpn 
                 cfelyipnnk 
                 gqlikackte 
                 adgrvvegnh 
                 mvyrisaptq 
               
               
                   
               
               
                 361 
                 eekdewiksi 
                 qaavsvdpfy 
                 emlaarkkri 
                 svkkkqeqp 
               
            
           
         
       
     
      In a related embodiment the invention provides a variant ARNO polyeptide having one or more of the following substitutions: K273A, K283A, R285A, Y296F, R278G, V279G, K280G, T281G, R306A, K344A, N355A, and/or H356A of SEQ ID NO:3.  
      PH Domains  
      Lipid binding domains that target intracellular membranes play a crucial role in the assembly of signaling and trafficking complexes and in membrane remodeling events such as vesicle budding, phagocytosis, and cell motility. The biological significance of membrane targeting is underscored by the prevalence of lipid binding domains, which rank amongst the most common domains in the eukaryotic proteome, and by the discovery of major proto-oncogene proteins and tumor suppressors containing essential lipid binding domains and/or lipid metabolic activities that regulate membrane association (1-4), There are several major classes of lipid binding domains including pleckstrin homology (PH), FYVE (acronym of Fabl, YOTB, Vacl, and EEA1), plant homeodomain (PHD), phox homology (PX), and C2 (named for homology with protein kinase C, PKC) domains as well as variety of smaller domain families and peptide motifs. The variation in physical properties and recognition mechanisms between and within families is striking.  
      The “pleckstrin homology” (PH) domain is a domain of about 100 residues that is present in a wide range of proteins involved in intracellular signaling or as constituents of the cytoskeleton. The pleckstrin homology domain is given PFAM accession number PF00169.  
      Lipids, Head Groups, and Phosphoinositides  
      Membranes consisting of phospholipid bilayers represent a ubiquitous component of all cells. With a large repertoire of chemically distinct lipids, the composition of biological membranes is highly complex and variable, depending both on the type of cell and organelle of interest. Lipid composition also varies within organelles, giving rise to microdomains with distinct physiochemical properties that reflect both the stereochemical and electrostatic characteristics of the head group as well as the length, saturation, and branching of the hydrocarbon chains. The more abundant phospholipids include phosphatidyl choline (PC), phosphatidyl ethanolamine (PE) and phosphatidyl serine (PS). PC and PE are neutral zwitterions, whereas PS bears a net negative charge. Though less abundant, mono and polyphosphorlyted derivatives of phosphatidyl inositol (PtdIns), collectively referred to as ‘phosphoinositides’, play a disproportionately critical role as targets for the known lipid binding domains. Several physiochemical properties that distinguish inositol from other common lipid head groups may well have contributed to the convergent evolution of functionally related yet structurally distinct phosphoinositide binding domains. With five equatorial hydroxyl substituents and a single axial hydroxyl group at the D2 position, the semi-rigid cyclohexane based ring of D-myo inositol represents a prominent landmark against the backdrop of typically smaller head groups. Reversible phosphorylation of the D3, D4, and/or D5 hydroxyl groups transforms an otherwise weakly anionic phospholipid, with a net negative charge of −1, into seven distinct derivatives: three monophosphates, three bisphosphates, and a single trisphosphate, with high net negative charges of −3, −5, and −7, respectively. The extensive literature on phosphoinositide metabolism by lipid kinases and phosphatases is covered in two recent reviews (5, 6). Given a high negative charge density, distributed over 2-4 phosphates in close proximity, it is not surprising that a strong positive electrostatic potential should be a common feature of the various domains that recognize phosphoinositides. What is more remarkable, in view of the pseudo-symmetry of the D-myo-inositol head group, is the high degree of stereochemical selectivity that lipid binding domains have evolved to distinguish even the most structurally similar phosphoinositides.  
      PH Domains  
      PH domains comprise one of the largest and most intensively investigated families of lipid binding domains (7-9). They were initially identified in signaling, cytoskeletal and metabolic proteins as evolutionarily conserved modules of −120 amino acids with weak homology to pleckstrin, a protein kinase C (PKC) substrate in platelets (10-13). As estimated by the human genome project, there are over 250 proteins containing one or more PH domains, making them one of the most common domains (14, 15). Of the PH domains that have been characterized, the majority bind weakly to phosphoinositides with little or no selectivity. An elite subset representing 10-20% of PH domains exhibit relatively high affinity (Kd for the head group in the low uM to nM range) and varying degrees of specificity for polyphosphoinositides (16-19).  
      Despite high sequence variability, NMR and crystal structures of more than dozen different PH domains have established a canonical core fold consisting of a seven stranded partly open p barrel, capped at one end by a C-terminal a helix (7, 20-28). Outside the core regions, the loops connecting the various secondary structural elements are best characterized as hypervariable with respect to composition, length, and structure, although similarities are apparent within sub-families. As discussed below, the hypervariable loops play a critical role in determining the functional properties, in particular the diverse affinity and specificity for phosphoinositides. Nevertheless, one property characteristic of PH domains that bind phosphosphoinositides, with either high or low affinity/selectivity, is a strongly dipolar electrostatic potential, with the positive lobe typically centered near the open end of the central p barrel (29, 30). This bulk electrostatic property accounts, at least in part, for the weak phosphoinositide affinities and specificities of many PH domains that correlate directly with the net charge of the head group (18). In these cases, preferences for phosphoinositides over other acidic lipids presumably derive from the higher negative charge density of mono- and polyphosphoinositides rather than stereochemical determinants.  
      A significant number of PH domains have be shown to bind polyphosphoinositides with relatively high affinity and with specificities dependent on the arrangement of phosphate groups attached to the inositol ring. These include the PtdIns(4,5)P2 specific PLC8 PH domain as well as the PtdIns(3,4,5)P3 specific PH domains of Bruton&#39;s Tyrosine Kinase (Btk) and General Receptor for 3-Phosphoinositides (Grp1) (16-19, 31-33). The PH domains of Dual Adapter for Phosphotyrosine and 3-Phosphoinositides (Dapp1) and the protein kinase B proto-oncogene (PKB/Akt) are promiscuous for Ptdms(3,4,)P 2  and PtdIns(3,4,5)P3 yet discriminate against PtdIns(4,5)P 2  (1, 17-19, 34-36). In a peculiar evolutionary twist, splice variants within the Grp1 family of PH domains, in which a single glycine residue is inserted at the N-terminus of the p1/p2 loop, bind promiscuously to either PtdIns(4,5)P 2  or PtdIns(3,4,5)P 2  (37, 38). Several of these PH domains have the property of binding with higher affinity to the head group than to the corresponding lipid (18). At least for the PH domains of Grp1, Btk, Dapp1, PKB/Akt, and PLC8, which target the plasma membrane, the affinity for the head group appears to be sufficient to drive membrane association, although other interactions may influence the precise localization within membrane microdomains.  
      Crystal structures of the aforementioned PH domains in complex with inositol polyphosphates provide insight into the determinants of phosphoinositide recognition (22, 23, 28, 39, 40). These PH domains conserve a basic signature motif, K—X m —(R/K)—X—R—X n —(Y/N), with the first lysine located near the C-terminus of the (β 1 strand, the (R/K)—X—R sequence near the N-terminus of the β 2 stand, and a tyrosine residue in the β3 strand (17-19, 22, 23}. In a variation on theme, the PKB/Akt PH domain substitutes the signature tyrosine with a functionally analogous asparagine residue from the β 3/β 4 loop (28). The first and third basic residues of the signature motif line the most deeply buried and positively charged region of the binding site and, together with the signature tyrosine/asparigine residue, mediate stereochemically equivalent interactions with either the 3- and 4-phosphates (Grp1, Btk, Dapp1, and PKB/Akt) or the 4-and 5-phosphates (PLCδ). Mutational analyses indicate that the signature residues, in particular the first and third basic residues, are critical but not sufficient for head group binding (17, 19, 31, 32, 41). With the exception of the PLCδ PH domain, the majority of the interactions with the head group are mediated by basic and polar residues from three ‘specificity determining regions’ (SDKs) corresponding to the hypervariable β 1/β 2, β 3/β 4, and β 6/β 7 loops, which flank the phospoinositide binding site at open end of the β barrel. Main chain NH groups in β 1/β2 loop mediate interactions with either the 5-phosphate (Btk and Grp1) or the 1-phosphate (PKB/Akt), reminiscent of P-loop interactions with phosphate groups in nucleotide binding proteins.  
      An important lesson from these studies is that similar specificities can be achieved through quite distinct structural mechanisms. For example, the relatively long (11 residue) P1/P2 loop in the Btk PH domain accounts for all of the interactions with the 5-phosphate and half of the contacts with the 4-phosphate. In the Grp1 PH domain, a twenty residue insertion in the P6/P7 loop adopts a P hairpin structure, which straddles the 4- and 5-phosphates, thereby compensating for a short (6 residue) p1/p2 loop. Equally significant structural variations are observed between the Dapp1 and PKB/Akt PH domains.  
      Grp1 Family PH Domains  
      The highly homologous proteins Grp1, ARNO (Arf nucleotide binding site opener), and cytohesin define a functionally related family with a modular domain architecture consisting of an N-terminal heptad repeat, a domain with exchange activity for Arf GTPases, a PH domain, and a C-terminal polybasic sequence. Alternative splice variants of ARNO and cytohesin give rise to full length proteins that differ only in the number glycine residues at the N-terminus of the β 1/β 2 loop in the PH domain. The ‘diglycine’ (2G) variants exhibit a strong selectivity for PtdIns(3,4,5)P 3 , with a 30 fold higher affinity compared with that for PtdIns(4,5)P2- In contrast, the ‘triglycine’ (3G) variants, which contain a glycine insertion relative to the diglycine variants, bind both PtdIns(4,5)P2 and PtdIns(3,4,5)P3 with comparable affinity.  
      Polypeptides  
      An “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the variant PH domain containing protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language “substantially free of cellular material” includes preparations of variant PH domain containing protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the language “substantially free of cellular material” includes preparations of variant PH domain containing protein having less than about 30% (by dry weight) of non-variant PH domain containing protein (also referred to herein as a “contaminating protein”), more preferably less than about 20% of non-variant PH domain containing protein, still more preferably less than about 10% of non-variant PH domain containing protein, and most preferably less than about 5% non-variant PH domain containing protein. When the variant PH domain containing protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.  
      The term “GAC family proteins” in intended to include polypeptides that are homologous to the GRP1/ARNO/Cytohesion polypeptides. Further, this term is intended to include polypeptides that are homologous to the PH domain from the GAC family of proteins, or that contain a PH domain that is homologous to a PH domain from a GAC family polypeptide.  
      The term “PH domain containing protein” is intended to include polypeptides that naturally have a PH domain or that have been genetically engineered to have a PH domain (e.g., chimeric or fusion proteins). Proteins that naturally have a PH domain include, but are not limited to, Grp1, Btk, Dapp1, PKB/Adk, and PCLδ.  
      The language “substantially free of chemical precursors or other chemicals” includes preparations of variant PH domain containing protein in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of variant PH domain containing protein having less than about 30% (by dry weight) of chemical precursors or non- variant PH domain containing chemicals, more preferably less than about 20% chemical precursors or non- variant PH domain containing chemicals, still more preferably less than about 10% chemical precursors or non- variant PH domain containing chemicals, and most preferably less than about 5% chemical precursors or non-variant PH domain containing chemicals.  
      In a preferred embodiment, the variant PH domain is a variant of the amino acid sequence shown in SEQ ID NO:5 or 6. In other embodiments, the variant PH domain containing protein is substantially identical to SEQ ID NO:5 or 6, and retains the functional activity of a Pleckstrin homology domain, yet differs in amino acid sequence. Accordingly, in another embodiment, the variant PH domain containing protein is a protein which comprises an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identical to SEQ ID NO:1 or 3, or the PH domain of SEQ ID NO:1 or 3 (SEQ ID NO:5 or 6). Further, the PH domain variant has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid insertions, deletions, or substitutions as compared to the wild-type sequence.  
      To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% of the length of the reference. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.  
      The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ( J. Mol. Biol . (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available online through the website of the Genetics Computer Group), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available online through the website of the Genetics Computer Group), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of Meyers, E. and Miller, W. ((1988)  Comput. Appl. Biosci.  4:11-17) which has been incorporated into the ALIGN program (version 2.0 or 2.0U), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.  
      The nucleic acid and protein sequences of the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990)  J. Mol. Biol.  215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to OP nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=100, wordlength=3 to obtain amino acid sequences homologous to OP protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997)  Nucleic Acids Res.  25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See the website of the National Center for Biotechnology Information.  
      Variants of the PH domain containing proteins can be generated by mutagenesis, e.g., discrete point mutation or truncation of a PH domain containing protein. Specific biological effects can be elicited by introducing mutations into the PH domain containing protein (see the Examples).  
      In one embodiment, a library of PH domain variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a gene library. A library of PH domain variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential PH domain sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of PH domain sequences therein. There are a variety of methods which can be used to produce libraries of potential PH domain variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential OP sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang, S. A. (1983)  Tetrahedron  39:3; Itakura et al. (1984)  Annu. Rev. Biochem.  53:323; Itakura et al. (1984)  Science  198:1056; Ike et al. (1983)  Nucleic Acid Res.  11:477.  
      Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of PH domain containing proteins. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a new technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify PH domain variants (Arkin and Youvan (1992)  Proc. Natl. Acad. Sci. USA  89:7811-7815; Delagrave et al. (1993)  Protein Eng.  6(3):327-331).  
      Nucleic Acid Molecules  
      One aspect of the invention pertains to nucleic acid molecules that encode variant PH domain containing proteins. As used herein, the term “nucleic acid molecule” is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.  
      A nucleic acid molecule of the present invention, e.g., a nucleic acid molecule that is a variant of SEQ ID NO:2 or 4 (Table III and IV, respectively) can be isolated using standard molecular biology techniques and the sequence information provided herein.  
               TABLE III                       GrP1 nucleic acid sequence                                        LOCUS   NM_004227 4482 bp mRNA linear       DEFINITION     Homo sapiens  pleckstrin homology, Sec7 and coiled-coil domains 3           (PSCD3), mRNA.       ACCESSION   NM_004227       VERSION   NM_004227.3 GI: 33946275       KEYWORDS   .       SOURCE     Homo sapiens  (human)       ORGANISM     Homo sapiens             Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi;           Mammalia; Eutheria; Primates; Catarrhini; Hominidae; Homo.       REFERENCE   1 (bases 1 to 4482)       AUTHORS   Ogasawara, M., Kim, S. C., Adamik, R., Togawa, A., Ferrans, V. J.,           Takeda, K., Kirby, M., Moss, J. and Vaughan, M.       TITLE   Similarities in function and gene structure of cytohesin-4 and           cytohesin-1, guanine nucleotide-exchange proteins for ADP-ribosylation factors       JOURNAL   J. Biol. Chem. 275 (5), 3221-3230 (2000)       MEDLINE   20119275       PUBMED   10652308       REFERENCE   2 (bases 1 to 4482)       AUTHORS   Venkateswarlu, K., Gunn-Moore, F., Oatey, P. B., Tavare, J. M. and           Cullen, P. J.       TITLE   Nerve growth factor- and epidermal growth factor-stimulated           translocation of the ADP-ribosylation factor-exchange factor GRP1           to the plasma membrane of PC12 cells requires activation of           phosphatidylinositol 3-kinase and the GRP1 pleckstrin homology           domain       JOURNAL   Biochem. J. 335 (Pt 1), 139-146 (1998)       MEDLINE   98416124       PUBMED   9742223       REFERENCE   3       AUTHORS   Franco, M., Boretto, J., Robineau, S., Monier, S., Goud, B., Chardin, P.           and Chavrier, P.       TITLE   ARNO3, a Sec7-domain guanine nucleotide exchange factor for ADP           ribosylation factor 1, is involved in the control of Golgi           structure and function       JOURNAL   Proc. Natl. Acad. Sci. U.S.A. 95 (17), 9926-9931 (1998)       MEDLINE   98374282       PUBMED   9707577       REFERENCE   4 (bases 1 to 4482)       AUTHORS   Klarlund, J. K., Guilherme, A., Holik, J. J., Virbasius, J. V., Chawla, A.           and Czech, M. P.       TITLE   Signaling by phosphoinositide-3,4,5-trisphosphate through proteins           containing pleckstrin and Sec7 homology domains       JOURNAL   Science 275 (5308), 1927-1930 (1997)       MEDLINE   97228176       PUBMED   9072969       COMMENT   REVIEWED REFSEQ: This record has been curated by NCBI staff. The           reference sequence was derived from CB988199.1, AJ223957.1,           BC028717.1 and BG620295.1.           On Aug 20, 2003 this sequence version replaced gi: 8670548.           Summary: This gene encodes a member of the PSCD (pleckstrin           homology, Sec7 and coiled-coil domains) family. PSCD family           members           have identical structural organization that consists of an           N-terminal coiled-coil motif, a central Sec7 domain, and a           C-terminal pleckstrin homology (PH) domain. The coiled-coil motif           is involved in homodimerization, the Sec7 domain contains           guanine-nucleotide exchange protein (GEP) activity, and the PH           domain interacts with phospholipids and is responsible for           association of PSCDs with membranes. Members of this family           appear to mediate the regulation of protein sorting and membrane           trafficking. This encoded protein is involved in the control of           Golgi structure and function, and it may have a physiological role           in regulating ADP-ribosylation factor protein 6 (ARF) functions,           in addition to acting on ARF1.           COMPLETENESS: complete on the 3′ end.       FEATURES   Location/Qualifiers       source   1..4482           /organism=“ Homo sapiens ”           /mol_type=“mRNA”           /db_xref=“taxon:9606”           /chromosome=“7”           /map=“7p22.2”       gene   1..4482           /gene=“PSCD3”           /note=“synonyms:GRP1, ARNO3”           /db_xref=“GeneID:9265”           /db_xref=“LocusID:9265”           /db_xref=“MIM:605081”       CDS   105..1304           /gene=“PSCD3”           /note=“cytohesin 3; ARF nucleotide-binding site opener 3;           general receptor of phosphoinositides 1;           go_component: membrane fraction [goid 0005624] [evidence           E] [pmid 9742223];           go_component: plasma membrane [goid 0005886] [evidence E]           [pmid 9742223];           go_function: phosphatidylinositol binding [goid 0005545]           [evidence E] [pmid 9742223];           go_function: ARF guanyl-nucleotide exchange factor           activity [goid 0005086] [evidence E] [pmid 9707577];           go_function: inositol-1,4,5-triphosphate receptor           activity           [goid 0008095] [evidence P] [pmid 9742223];           go_process: vesicle-mediated transport [goid 0016192]           [evidence E] [pmid 9707577]”           /codon_start=1           /product=“pleckstrin homology, Sec7 and coiled/coil           domains 3”           /protein_id=“NP_004218.1”           /db_xref=“GI:4758968”           /db_xref=“GeneID:9265”           /db_xref=“LocusID:9265”           /db_xref=“MIM:605081”           translation=“MDEDGGGEGGGVPEDLSLEEREELLDIRRRKKELIDDIERLKYE                 IAEVMTEIDNLTSVEESKTTQRNKQIAMGRKKFNMDPKKGIQFLIENDLLQSSPEDVA       QFLYKGEGLNKTVIGDYLGERDEFNIKVLQAFVELHEFADLNLVQALRQFLWSFRLPG       EAQKIDRMMEAFASRYCLCNPGVFQSTDTCYVLSFAIIMLNTSLHNHNVRDKPTAERF       IAMNRGINEGGDLPEELLRNLYESIKNEPFKIPEDDGNDLTHTFFNPDREGWLLKLGG       RVKTWKRRWFILTDNCLYYFEYTTDKEPRGIIPLENLSIREVEDPRKPNCFELYNPSH       KGQVIKACKTEADGRVVEGNHVVYRISAPSPEEKEEWMKSIKASISRDPFYDMLATRK                         RRIANKK”       misc_feature   297..848           /gene=“PSCD3”           /note=“Sec7; Region: Sec7 domain. The Sec7 domain is a           guanine-nucleotide-exchange-factor (GEF) for the           pfam00025           family”           /db_xref=“CDD:pfam01369”       misc_feature   897..1247           /gene=“PSCD3”           /note=“PH; Region: Pleckstrin homology domain. Domain           commonly found in eukaryotic signalling proteins. The           domain family possesses multiple functions including the           abilities to bind inositol phosphates, and various           proteins. PH domains have been found to possess inserted           domains (such as in PLC gamma, syntrophins) and to be           inserted within other domains. Mutations in Brutons           tyrosine kinase (Btk) within its PH domain cause X-linked           agammaglobulinaemia (XLA) in patients. Point mutations           cluster into the positively charged end of the molecule           around the predicted binding site for           phosphatidylinositol           lipids”           /db_xref=“CDD:smart00233”       polyA_signal   4444..4449           /gene=“PSCD3”       polyA_site   4469           /gene=“PSCD3”           /evidence=experimental                  
 
     
       
         
           
               
               
               
               
               
               
               
               
            
               
                   
               
               
                 1 
                 tgaggagccg 
                 cccggtcgcc 
                 tgcgcgctcc 
                 ctccggcggc 
                 gtccccagcc 
                 cgcggcccct 
                   
               
               
                   
               
               
                 61 
                 ctgctgccgg 
                 cccccggctc 
                 gccggctgcg 
                 ggagtggcct 
                 caagatggat 
                 gaagacggcg 
               
               
                   
               
               
                 121 
                 gcggcgaggg 
                 tggtggcgtg 
                 cctgaagacc 
                 tctcattaga 
                 agagagagaa 
                 gaacttctag 
               
               
                   
               
               
                 181 
                 acattcgtcg 
                 aagaaaaaag 
                 gaacttattg 
                 atgacattga 
                 gaggctgaaa 
                 tatgaaattg 
               
               
                   
               
               
                 241 
                 cagaggtgat 
                 gacagagatc 
                 gacaatctaa 
                 cttccgtaga 
                 ggagagcaaa 
                 acgactcaga 
               
               
                   
               
               
                 301 
                 ggaacaaaca 
                 gatagccatg 
                 ggaagaaaga 
                 aattcaacat 
                 ggatcccaaa 
                 aagggaattc 
               
               
                   
               
               
                 361 
                 agtttctaat 
                 agaaaatgac 
                 ctgctacaga 
                 gttccccaga 
                 agacgtcgcc 
                 cagttccttt 
               
               
                   
               
               
                 421 
                 ataaaggaga 
                 aggcctaaat 
                 aagaccgtca 
                 ttggggacta 
                 cctgggtgaa 
                 agggatgaat 
               
               
                   
               
               
                 481 
                 ttaatattaa 
                 agttcttcaa 
                 gcctttgttg 
                 aactccatga 
                 gtttgctgat 
                 ctcaaccttg 
               
               
                   
               
               
                 541 
                 tacaagcctt 
                 aaggcagttc 
                 ttatggagct 
                 tcaggctgcc 
                 cggggaggcg 
                 cagaagattg 
               
               
                   
               
               
                 601 
                 atcgcatgat 
                 ggaggctttc 
                 gcttctcgct 
                 actgcctgtg 
                 caaccccggg 
                 gtcttccagt 
               
               
                   
               
               
                 661 
                 ccacagacac 
                 gtgctacgtg 
                 ctgtcattcg 
                 ccatcatcat 
                 gctcaacacc 
                 agcctccaca 
               
               
                   
               
               
                 721 
                 accacaacgt 
                 gcgtgacaag 
                 cccacggcag 
                 aacggttcat 
                 cgccatgaac 
                 cgcggcatca 
               
               
                   
               
               
                 781 
                 acgagggcgg 
                 ggacctccct 
                 gaggagctgc 
                 tgaggaattt 
                 gtatgagagc 
                 attaagaacg 
               
               
                   
               
               
                 841 
                 agccatttaa 
                 gatcccggag 
                 gacgacggga 
                 acgacctgac 
                 ccacaccttc 
                 ttcaaccccg 
               
               
                   
               
               
                 901 
                 accgcgaggg 
                 ctggctcctg 
                 aagctgggag 
                 ggcgtgtgaa 
                 gacctggaag 
                 cgccggtggt 
               
               
                   
               
               
                 961 
                 tcatcctgac 
                 cgataactgc 
                 ctctattact 
                 ttgaatacac 
                 aacagataag 
                 gagcccaggg 
               
               
                   
               
               
                 1021 
                 gaatcatccc 
                 gttggaaaac 
                 ctcagcatca 
                 gggaggtgga 
                 ggacccccgg 
                 aaacccaact 
               
               
                   
               
               
                 1081 
                 gttttgagct 
                 ctacaatccc 
                 agccacaaag 
                 ggcaggtcat 
                 caaggcctgt 
                 aagactgagg 
               
               
                   
               
               
                 1141 
                 ccgacggccg 
                 cgtggtagag 
                 gggaaccatg 
                 tggtgtaccg 
                 gatctcagcc 
                 ccgagcccgg 
               
               
                   
               
               
                 1201 
                 aggagaagga 
                 ggagtggatg 
                 aaatccatca 
                 aagccagtat 
                 cagcagagat 
                 cccttctatg 
               
               
                   
               
               
                 1261 
                 acatgttggc 
                 aacgaggaaa 
                 cgaaggattg 
                 ccaataaaaa 
                 atagctttcc 
                 tggctaaaag 
               
               
                   
               
               
                 1321 
                 acccaggtaa 
                 aagacccaac 
                 cccagcagaa 
                 agacaccgcg 
                 ggcggcccct 
                 ccgcggaagg 
               
               
                   
               
               
                 1381 
                 cgtggcaggg 
                 aggcagtcgc 
                 cctgcggtgc 
                 aagctgctgc 
                 tccagagcat 
                 accgtggccc 
               
               
                   
               
               
                 1441 
                 aggtggtatc 
                 cccaaggcct 
                 cgtgccgtgg 
                 ctggggtcct 
                 gggaggtggt 
                 cgccctgcag 
               
               
                   
               
               
                 1501 
                 tgcaagctgc 
                 tgctccagag 
                 cgtaccgtgg 
                 cccagactga 
                 tcctcgaggc 
                 ctcctgccgt 
               
               
                   
               
               
                 1561 
                 ggctggggtc 
                 atggtcggct 
                 gcgcatgtcc 
                 agaagcattt 
                 ccttcctgcg 
                 accatcccgg 
               
               
                   
               
               
                 1621 
                 cgcccctagg 
                 gggagaagcc 
                 aggacagcag 
                 cttccgctgt 
                 ctccacagca 
                 gacacgggac 
               
               
                   
               
               
                 1681 
                 ggattccaca 
                 gacgggagcc 
                 tcattcgtac 
                 catgccaaac 
                 gcattcactc 
                 ggggcagtat 
               
               
                   
               
               
                 1741 
                 taaccgttct 
                 agaaagccac 
                 tgttttatag 
                 caaaacagga 
                 aaggaaaagc 
                 taccagtttt 
               
               
                   
               
               
                 1801 
                 ttattcagaa 
                 tttttctcag 
                 atatatagga 
                 ttatagcttt 
                 tatatgcctt 
                 tttatattct 
               
               
                   
               
               
                 1861 
                 gaaattataa 
                 caaaagatac 
                 tttctaacag 
                 tagtattttt 
                 agaatggcag 
                 ctataaagtt 
               
               
                   
               
               
                 1921 
                 aactcctgga 
                 cacaagtata 
                 tactgtgcac 
                 tgaaaaaata 
                 tccatctaca 
                 cagcacccaa 
               
               
                   
               
               
                 1981 
                 ggggagggct 
                 gggggcaccg 
                 gcacgggggc 
                 agcgtgcagc 
                 cctgccctgt 
                 caggctgtca 
               
               
                   
               
               
                 2041 
                 gacaagcccc 
                 ggggggcagc 
                 aggtgggctc 
                 gggacgggct 
                 gggggaggga 
                 cggccatggc 
               
               
                   
               
               
                 2101 
                 acttgggggc 
                 tccagggtga 
                 ctcccatgag 
                 gcctcccttc 
                 aaccaggctt 
                 tttggcccca 
               
               
                   
               
               
                 2161 
                 caaatacttt 
                 aagcaaatca 
                 ttaaaattat 
                 aacagttaat 
                 ggtttggggg 
                 tgtttaggct 
               
               
                   
               
               
                 2221 
                 gtaactgcta 
                 actcctagga 
                 aacagccttt 
                 tccctggaca 
                 cagatggtcc 
                 atacgctgag 
               
               
                   
               
               
                 2281 
                 ccacgtgaaa 
                 ctgctgatgt 
                 tttgtttaga 
                 tgcacacaca 
                 tggcagcgtt 
                 tcatacaggt 
               
               
                   
               
               
                 2341 
                 cagcaggtta 
                 gaccggcttt 
                 tgaccatatt 
                 catcgctatt 
                 taaaacctgt 
                 ggcaaaatga 
               
               
                   
               
               
                 2401 
                 acgcttattt 
                 tacagacttt 
                 ctaatttgac 
                 cagatttctt 
                 aatgaataga 
                 cacagaatta 
               
               
                   
               
               
                 2461 
                 actaaaaaca 
                 gtctcacccc 
                 atgtagtgcg 
                 ccgtgtcctg 
                 agagaggtgc 
                 cctccctacg 
               
               
                   
               
               
                 2521 
                 aggagggaag 
                 aacaggccct 
                 ggggtgcaga 
                 ggcccggcac 
                 gtagagaacc 
                 cagatagacg 
               
               
                   
               
               
                 2581 
                 ccggtggtgg 
                 aactggtcaa 
                 actccacgcc 
                 cgcctgggag 
                 gttgtcaggt 
                 tgctgtggat 
               
               
                   
               
               
                 2641 
                 gtaaggatag 
                 gaggtgccca 
                 gtgctccgct 
                 caaggaaggc 
                 tggatctggg 
                 ccccacctac 
               
               
                   
               
               
                 2701 
                 agagagggct 
                 cagggctgga 
                 ccgggggcat 
                 tgtgtgcttg 
                 ggccgacccg 
                 ggccggtggc 
               
               
                   
               
               
                 2761 
                 agacgctgtt 
                 ctctgtcggg 
                 agatttgcgt 
                 ccccaggacc 
                 ctgttacaca 
                 gtgggctgtt 
               
               
                   
               
               
                 2821 
                 gggttggtgg 
                 ctggcttttc 
                 ctctatggac 
                 ttcctcttcc 
                 tgccccacct 
                 gcataggcac 
               
               
                   
               
               
                 2881 
                 acacaccttg 
                 aatctgcacc 
                 ctctggaggg 
                 catctgtact 
                 cctgtgcaaa 
                 atgcccagtc 
               
               
                   
               
               
                 2941 
                 cagagacaaa 
                 acctcagact 
                 ttgtgcacct 
                 aggtttcctt 
                 ctcagcagcg 
                 gagactgttc 
               
               
                   
               
               
                 3001 
                 tttgagttgc 
                 cttgaagtgg 
                 aggccgagcg 
                 gctgcgggcc 
                 cttcgcctcc 
                 ctgcggctga 
               
               
                   
               
               
                 3061 
                 ccttgatgta 
                 gctttaagtc 
                 acactagact 
                 gcagaggggt 
                 ccgaggccag 
                 aaacccctgt 
               
               
                   
               
               
                 3121 
                 cctgcatcag 
                 actttcattc 
                 ccacgttctt 
                 aggctttgtt 
                 actgatacct 
                 caaatcggaa 
               
               
                   
               
               
                 3181 
                 gttttagttc 
                 tgagaaaggc 
                 aagtcagcgt 
                 tcttgaaatg 
                 cctgactggt 
                 agatatgcaa 
               
               
                   
               
               
                 3241 
                 ctctggcctc 
                 cagtcttcca 
                 tgaaaataaa 
                 tgctgcctgg 
                 acccccaccc 
                 agaccacaca 
               
               
                   
               
               
                 3301 
                 ctgacggccg 
                 gctccggcgg 
                 tgcccacccc 
                 tcaggctggc 
                 ccggcaccca 
                 agactggcca 
               
               
                   
               
               
                 3361 
                 cagccagctc 
                 tgtcagcatg 
                 ttgtgctcgg 
                 acaagctgtt 
                 tccttcttct 
                 gaccaaccca 
               
               
                   
               
               
                 3421 
                 ggtgtgacct 
                 ggggatgcag 
                 agctttctgt 
                 tttgggtgtt 
                 gggagaagca 
                 gcaggaagga 
               
               
                   
               
               
                 3481 
                 gtcgccagat 
                 gatcaagctc 
                 cccctttgct 
                 gtcatctgtg 
                 aatgagcttc 
                 gccaggtggt 
               
               
                   
               
               
                 3541 
                 gggcacctgg 
                 gagccatgca 
                 gaggctgtgg 
                 tgctgagtta 
                 gactccaggt 
                 actttgtggt 
               
               
                   
               
               
                 3601 
                 caaaggaaat 
                 cgcctagctc 
                 caggctgtgt 
                 taggacagta 
                 ttagcatgaa 
                 ggctgtgcga 
               
               
                   
               
               
                 3661 
                 ccatcatgcc 
                 tgctgatcct 
                 tgaggcaggc 
                 ctggtccaga 
                 aaactctggg 
                 tcagtgactg 
               
               
                   
               
               
                 3721 
                 cgcagggcca 
                 gccgctacca 
                 ggacggccct 
                 gaaacaggac 
                 acatctgttt 
                 tttgtccctc 
               
               
                   
               
               
                 3781 
                 accctgggca 
                 ggccgcgtca 
                 caatcacagt 
                 cctcctcctc 
                 cccaccctga 
                 cgtctgagcg 
               
               
                   
               
               
                 3841 
                 cagggcttga 
                 attgttagtc 
                 ccaactctgg 
                 ccaaagatac 
                 ttttttccag 
                 agacagaggc 
               
               
                   
               
               
                 3901 
                 caggaggcag 
                 tgaggggagc 
                 cccgcgggga 
                 ggcggcggcg 
                 actgccacag 
                 cccttccagc 
               
               
                   
               
               
                 3961 
                 ctgtcttgct 
                 ggccgccctg 
                 gttcatattt 
                 gagtttaatt 
                 gtactgaccc 
                 tggacccaga 
               
               
                   
               
               
                 4021 
                 taagcagcaa 
                 ctttgtgtct 
                 ttggggtcac 
                 agaacatttt 
                 ggggcagttt 
                 aatgtggtac 
               
               
                   
               
               
                 4081 
                 caaactgaaa 
                 ataggagcta 
                 tttatagatg 
                 gagcagcact 
                 tagtgcttca 
                 tagaaagcaa 
               
               
                   
               
               
                 4141 
                 tgcctatttt 
                 taaagttaca 
                 aacgcagata 
                 tctacataga 
                 tatgctttgc 
                 tgagaagtta 
               
               
                   
               
               
                 4201 
                 ggtctgtggt 
                 agaccagaaa 
                 ccacaaattg 
                 actttttttc 
                 ttagaaaata 
                 tttctatttg 
               
               
                   
               
               
                 4261 
                 cggtaaatat 
                 agtaatatgt 
                 aaataatgta 
                 catctgttga 
                 tttctggagt 
                 gtctgttatt 
               
               
                   
               
               
                 4321 
                 caatgatgta 
                 tatactccca 
                 cagctcgcat 
                 gaaggaacag 
                 cctctattga 
                 tacttggttg 
               
               
                   
               
               
                 4381 
                 taaagtgaag 
                 taagattgga 
                 gggtggatgg 
                 ctgtcagagc 
                 tcttgcagat 
                 actgtgttca 
               
               
                   
               
               
                 4441 
                 ctaaataaaa 
                 atcacatgta 
                 ttgttaaaaa 
                 aaaaaaaaaa 
                 aa 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE IIII 
               
               
                   
               
               
                   
               
               
                 ARNO Nucleic Acid sequence 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                 LOCUS 
                 NM_004228 1358 bp mRNA linear 
               
               
                 DEFINITION 
                   Homo sapiens  pleckstrin homology, Sec7 and coiled-coil domains 2 
               
               
                   
                 (cytohesin-2) (PSCD2), transcript variant 2, mRNA. 
               
               
                 ACCESSION 
                 NM_004228 
               
               
                 VERSION 
                 NM_004228.3 GI: 10880123 
               
               
                 KEYWORDS 
                 . 
               
               
                 SOURCE 
                   Homo sapiens  (human) 
               
               
                 ORGANISM 
                 
                   Homo sapiens 
                 
               
               
                   
                 Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; 
               
               
                   
                 Mammalia; Eutheria; Primates; Catarrhini; Hominidae, Homo. 
               
               
                 REFERENCE 
                 1 (bases 1 to 1358) 
               
               
                 AUTHORS 
                 Huh, M., Han, J. H., Lim, C. S., Lee, S. H., Kim, S., Kim, E. and Kaang, B. K. 
               
               
                 TITLE 
                 Regulation of neuritogenesis and synaptic transmission by msec7-1. 
               
               
                   
                 a guanine nucleotide exchange factor, in cultured Aplysia neurons 
               
               
                 JOURNAL 
                 J. Neurochem. 85 (1), 282-285 (2003) 
               
               
                 MEDLINE 
                 22529431 
               
               
                 PUBMED 
                 12641750 
               
               
                 REMARK 
                 GeneRIF: The overexpression of ARNO, another mammalian GEF, 
               
               
                   
                 produces extensive neuritogenesis in Aplysia neurons 
               
               
                 REFERENCE 
                 2 (bases 1 to 1358) 
               
               
                 AUTHORS 
                 Smith, J. S., Tachibana, I., Pohl, U., Lee, H. K., Thanarajasingam, U., 
               
               
                   
                 Portier, B. P., Ueki, K., Ramaswamy, S. , Billings, S. J., 
               
               
                   
                 Mohrenweiser, H. W., Louis, D. N. and Jenkins, R. B. 
               
               
                 TITLE 
                 A transcript map of the chromosome 19q-arm glioma tumor suppressor 
               
               
                   
                 region 
               
               
                 JOURNAL 
                 Genomics 64 (1), 44-50 (2000) 
               
               
                 MEDLINE 
                 20175430 
               
               
                 PUBMED 
                 10708517 
               
               
                 REFERENCE 
                 3 (bases 1 to 1358) 
               
               
                 AUTHORS 
                 Ogasawara, M., Kim, S. C., Adamik, R., Togawa, A., Ferrans, V. J., 
               
               
                   
                 Takeda, K., Kirby, M., Moss, J. and Vaughan, M. 
               
               
                 TITLE 
                 Similarities in function and gene structure of cytohesin-4 and 
               
               
                   
                 cytohesin-1, guanine nucleotide-exchange proteins for 
               
               
                   
                 ADP-ribosylation factors 
               
               
                 JOURNAL 
                 J. Biol. Chem. 275 (5), 3221-3230 (2000) 
               
               
                 MEDLINE 
                 20119275 
               
               
                 PUBMED 
                 10652308 
               
               
                 REFERENCE 
                 4 (bases 1 to 1358) 
               
               
                 AUTHORS 
                 Venkateswarlu, K., Oatey, P. B., Tavare, J. M. and Cullen, P. J. 
               
               
                 TITLE 
                 Insulin-dependent translocation of ARNO to the plasma membrane of 
               
               
                   
                 adipocytes requires phosphatidylinositol 3-kinase 
               
               
                 JOURNAL 
                 Curr. Biol. 8 (8), 463-466 (1998) 
               
               
                 MEDLINE 
                 98217355 
               
               
                 PUBMED 
                 9550703 
               
               
                 REFERENCE 
                 5 (bases 1 to 1358) 
               
               
                 AUTHORS 
                 Cherfils, J., Menetrey, J., Mathieu, M., Le Bras, G., Robineau, S., 
               
               
                   
                 Beraud-Dufour, S., Antonny, B. and Chardin, P. 
               
               
                 TITLE 
                 Structure of the Sec7 domain of the Arf exchange factor ARNO 
               
               
                 JOURNAL 
                 Nature 392 (6671), 101-105 (1998) 
               
               
                 MEDLINE 
                 98169075 
               
               
                 PUBMED 
                 9510256 
               
               
                 REFERENCE 
                 6 (bases 1 to 1358) 
               
               
                 AUTHORS 
                 Mossessova, E., Gulbis, J. M. and Goldberg, J. 
               
               
                 TITLE 
                 Structure of the guanine nucleotide exchange factor Sec7 domain of 
               
               
                   
                 human arno and analysis of the interaction with ARF GTPase 
               
               
                 JOURNAL 
                 Cell 92 (3), 415-423 (1998) 
               
               
                 MEDLINE 
                 98135767 
               
               
                 PUBMED 
                 9476900 
               
               
                 REFERENCE 
                 7 (bases 1 to 1358) 
               
               
                 AUTHORS 
                 Frank, S., Upender, S., Hansen, S. H. and Casanova, J. E. 
               
               
                 TITLE 
                 ARNO is a guanine nucleotide exchange factor for ADP-ribosylation 
               
               
                   
                 factor 6 
               
               
                 JOURNAL 
                 J. Biol. Chem. 273 (1), 23-27 (1998) 
               
               
                 MEDLINE 
                 98079021 
               
               
                 PUBMED 
                 9417041 
               
               
                 REFERENCE 
                 8 (bases 1 to 1358) 
               
               
                 AUTHORS 
                 Chardin, P., Paris, S., Antonny, B., Robineau, S., Beraud-Dufour, S., 
               
               
                   
                 Jackson, C. L. and Chabre, M. 
               
               
                 TITLE 
                 A human exchange factor for ARF contains Sec7- and 
               
               
                   
                 pleckstrin-homology domains 
               
               
                 JOURNAL 
                 Nature 384 (6608), 481-484 (1996) 
               
               
                 MEDLINE 
                 97100951 
               
               
                 PUBMED 
                 8945478 
               
               
                 REFERENCE 
                 9 (bases 1 to 1358) 
               
               
                 AUTHORS 
                 Kolanus, W., Nagel, W., Schiller, B., Zeitlmann, L. , Godar, S., 
               
               
                   
                 Stockinger, H. and Seed, B. 
               
               
                 TITLE 
                 Alpha L beta 2 integrin/LFA-1 binding to ICAM-1 induced by 
               
               
                   
                 cytohesin-1, a cytoplasmic regulatory molecule 
               
               
                 JOURNAL 
                 Cell 86 (2), 233-242 (1996) 
               
               
                 MEDLINE 
                 96319726 
               
               
                 PUBMED 
                 8706128 
               
               
                 COMMENT 
                 REVIEWED REFSEQ: This record has been curated by NCBI staff. The 
               
               
                   
                 reference sequence was derived from X99753.1 and U70728.1. 
               
               
                   
                 On Oct. 18, 2000 this sequence version replaced gi: 8670547. 
               
               
                   
                 Summary: Pleckstrin homology, Sec7 and coiled/coil domains 2 
               
               
                   
                 (PSCD2) is a member of the PSCD family. Members of this family have 
               
               
                   
                 identical structural organization that consists of an N-terminal 
               
               
                   
                 coiled-coil motif, a central Sec7 domain, and a C-terminal 
               
               
                   
                 pleckstrin homology (PH) domain. The coiled-coil motif is involved 
               
               
                   
                 in homodimerization, the Sec7 domain contains guanine-nucleotide 
               
               
                   
                 exchange protein (GEP) activity, and the PH domain interacts with 
               
               
                   
                 phospholipids and is responsible for association of PSCDs with 
               
               
                   
                 membranes. Members of this family appear to mediate the regulation 
               
               
                   
                 of protein sorting and membrane trafficking. PSCD2 exhibits GEP 
               
               
                   
                 activity in vitro with ARF1, ARF3, and ARF6. PSCD2 protein is 83% 
               
               
                   
                 homologous to PSCD1. 
               
               
                   
                 Transcript Variant: This transcript (2) is missing 3 bp in the PH 
               
               
                   
                 domain region, which results in a protein isoform missing a single 
               
               
                   
                 glycine residue. 
               
               
                 FEATURES 
                 Location/Qualifiers 
               
               
                 source 
                 1..1358 
               
               
                   
                 /organism=“ Homo sapiens ” 
               
               
                   
                 /mol_type=“mRNA” 
               
               
                   
                 /db_xref=“taxon:9606” 
               
               
                   
                 /chromosome=“19” 
               
               
                   
                 /map=“19q13.3” 
               
               
                 gene 
                 1..1358 
               
               
                   
                 /gene=“PSCD2” 
               
               
                   
                 /note=“synonyms: ARNO, CTS18.1, Sec7p-L” 
               
               
                   
                 /db_xref=“GeneID:9266” 
               
               
                   
                 /db_xref=“LocusID:9266” 
               
               
                   
                 /db_xref=“MIM:602488” 
               
               
                 CDS 
                 159..1358 
               
               
                   
                 /gene=“PSCD2” 
               
               
                   
                 /note=“pleckstrin homology, Sec7 and coiled/coil domains 
               
               
                   
                 2; cytohesin 2; 
               
               
                   
                 go_component: kinesin complex [goid 0005871] [evidence 
               
               
                   
                 IEA]; 
               
               
                   
                 go_component: membrane fraction [goid 0005624] [evidence 
               
               
                   
                 TAS] [pmid 9417041]; 
               
               
                   
                 go_component: plasma membrane [goid 0005886] [evidence 
               
               
                   
                 TAS] [pmid 9417041]; 
               
               
                   
                 go_function: ARF guanyl-nucleotide exchange factor 
               
               
                   
                 activity [goid 0005086] [evidence TAS] [pmid 9417041]; 
               
               
                   
                 go_function: guanyl-nucleotide release factor activity 
               
               
                   
                 [goid 0019839] [evidence IEA]; 
               
               
                   
                 go_process: actin cytoskeleton reorganization [goid 
               
               
                   
                 0007012] [evidence TAS] [pmid 9417041]; 
               
               
                   
                 go_process: endocytosis [goid 0006897] [evidence TAS] 
               
               
                   
                 [pmid 9417041]” 
               
               
                   
                 /codon_start=1 
               
               
                   
                 /product=“pleckstrin homology, Sec7 and coiled/coil 
               
               
                   
                 domains 2 isoform 2” 
               
               
                   
                 /protein_id=“NP_004219.1” 
               
               
                   
                 /db_xref=“GI:4758966” 
               
               
                   
                 /db_xref=“GeneID:9266” 
               
               
                   
                 /db_xref=“LocusID:9266” 
               
               
                   
                 /db_xref=“MIM:602488” 
               
               
                   
                 /translation=“MEDGVYEPPDLTPEERMELENIRRRKQELLVEIQRLREELSEAM 
               
               
                   
                 SEVEGLEANEGSKTLQRNRKMAMGRKKFNMDPKKGIQFLVENELLQNTPEEIARFLYK 
               
               
                   
                 GEGLNKTAIGDYLGEREELNLAVLHAFVDLHEFTDLNLVQALRQFLWSFRLPGEAQKI 
               
               
                   
                 DRMMEAFAQRYCLCNPGVFQSTDTCYVLSFAVIMLNTSLHNPNVRDKPGLERFVAMNR 
               
               
                   
                 GINEGGDLPEELLRNLYDSIRNEPFKIPEDDGNDLTHTFFNPDREGWLLKLGGRVKTW 
               
               
                   
                 KRRWFILTDNCLYYFEYTTDKEPRGIIPLENLSIREVDDPRKPNCFELYIPNNKGQLI 
               
               
                   
                 KACKTEADGRVVEGNHMVYRISAPTQEEKDEWIKSIQAAVSVDPFYEMLAARKKRISV 
               
               
                   
                 KKKQEQP” 
               
               
                 misc_feature 
                 195..320 
               
               
                   
                 /gene=“PSCD2” 
               
               
                   
                 /note=“Region: Coiled-coil domain” 
               
               
                 misc_feature 
                 336..887 
               
               
                   
                 /gene=“PSCD2” 
               
               
                   
                 /note=“Sec7; Region: Sec7 domain. The Sec7 domain is a 
               
               
                   
                 guanine-nucleotide-exchange-factor (GEF) for the pfam00025 
               
               
                   
                 family” 
               
               
                   
                 /db_xref=“CDD:pfam01369” 
               
               
                 misc_feature 
                 372..914 
               
               
                   
                 /gene=“PSCD2” 
               
               
                   
                 /note=“Region: Sec7 domain” 
               
               
                 misc_feature 
                 936..1283 
               
               
                   
                 /gene=“PSCD2” 
               
               
                   
                 /note=“PH; Region: Pleckstrin homology domain. Domain 
               
               
                   
                 commonly found in eukaryotic signalling proteins. The 
               
               
                   
                 domain family possesses multiple functions including the 
               
               
                   
                 abilities to bind inositol phosphates, and various 
               
               
                   
                 proteins. PH domains have been found to possess inserted 
               
               
                   
                 domains (such as in PLC gamma, syntrophins) and to be 
               
               
                   
                 inserted within other domains. Mutations in Brutons 
               
               
                   
                 tyrosine kinase (Btk) within its PH domain cause X-linked 
               
               
                   
                 agammaglobulinaemia (XLA) in patients. Point mutations 
               
               
                   
                 cluster into the positively charged end of the molecule 
               
               
                   
                 around the predicted binding site for phosphatidylinositol 
               
               
                   
                 lipids” 
               
               
                   
                 /db_xref=“CDD:smart00233” 
               
               
                 misc_feature 
                 942..1283 
               
               
                   
                 /gene=“PSCD2” 
               
               
                   
                 /note=“Region: PH domain” 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
               
               
            
               
                   
               
               
                 1 
                 ttccgaagga 
                 agagtctttt 
                 cagcgctgag 
                 gactggcgct 
                 gaggaggcgg 
                 cggtggctcc 
                   
               
               
                   
               
               
                 61 
                 cggggcgttt 
                 gagcgggctc 
                 acccgagccg 
                 cgggccaacg 
                 cggatccagg 
                 cccgactgcg 
               
               
                   
               
               
                 121 
                 ggaccgcccc 
                 ggattccccg 
                 cgggccttcc 
                 tagccgccat 
                 ggaggacggc 
                 gtttatgaac 
               
               
                   
               
               
                 181 
                 ccccagacct 
                 gactccggag 
                 gagcggatgg 
                 agctggagaa 
                 catccggcgg 
                 cggaagcagg 
               
               
                   
               
               
                 241 
                 agctgctggt 
                 ggagattcag 
                 cgcctgcggg 
                 aggagctcag 
                 tgaagccatg 
                 agcgaggtgg 
               
               
                   
               
               
                 301 
                 aggggctgga 
                 ggccaatgag 
                 ggcagtaaga 
                 ccttgcaacg 
                 gaaccggaag 
                 atggcaatgg 
               
               
                   
               
               
                 361 
                 gcaggaagaa 
                 gttcaacatg 
                 gaccccaaga 
                 aggggatcca 
                 gttcttggtg 
                 gagaatgaac 
               
               
                   
               
               
                 421 
                 tgctgcagaa 
                 cacacccgag 
                 gagatcgccc 
                 gcttcctgta 
                 caagggcgag 
                 gggctgaaca 
               
               
                   
               
               
                 481 
                 agacagccat 
                 cggggactac 
                 ctgggggaga 
                 gggaagaact 
                 gaacctggca 
                 gtgctccatg 
               
               
                   
               
               
                 541 
                 cttttgtgga 
                 tctgcatgag 
                 ttcaccgacc 
                 tcaatctggt 
                 gcaggccctc 
                 aggcagtttc 
               
               
                   
               
               
                 601 
                 tatggagctt 
                 tcgcctaccc 
                 ggagaggccc 
                 agaaaattga 
                 ccggatgatg 
                 gaggccttcg 
               
               
                   
               
               
                 661 
                 cccagcgata 
                 ctgcctgtgc 
                 aaccctgggg 
                 ttttccagtc 
                 cacagacacg 
                 tgctatgtgc 
               
               
                   
               
               
                 721 
                 tgtccttcgc 
                 cgtcatcatg 
                 ctcaacacca 
                 gtctccacaa 
                 tcccaatgtc 
                 cgggacaagc 
               
               
                   
               
               
                 781 
                 cgggcctgga 
                 gcgctttgtg 
                 gccatgaacc 
                 ggggcatcaa 
                 cgagggcggg 
                 gacctgcctg 
               
               
                   
               
               
                 841 
                 aggagctgct 
                 caggaacctg 
                 tacgacagca 
                 tccgaaatga 
                 gcccttcaag 
                 attcctgagg 
               
               
                   
               
               
                 901 
                 atgacgggaa 
                 tgacctgacc 
                 cacaccttct 
                 tcaacccgga 
                 ccgggagggc 
                 tggctcctga 
               
               
                   
               
               
                 961 
                 agctgggggg 
                 ccgggtgaaa 
                 acgtggaagc 
                 ggcgctggtt 
                 tatcctcaca 
                 gacaactgcc 
               
               
                   
               
               
                 1021 
                 tctactactt 
                 tgagtacacc 
                 acggacaagg 
                 agccccgagg 
                 aatcatcccc 
                 ctggagaatc 
               
               
                   
               
               
                 1081 
                 tgagcatccg 
                 agaggtggac 
                 gacccccgga 
                 aaccgaactg 
                 ctttgaactt 
                 tacatcccca 
               
               
                   
               
               
                 1141 
                 acaacaaggg 
                 gcagctcatc 
                 aaagcctgca 
                 aaactgaggc 
                 ggacggccga 
                 gtggtggagg 
               
               
                   
               
               
                 1201 
                 gaaaccacat 
                 ggtgtaccgg 
                 atctcggccc 
                 ccacgcagga 
                 ggagaaggac 
                 gagtggatca 
               
               
                   
               
               
                 1261 
                 agtccatcca 
                 ggcggctgtg 
                 agtgtggacc 
                 ccttctatga 
                 gatgctggca 
                 gcgagaaaga 
               
               
                   
               
               
                 1321 
                 agcggatttc 
                 agtcaagaag 
                 aagcaggagc 
                 agccctga 
               
            
           
         
       
     
      A nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to PH domain variant nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.  
      In still another preferred embodiment, an isolated nucleic acid molecule of the present invention comprises a nucleotide sequence which is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identical to the entire length of the nucleotide sequence shown in SEQ ID NO:2 or 4, or to the entire length of the PH domain of SEQ ID NO:2 or 4 (SEQ ID NO:5 and 6).  
      The invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO:2 or 4 or the PH domain encoded by SEQ ID NO:2 or 4.  
      Variants of PH domains include both functional and non-functional proteins. Functional variants are amino acid sequence variants of proteins containing PH domains that maintain the ability to bind a ligand or substrate (e.g., phosphoinostide). Functional variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NO:1 or 3, or substitution, deletion or insertion of non-critical residues in non-critical regions of the protein. In specific embodiments, the PH variants will have insertions, deletions, or substitutions in the loop regions that connect the β strands.  
      Non-functional variants are amino acid sequence variants of PH domain containing proteins that do not have the ability to either bind an PH domain ligand or substrate (e.g., phosphoinostide). Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:1 or 3, or a substitution, insertion or deletion in critical residues or critical regions.  
      Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding PH domain containing proteins that contain changes in amino acid residues that are not essential for activity. Such PH domain containing proteins differ in amino acid sequence from SEQ ID NO:1 or 3, yet retain biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identical to SEQ ID NO:1 or3.  
      A nucleic acid molecule encoding an variant PH domain containing protein, can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO:2 or 4, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into SEQ ID NO:2 or 4, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a PH domain containing protein is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a PH domain containing coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for PH domain biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NO:2 or 4, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.  
      Variants  
      The instant invention provides variants of PH domain containing proteins. In one embodiment the variants are variants of SEQ ID NO:1 or 3. In another embodiment the variants are variants of the PH domain of SEQ ID NO: 1 or 3 (SEQ ID NO:5 or 6). The variants are further described in the Proteins section above.  
      Variants polypeptides of the invention are variants of GAC family polypeptides that have altered binding properties compared to polypeptides with the wild-type sequence due to one or more insertion, deletion or substitution in the PH domain. In one embodiment, the variant polypeptide binds to one or more natural ligand with increased affinity. Alternatively, the variant polypeptide of the invention binds to one or more natural ligand with decreased affinity. In a third embodiment, the variant polypeptide of the invention binds to one ligand with increased or decreased affinity while binding to another ligand with decreased affinity. In a fourth embodiment, the variant polypeptide of the invention binds to one ligand with increased or decreased affinity while binding to another ligand with increased affinity. In a fifth embodiment, the variant polypeptide of the invention binds to one ligand with increased or decreased affinity while not changing the affinity for another ligand. The affinity of the variant can be measured as described herein. In certain embodiments the affinity can be changed by 10, 50, 100, 500, 1000, or 10000 times.  
      In specific embodiments the variants of the invention have inserted, deleted or substituted residues in the loops that connect the β strands. In certain embodiments, the insertions are one or more glycine residues.  
      In another embodiment the variant polypeptide has altered specificity for one or more ligands. In one embodiment, the variant polypeptide may be able to selectively bind to one phosphoinositide while not binding to other, whereas the wild-type sequence bind promiscuously to multiple phosphoinositides.  
      Assays  
      The invention provides variants of GAC family proteins. Once GAC family protein variants are made and expressed, the following assays can be used to test their ability to interact with ligand.  
      In one embodiment, the assay of the present invention is a cell-free assay in which a GAC family polypeptide (e.g., protein or variant) or biologically active portion thereof is contacted with a compound, e.g., a phosphoinositide, and the ability of the compound to bind to the GAC polypeptide is determined. Preferred biologically active portions of the GAC family polypeptides to be used in assays of the present invention include fragments which contain a PH domain. Binding of the test compound to the GAC family polypeptide can be determined either directly or indirectly as described herein. In a preferred embodiment, the assay includes contacting the GAC family polypeptide or biologically active portion thereof with a first compound which binds to a GAC family polypeptide to form an assay mixture, contacting the assay mixture with a second compound, and determining the ability of the second compound to interact with a GAC family polypeptide, wherein determining the ability of the second compound to interact with a GAC family polypeptide comprises determining the ability of the second compound to preferentially bind to a GAC family polypeptide or biologically active portion thereof as compared to the known compound. First and second compounds are, for example, different phosphoinositides.  
      In another embodiment, the assay is a cell-free assay in which a GAC family polypeptide, variant thereof or biologically active portion thereof is contacted with a compound and the ability of the compound bind to the GAC family polypeptide, variant thereof or biologically active portion thereof is determined. Determining the ability of the test compound to modulate an activity of a GAC family polypeptide or a variant thereof, e.g., the ability to participate in cell survival, membrane trafficking, insulin regulation, cell adhesion, cell migration and cytoskeletal dynamics can be accomplished, for example, by determining the ability of the GAC family polypeptide, or variant thereof to bind to a GAC family polypeptide target molecule, e.g., a phosphoinositide, by one of the methods described above for determining direct binding. Determining the ability of a GAC family polypeptide, or variant thereof, to bind to a GAC family protein target molecule can also be accomplished using a technology such as real-time Biomolecular Interaction Analysis (BIA). Sjolander, S. and Urbaniczky, C. (1991)  Anal. Chem.  63:2338-2345 and Szabo et al. (1995)  Curr. Opin. Struct. Biol.  5:699-705. As used herein, “BIA” is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.  
      In an alternative embodiment, determining the ability of the test compound to modulate the activity of a GAC family polypeptide can be accomplished by determining the ability of the GAC family polypeptide or variant thereof to further modulate the activity of a downstream effector of a GAC family protein target molecule.  
      In yet another embodiment, the cell-free assay involves contacting a GAC family polypeptide , variant thereof or biologically active portion thereof with a known compound which binds the GAC family polypeptide to form an assay mixture, contacting the assay mixture with a compound, and determining the ability of the test compound to interact with the GAC family polypeptide, wherein determining the ability of the test compound to interact with the GAC family polypeptide comprises determining the ability of the GAC family polypeptide to preferentially bind to or modulate the activity of a GAC family target molecule.  
      The above cell-free and cell based assays exemplify the utility of the variant polypeptides of the invention. In particular, variant polypeptides of the invention can be used in any assay suitable for detecting interaction between ligands (e.g., phophotidylinositides) and PH domains, for example, those described above, as well as other suitable art-recognized assays. Direct assays (e.g., direct binding and/or activity assays) as well as indirect assays (e.g., assays for downstream effects, for example, signaling effects or resulting cellular effects) are within the scope of the invention. An exemplary use for the variant polypeptides of the invention is in the detection of specific phosphotidylinositides. For example, a variant polypeptide having an altered specificity for a given phosphotidylinositide can be used as a detection reagent for said phosphotidylinositide, preferably in a manner that excludes detection of other phosphotidylinositides. Alternatively, variant polypeptides of the invention can be used as control reagents, for example, as positive, negative or specificity control reagents. Other uses for the variant polypeptides of the invention will be apparent from the following Examples and claims.  
      Exemplification  
      The invention is further illustrated by the following examples which should not be construed as limiting.  
     EXAMPLE  
     Example 1  
     Ability of Variant Grp1 and ARNO to Bind IP  
      Variant Grp1 and ARNO polypeptides were constructed. Each variant was tested for the ability to bind to the natural IP ligand.  
      Constructs of GST tagged Grp1(2G) were titrated with IP4. The dissociation constant (Kd) for each interaction was determined and compared to the wild type. The relative Kds are plotted for each Grp1 mutant in  FIG. 2A .  
      Constructs of GST tagged ARNO (3G) were titrated with IP3 or IP4. The dissociation constant (Kd) for each interaction was determined and compared to the wild type. The relative Kds are plotted for each ARNO mutant in  FIGS. 2B  and C, respectively. The V279G mutant bound IP3 and IP4 with higher affinity than wild type.  
     Example 2  
     Determination of Alternate Binding Modes of PtdIns(4,5)P2 and PtdIns(3,4,5)P3  
      The crystal structures of the 3G variant of the ARNO PH domain bound to the head groups of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 has been determined. The structures reveal two distinct modes of binding, with different head group orientations and different networks of interactions with amino acid residues in the binding site. Although the orientation of the PtdIns(3,4,5)P3 head group is nearly identical to that observed previously for the 2G Grp1 PH domain, the orientation and mode of PtdIns(4,5)P2 binding to the 3G ARNO PH domain is entirely novel. On the basis of the structural data, a systematic, quantitative, comprehensive mutational analysis of all residues, both conserved and non-conserved, that mediate interactions with the phosphoinositide head groups as well as other residues in the adjacent β1/β2 loop that do not contact the head group directly has been preformed. These studies reveal that some of the variant Grp1 and ARNO PH domains, differing by single amino acid substitutions, have altered specificities as well as affinities for phosphoinositide head groups. The extensive structural, mutational, and binding data provide the information necessary to design and optimize variant Grp1/ARNO/Cytohesin family PH domains with altered binding affinity and/or specificity. Such engineered PH domains can be used as biochemical reagents for detection of specific phosphoinositides in in vitro and/or cell based assays. In addition, a number of stable mutants of the Grp1 and ARNO PH domains have been identified, which are unable to bind phosphoinositides. These mutant proteins can be used as key controls to determine the extent of and correct for non-specific background binding. It is possible to introduce mutations that reduce non-specific background binding without affecting the affinity and specificity for phosphoinositides. With respect to practicality and feasibility, it has been: i) established that the Grp1 and ARNO PH domains can be efficiently prepared as GST-fusion proteins in high yield (20 mg/L of  E. coll  culture) and purity (&gt;95%) in a single purification step; ii) determined that these GST fusion proteins behave homogenously and bind phosphoinositide head groups with 1:1 stoichiometry; and iii) determined that the GST fusions of Grp1 are highly tolerant of non-conservative amino acid substitutions within and adjacent to the head group binding site such that a variety of engineered Grp1 PH domains can be reliably produced in high yield and purity. Finally, given the efficiency/reliability of expression and purification, the production of GST fusions of wild type as well as variant Grp1 and ARNO PH domains is readily scaleable.  
      This is the first and currently the only observation of different binding modes for different head groups in the context of the same PH domain. Furthermore, it has been shown, at least in the case of the head group of PtdIns(3,4,5)P3, that the mode of binding is not altered by structural changes in the P1/P2 loop that dramatically alter specificity. It has further been shown that non-naturally occurring single amino acid substitutions, as well as naturally occurring single amino acid substitutions, alter both the affinity and specificity for phosphoinoisitides. Moreover, it has been demonstrated that the Grp1 and ARNO PH domains are amenable to crystallization in multiple forms including the unliganded form (2G and 3G Grp1), the complex with the head group of PtdIns(4,5)P′2 (3G ARNO), and the complex with the head group of PtdIns(3,4,5)P3 (2G Grp1 and 3G ARNO). In addition, it has been established that the phosphoinositide binding properties of Grp1 family PH domains are independent of the nature of the N-terminal fusion tag (GST or hexahistidine). Thus, the Grp1 family PH domains are uniquely suitable for structure based engineering of PH domains with novel affinities and specificities for phosphoinositides.  
      The structure of the ARNO PH domain reveals a novel mode of phosphoinositide binding. ARNO binds PtdIns(4,5)P2 in a rotated orientation and position when compared to previously characterized PH domains. The inositol ring of PIP2 is bound in a similar orientation to PIP3 in the Grp1 and Btk PH domains. However, the phosphates make different contacts than the highly homologous Grp1 . The 4-phosphate is contacted by the pocket that would recognize the 3-phosphate in Grp1. The 5-phosphate is contacted by the pocket that would recognize the 4-phosphate. Meanwhile, the residues that would make contact with the 5-phosphate in Grp1 are not contacting the ligand. The extra glycine in the β1/β2 loop of ARNO creates a longer loop that can accommodate PIP3 or PIP2 with little variation in specificity. However, the shorter loop in Grp1 brings a valine group too close to the inositol ring of PIP2 for a favorable interaction. This may explain the strong specificity Grp1 exhibits for PIP3 over PIP2. The presence of sulfate ions in the unliganded Grp1 (3G) structure in place of the phosphates suggest a preset placement for recognizing phosphoinositides in a limited binding orientation. Testing phosphoinositide binding in mutants of the Grp 1 and ARNO PH domains using isothermal calorimetry validate the necessity of certain residues for phosphoinositide binding. The presence of valine in the C terminus of the β1/β2 loop and a third glycine in the N terminus of the β1/β2 loop strongly affect phosphoinositide recognition.  
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 Equivalents 
 
       
    
      Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein. Such equivalents are intended to be encompassed by the scope of the following claims.  
      Incorporation by Reference  
      All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.