Patent Application: US-29019802-A

Abstract:
the present invention features isolated , purified , or enriched nucleic acid encoding a sirp polypeptide and isolated , purified , or enriched sirp polypeptide and uses thereof .

Description:
the present invention relates to sirp polypeptides , nucleic acids encoding such polypeptides , cells , tissues and animals containing such nucleic acids , antibodies to such polypeptides , assays utilizing such polypeptides , and methods relating to all of the foregoing . those skilled in the art will recognize that many of the methods described below in relation to sirp1 , sirp4 , shp - 2 , shp - 1 and grb2 could also be utilized with respect to the other members of this group . various other features and aspects of the invention are : nucleic acid encoding a sirp polypeptide ; a nucleic acid probe for the detection of sirp ; probe based method and kit for detecting sirp ; dna constructs comprising a sirp nucleic acid molecule and cells containing these constructs ; purified sirp polypeptides ; sirp antibody and hybridoma ; an antibody based method and kit for detecting sirp ; isolation of compounds which interact with sirp ; compositions ; disruption of protein complexes ; antibodies to complexes ; pharmaceutical formulations and modes of administration ; identification of agents ; purification and production of complexes ; derivatives of complexes ; and evaluation of disorders . all of these aspects and features are explained in detail with respect to another protein involved with signal transduction , pyk - 2 , in pct publication wo 96 / 18738 , which is incorporated herein by reference in its entirety , including any drawings . those skilled in the art will readily appreciate that such description can be easily adapted to sirp as well , and is equally applicable to the present invention . the examples below are non - limiting and are merely representative of various aspects and features of the procedures used to identify the full - length nucleic acid and amino acid sequences of a series of sirp proteins . experiments demonstrating sirp expression , interaction and signaling activities are also provided . mm5 / c1 , rat1 - ir , a431 or human fibroblast cells were grown until confluency , starved for 18 hours in serum - free medium , and either left untreated or were pov —( 1 mm sodium orthovanadate , 3 mm h 2 o 2 ), insulin —( 100 nm ), egf —( 1 nm ), or pdgf —( 100 pm ) stimulated for different time intervals as indicated . sirp4 , shp - 2 ( vogel , et al ., science 259 : 1611 - 1614 ( 1994 )) or shp - 2c463a mutant ( stein - gerlach , et al . j . biol . chem . 270 : 24635 - 24637 ( 1995 )) cdnas were transiently cotransfected in bhk - ir , bhk - egfr or bhk - βpdgfr cells using the calcium precipitation method ( chen , et al . mol . cell . biol . 7 : 2745 - 2752 ( 1987 )). after stimulation , cells were lysed in buffer containing 50 mm hepes , ph 7 . 5 , 150 mm nacl , 1 % triton x - 100 , 10 % glycerol , 1 mm pov , 1 mm edta , 1 mm pmsf , 1 mg / ml leupeptin , 1 mg / ml aprotinin . shp - 2 immunoprecipitations were performed with polyclonal anti - shp - 2 antibodies ( vogel , et al ., science 259 : 1611 - 1614 ( 1994 )). overexpressed sirp4 or endogenous sirp4 - like proteins were immunoprecipitated by polyclonal anti - ex1 antibodies raised by immunizing rabbits with a gst - fusion protein containing the ex1 fragment ( fig2 ). western blots were labeled with monoclonal anti - phosphotyrosine antibodies 5e2 ( fendly , et al ., cancer res . 50 : 1550 - 1558 ( 1990 )), and after stripping , reprobed with monoclonal anti - shp - 2 antibodies ( transduction laboratories ), or polyclonal anti - sirp4 - ct antibodies , raised against a gst - fusion protein containing the c - terminal part of sirp4 ( amino acids 336 - 503 ). for immunolabeling goat anti - mouse or - rabbit horseradish peroxidase conjugates ( bio - rad ) and the ecl detection system ( amersham ) were used . to obtain 293 cells stably expressing sirp4 ( 293 / sirp4 ), cells were transfected with sirp4 cdna in plxsn ( miller , et al . biotechniques 7 : 980 - 988 ( 1989 )) using the calcium precipitation method , followed by selection with g418 ( 1 mg / ml ). sirp4 was immunoprecipitated from quiescent or pov - stimulated ( 1 mm ) 293 / sirp4 cells with polyclonal anti - ex1 antibodies . subsequently , crude lysates of [ 35 s ]- methionine labeled 293 cells expressing different sh2 domain containing proteins were added to the affinity matrix and incubated for 2 h at 4 ° c . the is immunocomplexes were washed , separated by sds - page and analyzed by autoradiography . to perform in vitro deglycosylation shp - 2 immunocomplexes or the 110 kda protein preparation were first denatured in the presence of 1 % sds at 100 ° c . for 5 min . deglycosylation was done in potassium phosphate buffer ( 40 mm , ph 7 . 0 ), containing 20 mm edta , 1 % b - mercaptoethanol , 1 % triton x - 100 and 0 . 5 unit of endoglycosidase f / n - glycosidase f ( boehringer mannheim ) at 37 ° c . for 16 hours . approximately 10 10 rat1 - ir cells were used to purify the 110 kda protein . starved rat1 - ir cells were insulin - stimulated ( 100 nm ) for 10 min , washed briefly with ice - cold hypotonic buffer containing 20 mm hepes , ph 7 . 5 , 1 mm pov , 1 mm edta , 1 mm pmsf , 1 mg / ml leupeptin , 1 mg / ml aprotinin , scraped into the same buffer and homogenized . obtained cell extracts were pelleted at 1000 rpm for 15 min , and supernatants were spun at 48 . 000 g for 1 hour . membranes were solubilized in lysis buffer as described above . hir was depleted from membrane extracts using an affinity column with monoclonal anti - hir antibody 83 - 14 ( redemann et al ., mol . cell . biol . 12 : 491 - 498 ( 1992 )), covalently coupled to protein a - sepharose beads ( pharmacia ). depleted extracts were applied onto a wga - agarose 6 mb column ( sigma ), and glycoproteins were eluted with 0 . 3 m n - acetyl - glucosamine in hntg ( 20 mm hepes ( ph 7 . 5 ), 150 mm nacl , 0 . 1 % triton x - 100 , 10 % glycerol , 1 mm pov ). after concentration protein extracts were applied onto an anti - phosphotyrosine antibody column ( sigma ). bound proteins were eluted with 20 mm phosphotyrosine in hntg . the eluate was subjected to sds - page , proteins were transferred to a pvdf membrane ( millipore ) and stained with coomassie blue . the protein of 110 kd apparent molecular weight was microsequenced . the following five tryptic peptides were obtained : piysfiggehfpr , ivepdteik , ygfspr , ikevahvnlevr , vaagdsat . to produce retroviruses expressing plxsn , wild type sirp4 and mutated sirp4 constructs , bosc 23 cells were transiently transfected by expression plasmids as described ( pear , et al . proc . natl . acad . sci . 90 : 8392 - 8396 ( 1993 )). to obtain nih3t3 cells stably expressing wild type sirp4 , sirp4 - 4y or sirp4 - dct mutants subconfluent nih3t3 cells ( 10 5 cells per 6 cm dish ) were incubated with supernatants of transfected bosc 23 cells for 4 h in the presence of polybrene ( 4 mg / ml ), followed by selection with g418 ( 1 mg / ml ). to perform focus formation assays cell lines 3t3 / plxsn , 3t3 / sirp4 , 3t3 / sirp4 - 4y or 3t3 / sirp4 - dct were superinfected for 4 hours with equal volumes of v - fms - virus supernatant ( 10 5 cells / 6 cm dish ). cells were cultivated for 14 days in 4 % fcs with medium change every second day . cell foci were stained with crystal violet ( 0 . 1 % crystal violet , 30 % methanol ). western blot of mammalian cells with anti - phosphotyrosine antibodies and anti - shp - 2 antibodies was used to identify tyrosine phosphorylated shp - 2 associated proteins . western blots containing anti - shp - 2 immunoprecipitates from starved or pov - treated mouse mm5 / c1 mammary carcinoma , rat fibroblast rat1 - ir or human epidermal carcinoma a431 cells were incubated with anti - phosphotyrosine antibodies or anti - shp - 2 antibodies . samples were deglycosylated with or treated without endoglycosidase f / n - glycosidase f ( endo . f / f ). as a control , insulin - stimulated rat1 - ir cell lysates were immunoprecipitated with preimmune rabbit serum ( ans ). samples from each purification step ( i . e ., solubilized crude membrane extract , hir - depleted extracts , concentrated eluate from wga - agarose beads , and eluate from anti - phosphotyrosine antibody column ) were analyzed by 10 % sds - page and visualized by silver staining and in western blots using monoclonal anti - phosphotyrosine antibodies . a major tyrosine phosphorylated protein was revealed in analysis of anti - shp - 2 immunoprecipitates from both pervanadate ( pov ) and growth factor stimulated cells . this phosphoprotein migrated at 120 kda , 110 kda and 90 kda positions in mouse mammary tumor ( mm5 / c1 ) cells , rat1 cells overexpressing the human insulin receptor ( rat1 - ir ), and human epidermoid carcinoma ( a431 ) cells , respectively . upon in vitro deglycosylation , this glycoprotein was reduced to 65 kda apparent molecular weight ( mw ) in all cases . this indicated that the same shp - 2 binding protein of 65 kda was differentially glycosylated in a species specific manner . in some cell lines such as a431 , other tyrosine phosphorylated proteins in the 90 - 120 kda range remained unaffected by the deglycosylation treatment . these proteins may represent gab1 and / or the human homologue of the drosophila dos protein . insulin treated rat1 - ir were used to purify the 110 kda shp - 2 binding glycoprotein using standard chromatography procedures . approximately 4 mg of the glycoprotein that copurified with shp - 2 were obtained and subject to microsequence analysis . this yielded five peptide sequences : piysfiggehfpr , ivepdteik , ygfspr , ikevahvnlevr , vaagdsat . computer aided search in the est database led to the identification of a 305 bp rat sequence ( accession nr . : h31804 ) and subsequent human cdna fragment of 2 kb ( embl databank , accession nr . : u6701 ) containing matching and homologous sequences , respectively . specific primers flanking the very 5 ′ portion of this sequence were used to amplify a 360 bp human dna fragment ( encoding ex1 in fig2 ) which was used to screen a human placenta cdna library . several positive clones were isolated . one clone of 2 . 4 kb encoded a polypeptide of 503 amino acids designated sirp4 ( for signal regulating protein 4 ) with a calculated mass of 57 , 000 . the deduced sequence identifies sirp4 as a transmembrane protein with three ig - like domains and a cytoplasmic portion containing four potential tyrosine phosphorylation sites and one proline - rich region . a second cdna clone , sirp1 , is also identified . this protein is highly homologous to sirp4 within the ig - like domains ( ig - 1 : 83 %; ig - 2 : 88 %; ig - 3 : 83 %), but displays striking sequence divergence at the amino terminus and upstream of the transmembrane domain which gives rise to a shorter protein that still contains a transmembrane - like region but lacks the cytoplasmic c - terminal portion . sirp4 and sirp1 are members of a novel protein family . this protein family has a variety of distinct sequence isoforms as evidenced by comparison of fifteen cdna and genomic sequences within the first ig - like domain ( fig2 ). two major classes exist in sirp family distinguished by the presence or absence of a cytoplasmic shp - 2 binding domain . sirp4 binds to shp - 2 and serves as a substrate for shp - 2 , ir , egfr , and βpdgfr the identity of sirp4 as shp - 2 binding protein and substrate was confirmed by expression of the sirp4 cdna either alone or in combination with shp - 2 or an enzymatically inactive mutant shp - 2c463a in bhk cells . bhk cells stably express human egf -, insulin - or βpdgf receptors . immunoprecipitations were performed with a polyclonal antibody raised against a gst - fusion protein containing the extracellular ex1 region ( fig2 ). western blots containing anti - sirp4 immunoprecipitations from quiescent or ligand - stimulated bhk - ir , bhk - egfr or bhk - βpdgf cells were labeled with anti - phosphotyrosine , anti - shp - 2 and anti - sirp4 antibodies , respectively . anti - sirp4 immunoprecipitation revealed a tyrosine phosphorylated protein of 85 - 90 kda upon ligand stimulation which associated with shp - 2 . the results suggested sirp4 to be a direct substrate of shp - 2 since expression of the shp - 2 mutant shp - 2c463a led to a significant increase in its phosphotyrosine content ( even in starved cells ) while coexpression of wt shp - 2 resulted in dephosphorylation . the mw of overexpressed sirp4 matches that of the endogenous protein detected in shp - 2 immunoprecipitates from a431 cells . endogenous sirp4 - like proteins were immunoprecipitated from untreated or egf - stimulated a431 cells , from quiescent or pdgf - treated human fibroblasts , or from starved or insulin - stimulated hbl - 100 cells . as a control , ligand - stimulated cell lysates were immunoprecipitated with preimmune rabbit serum ( ans ). immunoblots were probed with monoclonal anti - phosphotyrosine and monoclonal anti - shp - 2 antibodies . polyclonal anti - ex1 antibodies immunoprecipitate a protein of 85 - 90 kda apparent mw from a431 , hbl - 100 tumor cells and human fibroblasts . this protein was tyrosine phosphorylated upon egf , insulin or pdgf stimulation , respectively , and coprecipitated with shp - 2 in a ligand dependent manner . these data indicate the existence of sirp4 in several human cell lines where sirp4 serves as a substrate for insulin -, egf - and βpdgf receptors , binds shp - 2 in its tyrosine phosphorylated form and serves as a substrate for the phosphatase activity of shp - 2 . the interaction of shp - 2 with sirp4 likely involves one or both sh2 domains of shp - 2 as suggested by the requirement of phosphotyrosine residues and the abrogation of detectable association by mutation of critical residues in shp - 2 sh2 domains . in vitro binding assays were performed to determine whether sirp4 is able to interact with other sh2 domain - containing proteins . sirp4 - associated [ 35 s ]- methionine labeled proteins were resolved on sds - page and detected by autoradiography . the result shows that sirp4 associates with both shp - 1 and grb2 but not p85 , shc , grb7 , plc - g , c - src , nck , vav , gap , or isgf - 3 . a catalytically inactive shp - 1 mutant has recently been shown to bind an as yet unidentified tyrosine phosphorylated protein of 90 - 95 - kda in human 293 cells . this tyrosine phosphorylated protein is likely to be sirp4 or one of its family members . to investigate the biological function of sirp4 , three stable transfectants of nih3t3 cells were constructed to express wild type sirp4 or sirp4 mutants carrying either point mutations of the putative shp - 2 tyrosine binding sites ( sirp4 - 4y ) or a deletion of most of the cytoplasmic region ( sirp4 - dct ). ligand - stimulated [ 3 h ]- thymidine incorporation of nih3t3 cells expressing empty vector ( 3t3 / plxsn ), wild type sirp4 ( 3t3 / sirp4 ), sirp4 - 4y ( 3t3 / sirp4 - 4y ) or sirp4 - dct ( 3t3 / sirp4 - dct , amino acids 402 - 503 are deleted ) mutants . cells were grown to confluence in 24 - well dishes ( nunc ), starved for 24 h in dmem / 0 . 5 % fcs , stimulated with different concentrations of insulin or egf for 18 h , then incubated with 0 . 5 mci [ 3 h ]- thymidine per well for 4 h . incorporation into dna was determined as described ( redemann , et al . mol . cell . biol . 12 : 491 - 498 ( 1992 )). upon stimulation of cells with insulin , egf and pdgf , control cells showed growth factor - induced dna synthesis as measured by [ 3 h ]- thymidine incorporation . overexpression of sirp4 led to a decrease of [ 3 h ]- thymidine incorporation . in contrast , both sirp4 mutants had nearly no effect on dna synthesis . the observed inhibitory effect on dna synthesis must be connected to sirp4 tyrosine phosphorylation and / or its association with shp - 2 since wt sirp4 became tyrosine phosphorylated and bound to shp - 2 upon ligand stimulation , and sirp4 mutants did not . sirp4 effected growth inhibition upon insulin or egf stimulation is correlated with reduced map kinase activation in 3t3 / sirp4 cells . 3t3 / plxsn , 3t3 / sirp4 or 3t3 / sirp4 - 4y cells were starved for 18 hours in dmem / 0 . 5 % fcs and stimulated with insulin or egf for the time indicated . map kinase was detected in western blots by using polyclonal erk1 and erk2 antibodies ( santa cruz ). in contrast , expression of sirp4 mutants defective in shp - 2 binding had no effect on map kinase activation . similar observations were made upon stimulation of the cells with pdgf . these data strongly indicate that sirp4 represents a novel regulatory - element in the pathway that leads to map kinase activation . we next determined the consequence of sirp4 overexpression on oncogene mediated transformation of nih3t3 cells . to examine the ability of sirp4 to influence the formation of cell foci , subconfluent 3t3 / plxsn , 3t3 / sirp4 , 3t3 / sirp4 - 4y or 3t3 / sirp4 - dct cells were infected with v - fms virus supernatants . as measured by focus formation , transformation by a v - fms retrovirus was significantly suppressed in cells overexpressing wt sirp4 but not in cells expressing mutant sirp4 . previous reports have described certain shp - 2 binding proteins of 110 - 130 kda apparent mw in mouse , rat or hamster cells . tyrosine hyperphosphorylation of these proteins was observed when an enzymatically inactive shp - 2 mutant was overexpressed . in addition , disruption of shp - 2 function induced a variety of negative effects on growth factor - induced cellular signals . our experiments strongly indicate that these proteins belong to the sirp family and that the biological effects previously observed are due to the function of these sirp proteins . without being bound by any theory , applicant proposes that tyrosine docking sites on sirp proteins for either shp - 2 and / or other sh2 proteins such as shp - 1 or grb2 play a significant role since the inhibitory effect of sirp4 on nih3t3 cell proliferation and transformation depends on phosphorylation of tyrosines . one or both of the shp phosphatases may tightly regulate the sirp4 phosphorylation state . sirp4 may also act in its phosphorylated state as a “ trapping ” protein that sequesters shp - 2 from activated rtks . the sequestion makes shp - 2 unavailable for other positive regulatory functions such as an adapter which recruits the grb2 - sos complex to activated receptors . such a function is supported by the observation that shp - 2 has higher affinity to the tyrosine phosphorylated form of sirp4 than to autophosphorylated insulin and egf receptors ( yamauchi , et al ., j . biol . chem . 270 : 17716 - 17722 , yamauchi , et al . j . biol . chem . 270 : 14871 - 14874 ( 1995 )). a third possibility is based on the membrane - spanning structural features of the sirp4 variant . the high degree of sequence diversity within the ig - domains is reminiscent of immunoglobulin variable regions and suggests a role of extracellular determinants in the sirp related signal transduction . structurally defined interaction of sirp with specific receptors , soluble ligands , extracellular matrix components or other factors may result in specific regulatory consequences for intracellular signaling events . all publications referenced are incorporated by reference herein , including the nucleotide sequences , amino acid sequences , drawings and tables in each publication . all the compounds disclosed and referred to in the publications mentioned above are incorporated by reference herein , including those compounds disclosed and referred to in articles cited by the publications mentioned above . other embodiments of this invention are disclosed in the following claims . as will be obvious to those skilled in the art , may variations and modifications may be made without departing from the spirit and scope of the invention . ser ala thr leu arg cys ala met thr ser leu ile pro val gly pro ile met trp phe 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