Abstract:
The present invention provides fusion proteins including a green fluorescent protein inserted into the internal amino acid sequence of a Gαs protein and further provides method of using the fusion protein construct to follow activation of a G-protein receptor by a candidate drug.

Description:
This application is a national stage application of PCT/US02/21484 filed Jul. 3, 2002, which claims priority to U.S. Ser. No. 60/303,622 filed Jul. 6, 2001. 
    
    
     This invention was made with government support under MH39595-10, AG15482 and awarded by the National Institute of Health (NIH). The Government has certain rights in the invention. 
    
    
     The present invention relates a protein that is constructed by adding a green fluorescent protein designated GFP that is internal to the amino acid sequence of a G protein, in particular the Gαs protein. The resulting fusion protein is a non-radioactive marker used, for example, for high throughput screening of G protein-coupled receptor drug targets. 
     BACKGROUND OF THE INVENTION 
     A family of heterotrimeric nucleotide-binding proteins that bind to guanine (G proteins) transduces chemical and sensory signals across the plasma membrane by sequential interactions with receptor and second messenger-generating effectors. Because of the wide array of cellular processes that are mediated by G proteins, the study of G protein function and regulation is a significant area of research in the signal transduction field. There are reports containing suggestions of an important function for G protein at cellular locations other than the plasma membrane. Certain G proteins were detected at intracellular membranes, for example, the Golgi complex, whereas others associate with cytoskeletal structures, for example, microtubules and microfilaments. The mechanisms that govern the cellular destinations of G proteins and the relative proportions of G proteins that traffic to subcellular compartment are just beginning to be revealed. 
     G proteins are reported to couple the receptors for hormones or neurotransmitters to intracellular effectors such as adenylyl cyclase or phospholipase C. Twenty forms of the α-subunit of G proteins were identified and each is involved in the conveyance of multiple hormonal neurotransmitter signals from the outside of the cell to the effects that those hormones and neurotransmitters have on the inside of the cell. 
     G proteins may leave the membrane in response to neurotransmitter or hormone signals, but this has been very difficult to prove. 
     GFP, an autofluorescent protein of 238 amino acids, is a reporter molecule useful to monitor gene and protein expression and to observe the dynamics of protein movements within the living cell. Fusing GFP to another protein of interest allows time-course studies to be performed on living samples in real time. Accounts of GFP fusion proteins include receptors, secretory proteins, cytoskeleton proteins and signaling molecules. Presently, GFP fusion proteins are constructed by generating an expression construct that contains GFP fused in frame to either the N-amino or C-carboxyl terminus of the protein of interest. However, this attachment may alter the function of the protein fused with GFP consequently may not give results reflective of the natural state. 
     SUMMARY OF THE INVENTION 
     Fusion of a GFP protein at either NH 2  or COOH ends of Gαs protein subunits is not acceptable because the NH 2  region is important for association with Gαs protein βγ subunits, and the COOH terminal is required for interaction with receptors. Consequently, a biologically active Gαs-GFP that incorporated GFP at some other positions of the molecule was developed. Suitable regions for insertion of a GFP sequence are those regions that are free of interactions with receptors or effectors. 
     A fusion protein was constructed by inserting an amino acid sequence of a green fluorescent protein designated GFP, into the interior of an amino acid sequence of a G-protein, in particular the Gαs protein. Although, green fluorescent proteins have been inserted at either end of G-proteins, a method was needed to insert GFP into the internal amino acid sequence of a G-protein without altering the biological activity of the protein. 
     Green fluorescent protein (GFP) was inserted within the internal amino acid sequence of Gαs to generate a Gαs-GFP fusion protein. The fusion protein maintained a bright green fluorescence and was also identified by antibodies against Gαs or GFP, respectively. The cellular distribution of Gαs-GFP was similar to that of endogenous Gαs. Gαs-GFP was tightly coupled to the β adrenergic receptor to activate the Gαs effector, adenylyl cyclase. Activation of Gαs-GFP by cholera toxin caused a gradual displacement of Gαs-GFP from the plasma membrane throughout the cytoplasm in living cells. Unlike the slow release of Gαs-GFP induced by cholera toxin, the β adrenergic agonist isoproterenol caused a rapid partial release of Gαs-GFP into the cytoplasm. At 1 min after treatment with isoproterenol, the extent of this Gαs-GFP release from plasma membrane was maximal. Translocation of Gαs-GFP induced by isoproterenol suggested that the internalization of Gαs might play a role in signal transduction by interacting with effector molecules and cytoskeletal elements at multiple cellular sites. 
     Uses for the Gαs-GFP fusion construct of the present invention include: 
     1. G proteins from the intracellular plasma membrane move in response to activation by an antagonist. Following the activation of a G protein and discovering the time course for that activation. The occupancy of a receptor by an agonist is only the first step in a signaling cascade. The intracellular processes might be activated at different rates or, at specific areas within a cell. Gαs-GFP is useful because it can be followed in real time as events take place without disrupting natural progress of events. 
     2. Tracking protein functions in living cells. 
     3. As a non-radioactive marker for high throughput screening of G-proteins coupled receptor drug targets, following the course of activation of a putative receptor or a putative ligand. For example, if a drug company has a candidate that it believes activates G protein coupled receptors in a functional sense, the Gαs-GFP fusion construct is useful as a high throughput screen, because a change in fluorescence in response to the application of an agonist is detectable. Conversely, the activity of an antagonist is visualized by adding it in 96 well plates, and screening significant numbers of samples on a fluorimeter to determine which compounds block the expected fluorescence change. Gαs-GFP could be used in combination with a fluorescent receptor such as that developed by the Biosignal Corporation in Montreal. To do this, cells are transfected with fluorescent receptors and Gαs-GFP. A ligand which activated the receptor in such a way that the G protein was also activated should decrease the fluorescence of GFP induced by the emitted light from the receptor (fluorescence resonance energy transfer-FRET). Thus, a number of candidate compounds may be screened for receptor and G protein activation by conducting these assays in e.g. 96 well plates. 
     4. The use of green fluorescent protein (GFP) in the study of cellular signaling allows not only the observation of G protein trafficking, but the opportunity to study the dynamics of G proteins in real time as well as their function. 
     Other molecules may be modified in the same way, for example the other of the 20 G protein α subunits. Insertion sites for GFP are determined by an analysis of the sequence. None of the Gαs can be modified by adding GFP to either the amino or carboxy terminus because their function would be destroyed. Putting the GFP in the internal regions does not harm the effects of the protein, but rather bestows on its new properties. Several other signaling molecules may be suitable candidates for the fusion proteins of the present invention. 
     5. Gαs-GFP is modified in such a way that it will couple to other receptors. Modification of amino acids near the carboxy terminal generates a fluorescent Γα that is capable of coupling to receptors which normally couple to Gαi, Gαo or Gαq (Conklin, et al., 1996). This will allow the same fluorescent G protein to assess potency and efficacy of putative agonists and agonists for a large number of G protein coupled receptors. 
     The 5 C terminal residues of Gαs are QYELL (SEQ ID NO: 3). They are replaced with DCGLF (SEQ ID NO: 4) for Gi1 or Gi2, with ECGLY (SEQ ID NO: 5) for Gi3, with RCGLY (SEQ ID NO: 6) for Go, and with EYNLV (SEQ ID NO: 7) for Gq. 
     COS1 or HEK293 cells are suitable because they are easy to transfect. These or comparable cells are co-transfected with GFP-Gαs (either in its native form or engineered to couple to a receptor which normally couples to Gi or Gq) and the desired receptor. Putative agonists are screened by monitoring loss of fluorescence from the membrane. High-throughput fluorescence monitoring instruments that are known to those of skill in the art are used for this purpose. Putative antagonists are screened by assessing their ability to block the effects of known receptor agonists to evoke this phenomenon. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows Gαs fusion protein cDNA construction. (A) shows a schematic of Gαs-GFP. Gαs-GFP defines the fusion protein in which GFP was inserted within the NH 2 -terminal domain of the long Gαs. (B) presents a model of Gαs-GFP. The structure of GFP is shaded. The Gαs subunit structure is that of Gαs-GTPγS. 
         FIG. 2  shows expression of the Gαs-GFP fusion in COS-1 cells. COS-1 cells were lysed 24 h after transiently transfecting with Gαs-GFP (3 μg DNA). 30 μg protein was loaded, separated by SDS-PAGE gel, detected with polyclonal antibody against Gαs (panel B) or monoclonal antibody against GFP (Panel A), as indicated. Lane 1 represents a lysate from cells transfected with Gαs-GFP, Lane 2 is the lysate from control cells. 
         FIG. 3  shows Gαs-GFP is associated with the plasma membrane in transfected cells. 24 h post-transfection, cells were observed by confocal microscopy at 37° C. A computer-generated cross section of a typical cell is displayed on the top (x-z plane) and on the right (y-z plane). Each image shown is representative of at least 20 cells subjected to a z-scan analysis. Similar results were obtained with COS-1, PC12, and HEK 293 cells. 
         FIG. 4  shows subcellular distribution of Gαs-GFP in COS-1 cells. Particulate and soluble fractions were isolated from cells transfected with Gαs-GFP constructs 24 h post transfection as described herein. 20 μg protein was loaded, separated by SDS-PAGE gel and detected with a polyclonal antibody against the C-terminal peptide of Gαs. Lanes 1 and 2 represent the soluble portion from the control cells or cells transfected with Gαs-GFP, respectively. Lanes 3 and 4 indicate the particulate fraction from control cells or cells transfected with Gαs-GFP, respectively. 
         FIG. 5  shows Gαs-GFP binding to AAGTP. COS-1 cells were co-transfected with cDNA encoding Gαs-GFP (1 μg) and β-adrenergic receptor (4 μg). (A) Shows cell membranes prepared 24 h post-transfection and incubated with  32 P AAGTP in the presence and absence of isoproterenol (as indicated). Proteins were resolved by SDS-PAGE and autoradiography. Results shown are from one of four similar experiments. (B) Presents densitometric analysis of Gαs-GFP binding to AAGTP. Densitometric analysis of four independent experiments were carried out and displayed in densitometric units. [Shown is the mean±Standard error, n=4, ** indicates significant difference from control treated without ISO (P&lt;0.01]. 
         FIG. 6  shows Gαs-GFP activates adenylyl cyclase. Cells were transfected with GFP (control) or Gαs-GFP, respectively and assayed for cAMP formation in the presence or absence of isoproterenol (ISO: 50 μM) as indicated. (A) control cells in the absence of ISO. (B) Gαs-GFP transfected cells in the absence of ISO. C. control cells with ISO. D. Gαs-GFP transfected cells treated with ISO. The values shown are mean±standard error of nine samples from three experiments. Identical levels of Gαs-GFP in each group were determined by western blotting. ** indicates significant difference from control cells treated without ISO; (P&lt;0.01). 
         FIG. 7  demonstrates cholera toxin treatment translocates Gαs-GFP in living PC12 cells. (A) 24 h post-transfection with Gαs-GFP, media was replaced as described in Methods and living cells were viewed by confocal microscopy at 37° C. Cells were initially imaged (0 min), cholera toxin (3 μg/ml) was added and cells were observed for 1 h. Bar=10 μm. (B) computer-generated cross section of the whole cell after completion of the one hour, is displayed on the top (x-z plane) and on the right (y-z plane). Results shown are from one of four comparable experiments. Observation of other cell lines (COS-1 and HEK 293) showed similar results for response to cholera toxin. 
         FIG. 8  shows isoproterenol-stimulated rapid internalization of Gαs-GFP in living COS cells. Cells were transfected with Gαs-GFP and observed 24 h later at 37° C. with confocal or digital fluorescent microscopy. (A) cells were treated with or without isoproterenol (20 μM), and images were captured every 5 seconds (A video scan; showed COS-1 cell treatment with ISO for 2 min. and; shown control COS-1 cell for 2 min). Arrows indicate areas where membrane-bound Gαs-GFP released from plasma membrane significantly. Clusters of Gαs-GFP form subjacent to the plasma membrane (indicates by open arrowhead). (B) Observation of Gαs-GFP release from plasma membrane using confocal microscopy. Arrows display regions where Gαs-GFP released from plasma membrane significantly. The arrowheads indicate the sites where the Gαs-GFP was inserted after the 2 minutes time point. Bar=10 mm. These results are typical of 40 of 58 cells observed during the course of 15 experiments. Approximately 70% of the cells showed internalized Gαs-GFP in response to isoproterenol [ISO]. Thirty percent did not show a significant response to this agonist. 
         FIG. 9A-9C  are cDNA (nucleotide) (SEQ ID NO: 1) and its complement and amino acid sequences (SEQ ID NO: 2) of the Gαs-GFP. The letters in a box indicate the start codon for Gαs-GFP. The circled letters form the stop codon for Gαs-GFP. A, G, T and C are abbreviations of Adenine, Guanine, Thymine and Cytosine, respectively. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Construction of Gαs-GFP 
     Full length cDNAs encoding Gαs were excised from the PcDNA-1 vector by digesting with Sam I and Xba I restriction enzymes. The full length EGFP cDNA was obtained by PCR from the PEGFP-N3 using appropriate primers (sense 5′ GGAATTCATGAGCAAGGGCGAGGAACTG-3′ (SEQ ID NO: 8); antisense 5′-GCTCTAGACGACTTGTACAGCTCGT-3′) (SEQ ID NO: 9) and adding restriction sites to its cDNA (EcoR I at the initiation codon and Xba I at end of cDNA). To insert the EGFP within the sequence of Gαs, the first fragment of Gαs (from 1 to 71 amino acids) was amplified by PCR with restriction sites for Kap 1 at initiation codon and EcoR I at end of the fragment. The cDNA of the fragment was cloned into PcDNA3 vector by the Kap 1 and EcoR 1 restriction sites using primers (sense 5′GGGTACCATGGGCTGCCTCGGCAACA-3′ (SEQ ID NO: 10); antisense 5′-GGAATTCGTCCTCTTCGCCGCCCTTCT-3′) (SEQ ID NO: 11). Modified 7 EGFP cDNA was spliced into the first fragment of Gαs by EcoR 1 and Xba 1 restriction sites on PcDNA3 to get the fusion cDNA sequence of the first fragment of Gαs and EGFP. The second fragment of Gαs (from 82 to 394 amino acids) was also obtained using PCR with appropriate primers. The sense primer contained a part of a sequence overlapping with the 3′ end of EGFP (5′-CAGAGCTGGACAAGTCCAACAGCGATGGTGAGAA-3′) (SEQ ID NO: 12). The anti-sense primer contained an additional Xba 1 restriction site (5′-GCTCTAGACGACTTGTACAGCTCGT-3′) (SEQ ID NO: 9) The presenion cDNA fragment described above was amplified by PCR. The Gαs-GFP fusion fragment and the second fragment of Gαs were also linked using PCR strategy. The full length Gαs-GFP was cloned into PcDNA3 at Kap 1 and Xba 1 restriction sites. All DNA manipulations, including ligations, PCR, bacterial transformation were carried out using procedures disclosed herein. Plasmid purification was done using “plasmid purification kit” following the manufacture instruction (QIAGEN). 
     Ligation Protocol 
     1. In a 1.5 ml microfuge tube, cut 10 mL expression vector with the desired restricted enzyme in a total volume of 20 mL for 2 h at 31° C. 
     2. Loading the sample into 1% agarose gel, run the gel applying a voltage of 100 V. Run the gel long enough to resolve the fragments of interest. 
     3. Turn off the power supply and remove the gel from the apparatus. 
     4. Using “Gel Extraction Kit” (QIAGEN), purify fragments from gel. 
     5. In 0.5 ml microfuge tube, mix the fragments of vector (0.03 mg) and relevant inserts, add 5 mL 4′ ligation buffer (GIBCOBRL), incubating in a total volume of 20 mL at 14° C. overnight with 0.1 units T 4  ligase. 
     6. Take out 10 mL to transformation. 
     Polymerase Chain Reaction 
     1. In 0.5 ml thin wall tube mix the following ingredients. 
                                                     10′ buffer (GIBCOBRL)   5   μL           MgCl 2  (GIBCOBRL)   5   μL           primer 1   1   μL           primer 2   1   μL           template DNA   0.5   μL           4 dNTP mix (GIBCOBRL)   10   μL           H 2 O   26.5   μL           Taq polymerase (GIBCOBRL)   1   μL               50   mL                        
2. Spin down one time for 15 seconds and put one-drop mineral oil in tube.
 
3. Turn on the automated thermal cycler.
 
4. First denature simples 2 min at 94° C., then run program for 35 cycles.
         Denature 90 seconds   Anneal 50 seconds at 58° C.   Extend 1 min at 72° C.
 
When cycles finish, 7 min perform extra-extend at 72° C.
 
5. Run gel and purify the DNA with “PCP Purification Kit” (QIAGEN).
 
Transformation Protocol
 
1. Add 5 ml of LB medium (10 g tryptone, 5 g yeast extract, 10 g NaCl in 1 L H 2 O) to sterile 10 ml tube.
 
2. Scrape HB 101 bacterial cells (one colony) from stock plate with loop. Transfer cells to medium and shake bacterial cells off loop. Put the tube in shaking incubator at 31° C. for 12 h.
 
3. Spin down bacterial cells at 2000×g for 3 min at room temperature.
 
4. Gently resuspend pellet of bacterial cells in 1 ml 50 mM CaCl 2 , incubate for 40 min on ice.
 
5. Spin down again at 2000×g for 3 min at 4° C. Resuspend pellet of bacterial cells in 100 ml 50 mM CaCl 2 .
 
6. In 1.5 ml sterile microfuge tube, add 10 mL ligated plasmid vector, then mix it with 100 mL competent bacterial cells.
 
7. Incubate the mixture on ice for 20 min and then transfer tube to 42° C. for heat shock for 30 seconds.
 
8. Take the mixture, and add to plate (with antibiotic), agar side top incubating at 37° C. overnight.
       

     Three Gαs-GFP fusion constructs were made and expressed in COS-1 cells. In the Gαs-NGFP expression vector, in which the GFP was spliced to the N-amino terminus of Gαs sequence, the fusion protein could not associate with the plasma membrane of cells (see  FIG. 1 ,  FIG. 2A ). The attachment of palmitate at Cys-3 of Gαs is required both for its membrane association and for its ability to mediate hormonal stimulation of adenylyl cyclase. A sequence motif that serves as a predictor for a subset of palmitoylated proteins is Met-Gly-Cys at the amino terminus of a protein. This motif found in the Gi and Gαs subfamily of G-protein subunits and other proteins such as receptor tyrosine kinases. The GFP connected with the amino terminus of Gαs may affect the palmitoylation of Cys-3. A GFP tagged COOH terminal of Gαs, Gαs-CGFP was also constructed. Although this attached to the membrane, it did not respond to hormone activation. 
     Gαs exists as a short and a long splice variant. Compared with short Gαs, long Gαs contains an additional 15 amino acids inserted at position 72 of the polypeptide chain, and there is an exchange of glutamate for apartate at position 71. Although there has been some indication that subtle differences between short Gαs and long Gαs exist, the general function of the two forms is similar. No substantial difference in the function of the two forms has been detected. Furthermore, the yeast Gαs, GPA1, has an “extra loop” in this region as well. Levis et al. (1992) modified the long Gαs form at a site (residues 77-81) within the 15 amino acid insert to confer upon it recognition by an antibody directed against a well-defined peptide of the influenza hemaglutinin (HA). Addition of the HA epitope did not alter the ability of wild type Gαs to mediate hormonal stimulation of adenylyl cyclase or to attach to cell membranes. Given the possibility that this region was “inert”, a Gαs-GFP2 fusion protein was constructed by replacing the residues (72-81) within the long Gαs with a GFP sequence (see  FIG. 1 ). A western blot of membrane and cytosolic fractions ( FIG. 2B ), probed with an anti-Gαs polyclonal or anti-GFP monoclonal antibody, shows that Gαs-GFP2 is expressed in COS-1 cells with a distribution comparable to that of intrinsic Gαs. These results indicate that the GFP in the Gαs-GFP2 should not alter the attachment of Gαs to membranes. In addition, the fluorescence of GFP in Gαs-GFP2 is visual and stable with UV irradiation. 
     Based on the α-carbon model of the α-subunit of the retinal G-protein transducin, the sequence within which the 15 amino acid insert is localized in the long Gαs serves as a linker between the ras-like domain and the α-helical domain. The guanine nucleotide-binding site is embedded between these two domains. Thus, the change in this linker sequence might be expected to diminish the ability of binding to guanine nucleotides of Gαs. To study this, COS-1 cells were co-transfected with Gαs-GFP2 and β-adrenergic receptor cDNA. COS1 membranes were incubated with the photoaffinity GTP analog  32 P AAGTP as in the presence and absence of a beta adrenergic agonist. Labeling of membranes from the transfected COS-1 cells was accomplished by incubating with 0.1 mM [ 32 P] AAGTP for 5 min at 23° C., followed by treatment with isoproterenol (ISO) for 3 min. Gαs-GFP2 in COS-1 bound [ 32 P] AAGTP in response to ISO ( FIG. 3 ). This result dramatically and unexpectedly demonstrated that the insertion of GFP into the linker sequence between two domains of Gαs does not disrupt agonist-induced guanine nucleotide exchange. 
     Cholera toxin activates Gαs by directly ADP ribosylating arginine 201 of Gαs and inhibiting the intrinsic GTPase. Thus, cholera toxin locks Gαs in the activated state. After, cholera toxin-activated was no longer observed at the plasma membrane, but instead it was distributed throughout the cytoplasm. Increased solubility of Gαs may correlate with activation-induced depalmitoylation of Gαs, but it is not absolutely clear that the removal of the lipid group is necessary for cytosolic translocation.  FIG. 4  shows that the Gαs-GFP on the cellular membrane is internalized gradually subsequent to treatment of cells with cholera toxin. Cholera toxin activation of Gαs-GFP also provides further evidence that the fusion protein is capable of normal pysiological function. 
     The physiologic consequences of β-adrenergic receptor activation of Gαs were observed by examining the response of Gαs-GFP cos1 cells to isoproterenol. The rapid translocation of Gαs from membrane to cytoplasm was clearly delineated. 
     To determine whether Gαs-GFP was fully physiologically active, tests were performed to see if the fusion protein was capable of activating adenylyl cyclase. By measurement of cAMP accumulation in COS-1 cells transfected in different conditions, the overexpression of Gαs-GFP was found not to alter the base level of cAMP in cells. Isoproterenol treated cells showed the cAMP production in Gαs-GFP cells to be significantly higher than cells transfected with GFP-vector alone ( FIG. 6 ). 
     Thus, assay of subcellular distribution and signaling function shows in vitro and in vivo that the GFP insertion into the Gαs amino acid sequence does not substantially affect normal function of Gαs. The study indicates a new approach to constructing GFP fusion protein and the study of G protein molecular signaling transduction in cells. 
     DOCUMENTS CITED 
     
         
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