Rescue of Functional Interactions between the α2A-Adrenoreceptor and Acylation-resistant Forms of Gi1α by Expressing the Proteins from Chimeric Open Reading Frames
1997; Elsevier BV; Volume: 272; Issue: 39 Linguagem: Inglês
10.1074/jbc.272.39.24673
ISSN1083-351X
Autores Tópico(s)Glycosylation and Glycoproteins Research
ResumoCo-expression of the α2A-adrenoreceptor with a pertussis toxin-resistant (C351G), but not with an also palmitoylation-resistant (C3S/C351G), form of the α subunit of Gi1 resulted in agonist-induced, pertussis toxin-independent, GTP hydrolysis. Construction and expression of a chimeric fusion protein between the receptor and C351G Gi1α generated a membrane protein in which the G protein element was activated by receptor agonist. An equivalent fusion protein containing C3S/C351G Gi1α rescued the ability of receptor agonist to activate this mutant. Fusion proteins of a palmitoylation-resistant (C442A) α2A-adrenoreceptor and either C351G or C3S/C351G Gi1α also responded effectively to agonist. Myristoylation resistant (G2A/C351G) and combined acylation-resistant (G2A/C3S/C351G) mutants of Gi1α are cytosolic proteins. Expression of these as chimeric α2A-adrenoreceptor-G protein fusions restored membrane localization and activation of the G protein by receptor agonist. These studies demonstrate the general utility of generating chimeric fusion proteins to examine receptor regulation of G protein function and that the lack of functional activation of acylation-negative G proteins by a co-expressed receptor is related to deficiencies in cellular targeting and location rather than an inherent incapacity to produce appropriate protein-protein interactions and signal transmission. Co-expression of the α2A-adrenoreceptor with a pertussis toxin-resistant (C351G), but not with an also palmitoylation-resistant (C3S/C351G), form of the α subunit of Gi1 resulted in agonist-induced, pertussis toxin-independent, GTP hydrolysis. Construction and expression of a chimeric fusion protein between the receptor and C351G Gi1α generated a membrane protein in which the G protein element was activated by receptor agonist. An equivalent fusion protein containing C3S/C351G Gi1α rescued the ability of receptor agonist to activate this mutant. Fusion proteins of a palmitoylation-resistant (C442A) α2A-adrenoreceptor and either C351G or C3S/C351G Gi1α also responded effectively to agonist. Myristoylation resistant (G2A/C351G) and combined acylation-resistant (G2A/C3S/C351G) mutants of Gi1α are cytosolic proteins. Expression of these as chimeric α2A-adrenoreceptor-G protein fusions restored membrane localization and activation of the G protein by receptor agonist. These studies demonstrate the general utility of generating chimeric fusion proteins to examine receptor regulation of G protein function and that the lack of functional activation of acylation-negative G proteins by a co-expressed receptor is related to deficiencies in cellular targeting and location rather than an inherent incapacity to produce appropriate protein-protein interactions and signal transmission. A major mechanism for signal transduction across the plasma membrane involves seven transmembrane element G protein-coupled receptors (GPCRs) 1The abbreviations used are: GPCR, G protein-coupled receptor; G protein, guanine nucleotide binding protein; UK14304, 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine; ORF, open reading frame; bp, base pair(s).1The abbreviations used are: GPCR, G protein-coupled receptor; G protein, guanine nucleotide binding protein; UK14304, 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine; ORF, open reading frame; bp, base pair(s). and their activation of members of the family of αβγ heterotrimeric guanine nucleotide binding proteins (G proteins) (1Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4663) Google Scholar, 2Bourne H.R. Sanders D.A. McCormick F. Nature. 1991; 348: 125-132Crossref Scopus (1812) Google Scholar). Unlike the GPCRs, none of the individual G protein subunits contain transmembrane-spanning elements although the proteins are membrane-associated. In the case of the βγ complex, post-translational prenylation by the C15 farnesyl or C20 geranylgeranyl groups at a cysteine residue close to the C-terminal tail of the γ subunit followed by protein trimming and carboxymethylation acts to anchor the complex to the membrane (3Casey P.J. Science. 1995; 268: 221-225Crossref PubMed Scopus (726) Google Scholar, 4Wedegaertner P.B. Wilson P.T. Bourne H.R. J. Biol. Chem. 1995; 270: 503-506Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar). A key role in the targeting of specific G protein α subunits to the plasma membrane and their maintenance there has been ascribed to both co-translational myristoylation and post-translational palmitoylation (3Casey P.J. Science. 1995; 268: 221-225Crossref PubMed Scopus (726) Google Scholar, 4Wedegaertner P.B. Wilson P.T. Bourne H.R. J. Biol. Chem. 1995; 270: 503-506Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar, 5Mumby S.M. Heuckeroth R.O. Gordon J.I. Gilman A.G. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 728-732Crossref PubMed Scopus (210) Google Scholar, 6Jones T.L.Z. Simonds W.F. Merendino J.J. Brann M.R. Spiegel A.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 568-572Crossref PubMed Scopus (163) Google Scholar, 7Parenti M. Vigano M.A. Newman C.M.H. Milligan G. Magee A.I. Biochem. J. 1993; 291: 349-353Crossref PubMed Scopus (123) Google Scholar, 8Linder M.E. Middleton P. Helper J.R. Taussig R. Gilman A.G. Mumby S.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3675-3679Crossref PubMed Scopus (291) Google Scholar, 9Galbiati F. Guzzi F. Magee A.I. Milligan G. Parenti M. Biochem. J. 1994; 303: 697-700Crossref PubMed Scopus (41) Google Scholar, 10Degtyarev M.Y. Spiegel A.M. Jones T.L.Z. J. Biol. Chem. 1994; 269: 30898-30903Abstract Full Text PDF PubMed Google Scholar, 11Milligan G. Parenti M. Magee A.I. Trends Biochem. Sci. 1995; 20: 181-186Abstract Full Text PDF PubMed Scopus (283) Google Scholar, 12Wedegaertner P.B. Chu D.H. Wilson P.T. Levis M.J. Bourne H.R. J. Biol. Chem. 1993; 268: 25001-25008Abstract Full Text PDF PubMed Google Scholar, 13Li S. Okamoto T. Chun M. Sargiacomo M. Casanova J.E. Hansen S.H. Nishimoto I. Lisanti M.P. J. Biol. Chem. 1995; 270: 15693-15701Abstract Full Text Full Text PDF PubMed Scopus (554) Google Scholar). These acylations may also contribute to protein-protein interactions between the G protein α subunit and both receptors and the G protein βγ complex (12Wedegaertner P.B. Chu D.H. Wilson P.T. Levis M.J. Bourne H.R. J. Biol. Chem. 1993; 268: 25001-25008Abstract Full Text PDF PubMed Google Scholar, 14Edgerton M.D. Chabert C. Chollett A. Arkinstall S. FEBS Lett. 1994; 354: 195-199Crossref PubMed Scopus (41) Google Scholar). Addition of myristate occurs only on the α subunits of the Gi-family of G proteins because they contain the consensus sequence (MGXXXS) for the enzyme N-myristoyl-CoA transferase. The glycine that is found at codon 2 acts as the acceptor when it is exposed following removal of the initiator methionine. Palmitoylation of either one or two cysteine residues within the first ten amino acids of the α subunits occurs on all of the widely expressed G proteins (4Wedegaertner P.B. Wilson P.T. Bourne H.R. J. Biol. Chem. 1995; 270: 503-506Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar, 11Milligan G. Parenti M. Magee A.I. Trends Biochem. Sci. 1995; 20: 181-186Abstract Full Text PDF PubMed Scopus (283) Google Scholar). Although receptors can often be resolved from their cognate G proteins by detergent solubilization of membranes, in a number of examples such solubilization of membranes can result in the maintenance of receptor-G protein contacts (15Richardson A. Demoliou-Mason C. Barnard E.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10198-10202Crossref PubMed Scopus (38) Google Scholar, 16Tian W.-N. Duzic E. Lanier S.M. Deth R.C. Mol. Pharmacol. 1994; 45: 524-531PubMed Google Scholar, 17Georgoussi Z. Milligan G. Ziodrou C. Biochem. J. 1995; 306: 71-75Crossref PubMed Scopus (32) Google Scholar). The term "precoupled" is often used to describe such avid interactions between receptors and their cognate G protein. As a strategy to create physical precoupling of a receptor and its cognate G protein α subunit, Bertin et al. (18Bertin B. Friessmuth M. Jockers R. Strosberg A.D. Marullo S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8827-8831Crossref PubMed Scopus (108) Google Scholar) linked together the β2-adrenoreceptor and Gsα by generating a fusion protein between these open reading frames. This construct could be expressed, and addition of agonist for the receptor resulted in stimulation of adenylyl cyclase activity (18Bertin B. Friessmuth M. Jockers R. Strosberg A.D. Marullo S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8827-8831Crossref PubMed Scopus (108) Google Scholar). We have recently expanded this approach to the α2A-adrenoreceptor and the inhibitory G protein Gi1α and demonstrated that the addition of agonist to a fusion protein containing the wild-type sequences of both proteins results in activation of the G protein (19Wise A. Carr I.C. Milligan G. Biochem. J. 1997; 325: 17-21Crossref PubMed Scopus (47) Google Scholar). Although independent co-expression of the α2A-adrenoreceptor and Gi1α in COS cells allows functional interactions between the receptor and this G protein (20Wise A. Watson-Koken M.-A. Rees S. Lee M. Milligan G. Biochem. J. 1997; 321: 721-728Crossref PubMed Scopus (67) Google Scholar), no functional contacts can be measured following co-expression of the α2A-adrenoreceptor and acylation-deficient mutants of Gi1α (21Wise, A., Grassie, M. A., Parenti, M., Lee, M., Rees, S., and Milligan, G. (1997) Biochemistry, in pressGoogle Scholar) even though at least a C3S mutant can be localized to the membrane. We now demonstrate that agonist-induced signal transduction to the acylation-deficient mutants of Gi1α can be rescued by expressing these polypeptides as fusion constructs with the receptor and that effective agonist-induced signal transduction is unaffected by the palmitoylation potential of the receptor. All materials for tissue culture were supplied by Life Technologies, Inc. (Paisley, Strathclyde, Scotland). [3H]RS-79948–197 (90 Ci/mmol) was purchased from Amersham International. [γ-32P]GTP (30 Ci/mmol) was obtained from NEN Life Science Products. Pertussis toxin (240 μg/ml) was purchased from Speywood. All other chemicals were from Sigma or Fisons plc and were of the highest purity available. Oligonucleotides were synthesized on a Millipore Expedite Nucleic Acids Synthesis System. A pertussis toxin-resistant C351G form of rat Gi1α was generated (20Wise A. Watson-Koken M.-A. Rees S. Lee M. Milligan G. Biochem. J. 1997; 321: 721-728Crossref PubMed Scopus (67) Google Scholar) and linked to the porcine α2A-adrenoreceptor (22Guyer C.A. Horstman D.A. Wilson A.L. Clark J.D. Cragoe Jr., E.J. Limbird L.E. J. Biol. Chem. 1990; 265: 17307-17317Abstract Full Text PDF PubMed Google Scholar). To do so, the ORF of the α2A-adrenoreceptor DNA was amplified by polymerase chain reaction using the oligonucleotides: sense, 5′-TTGGTACCATGTATCCTTACGACGTTC-3′, and antisense, 5′-AAGAATTCCATGGCGATCCGTTTCCTGTCCCCACGGC-3′ (restriction sites for KpnI, EcoRI, and NcoI are underlined). The polymerase chain reaction-amplified fragment was digested with KpnI and EcoRI and ligated to pBluescript (Stratagene) through these restriction sites. Introduction of the NcoI site at the 3′-end of the ORF resulted in the C-terminal amino acid of the receptor being altered from Val to Ala and removal of the stop codon. The rat C351G Gi1α cDNA contains two NcoI sites, one straddling the ATG start codon and the other 268 bp downstream from this. This 268-bp fragment was removed from C351G Gi1α in pBluescript by digestion withNcoI, and the remaining C351G Gi1α pBluescript cDNA was religated. The shortened cDNA was excised from pBluescript with EcoRI and cloned into the EcoRI site of the α2A-adrenoreceptor in pBluescript, adjacent to the 3′-end of the receptor ORF. The 268-bp fragment was then inserted between the NcoI sites at the 3′-end of the α2A-adrenoreceptor ORF and at the 5′-end of the C351G Gi1α ORF. This resulted in production of an in-frame construct whereby the 3′-end of the α2A-adrenoreceptor ORF was exactly adjacent to the 5′-end of the C351G Gi1α ORF. The full fusion construct was then excised from pBluescript withKpnI and EcoRI and ligated into the eukaryotic expression vector pCDNA3. The same strategy was used to generate the various acylation-deficient chimeras. COS-7 cells were maintained in Dulbecco's modified Eagle's medium containing 10% (v/v) fetal calf serum, 2 mm L-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were seeded in 60-mm culture dishes and grown to 60–80% confluency (18–24 h) prior to transfection with pcDNA3 containing the relevant cDNA species using LipofectAMINE reagent (Life Technologies, Inc.) (20Wise A. Watson-Koken M.-A. Rees S. Lee M. Milligan G. Biochem. J. 1997; 321: 721-728Crossref PubMed Scopus (67) Google Scholar). For transfection, 2.5–2.8 μg of DNA were mixed with 10 μl of LipofectAMINE in 0.2 ml of Opti-MEM (Life Technologies, Inc.) and incubated at room temperature for 30 min prior to the addition of 1.8 ml of Opti-MEM. COS-7 cells were exposed to the DNA/LipofectAMINE mixture for 5 h. 2 ml of 20% (v/v) fetal calf serum in Dulbecco's modified Eagle's medium were then added to the cells. Cells were harvested 48 h after transfection. In all studies, the cells were treated with pertussis toxin (25 ng/ml) 24 h prior to cell harvest to eliminate possible interactions of the fusion proteins with endogenously expressed Gi-family G proteins (19Wise A. Carr I.C. Milligan G. Biochem. J. 1997; 325: 17-21Crossref PubMed Scopus (47) Google Scholar, 20Wise A. Watson-Koken M.-A. Rees S. Lee M. Milligan G. Biochem. J. 1997; 321: 721-728Crossref PubMed Scopus (67) Google Scholar). Plasma membrane-containing P2 particulate fractions were prepared from cell pastes that had been stored at −80 °C following harvest. Cell pellets were resuspended in 0.5 ml of 10 mm Tris-HCl, 0.1 mm EDTA, pH 7.5 (buffer A), and rupture of the cells was achieved with 50 strokes of a hand-held Teflon on-glass homogenizer followed by passage (10 times) through a 25-gauge needle. Cell lysates were centrifuged at 1000 × g for 10 min in a Beckman TJ-6 centrifuge to pellet the nuclei, and unbroken cells and P2 particulate fractions were then recovered by centrifugation of the supernatant at 200,000 ×g for 30 min in a Beckman TL 100 bench-top ultra-centrifuge using a Beckman TLA 100.2 rotor. P2 particulate fractions were resuspended in buffer A and stored at −80 °C until required. Binding assays were initiated by the addition of 2–4 μg of protein to an assay buffer (10 mm Tris-HCl, 50 mm sucrose, 20 mm MgCl2, pH 7.5) containing [3H]RS-79948–197 (23Gillard N.P. Linton C.J. Milligan G. Carr I.C. Patmore L. Brown C.M. Br. J. Pharmacol. 1996; 117: 298PGoogle Scholar) (1 nm).K d for this ligand at the fusion protein derived from the wild-type receptor and G protein = 0.35 nm(20Wise A. Watson-Koken M.-A. Rees S. Lee M. Milligan G. Biochem. J. 1997; 321: 721-728Crossref PubMed Scopus (67) Google Scholar). Nonspecific binding was determined in the presence of 100 μm idazoxan. Reactions were incubated at 30 °C for 45 min, and bound ligand was separated from free by vacuum filtration through GF/C filters. The filters were washed 3 times with 5 ml of assay buffer, and bound ligand was estimated by liquid scintillation spectrometry. Antiserum I1C (24Green A. Johnson J.L. Milligan G. J. Biol. Chem. 1990; 265: 5206-5210Abstract Full Text PDF PubMed Google Scholar) was produced in a New Zealand White rabbit using a conjugate of a synthetic peptide corresponding to amino acids 160–169 of the Gi1α subunit and keyhole limpet hemocyanin (Calbiochem) as antigen. The specificity of this antiserum for Gi1α has been demonstrated previously (24Green A. Johnson J.L. Milligan G. J. Biol. Chem. 1990; 265: 5206-5210Abstract Full Text PDF PubMed Google Scholar). Membrane samples were resolved by SDS-polyacrylamide gel electrophoresis using 10% (w/v) acrylamide gels containing 6m urea overnight at 100 V. Proteins were subsequently transferred to nitrocellulose (Schleicher & Schuell), probed with relevant antiserum, and visualized as described (25McKenzie F.R. Milligan G. Biochem. J. 1990; 267: 391-398Crossref PubMed Scopus (199) Google Scholar). High affinity GTPase assays were performed essentially as described previously (26Koski G. Klee W.A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 4185-4189Crossref PubMed Scopus (179) Google Scholar) using [γ-32P]GTP (0.5 μm, 60,000 cpm) and varying concentrations of UK14304 (up to 10 μm). Nonspecific GTPase was assessed by parallel assays containing 100 μm GTP. Expression of the porcine α2A-adrenoreceptor (22Guyer C.A. Horstman D.A. Wilson A.L. Clark J.D. Cragoe Jr., E.J. Limbird L.E. J. Biol. Chem. 1990; 265: 17307-17317Abstract Full Text PDF PubMed Google Scholar) in COS-7 cells allows agonist activation of endogenously expressed G proteins as measured by the capacity of the α2-adrenoreceptor agonist UK14304 to stimulate high affinity GTPase activity in membranes of these cells (20Wise A. Watson-Koken M.-A. Rees S. Lee M. Milligan G. Biochem. J. 1997; 321: 721-728Crossref PubMed Scopus (67) Google Scholar). Pertussis toxin treatment prior to cell harvest attenuates this effect, demonstrating it to reflect activation of Gi-family G proteins (20Wise A. Watson-Koken M.-A. Rees S. Lee M. Milligan G. Biochem. J. 1997; 321: 721-728Crossref PubMed Scopus (67) Google Scholar). A C351G mutation was introduced into Gi1α to render this protein insensitive to pertussis toxin (20Wise A. Watson-Koken M.-A. Rees S. Lee M. Milligan G. Biochem. J. 1997; 321: 721-728Crossref PubMed Scopus (67) Google Scholar). Co-expression of this modified G protein with the α2A-adrenoreceptor in COS-7 cells, followed by treatment of the cells with pertussis toxin, now resulted in a robust stimulation of high affinity GTPase activity upon addition of UK14304 (Fig.1). A C3S mutation of Gi1α prevents this polypeptide from acting as a substrate for post-translational palmitoylation (9Galbiati F. Guzzi F. Magee A.I. Milligan G. Parenti M. Biochem. J. 1994; 303: 697-700Crossref PubMed Scopus (41) Google Scholar, 10Degtyarev M.Y. Spiegel A.M. Jones T.L.Z. J. Biol. Chem. 1994; 269: 30898-30903Abstract Full Text PDF PubMed Google Scholar). Co-expression of C3S/C351G Gi1α and the α2A-adrenoreceptor failed to allow UK14304 stimulation of the GTPase activity of this mutated G protein (Fig. 1) although high levels of the G protein can be expressed in the membrane preparation (21Wise, A., Grassie, M. A., Parenti, M., Lee, M., Rees, S., and Milligan, G. (1997) Biochemistry, in pressGoogle Scholar). A G2A mutation of Gi1α prevents co-translational myristoylation of this protein (5Mumby S.M. Heuckeroth R.O. Gordon J.I. Gilman A.G. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 728-732Crossref PubMed Scopus (210) Google Scholar, 6Jones T.L.Z. Simonds W.F. Merendino J.J. Brann M.R. Spiegel A.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 568-572Crossref PubMed Scopus (163) Google Scholar), and the combination of G2A and C3S mutations totally eliminates acylation of the expressed polypeptide (9Galbiati F. Guzzi F. Magee A.I. Milligan G. Parenti M. Biochem. J. 1994; 303: 697-700Crossref PubMed Scopus (41) Google Scholar) and prevents membrane attachment. Co-expression of these forms of Gi1α, which also contained the C351G mutation with the α2A-adrenoreceptor, did not result in UK14304 stimulation of high affinity GTPase activity (21Wise, A., Grassie, M. A., Parenti, M., Lee, M., Rees, S., and Milligan, G. (1997) Biochemistry, in pressGoogle Scholar) as they were virtually entirely cytosolic (data not shown, but see Ref. 21Wise, A., Grassie, M. A., Parenti, M., Lee, M., Rees, S., and Milligan, G. (1997) Biochemistry, in pressGoogle Scholar). The open reading frames of each of C351G Gi1α, C3S/C351G Gi1α, G2A/C351G Gi1α, and G2A/C3S/C351G Gi1α were fused in-frame to the wild-type α2A-adrenoreceptor cDNA to create chimeric fusion proteins that contained the entire G protein α subunit sequence downstream of the receptor. This process introduced a single substitution of the receptor C-terminal Val by Ala and the maintained presence in the fusion proteins of the initiator Met of Gi1α (Fig. 2). Expression of each of these α2A-adrenoreceptor-C351G Gi1α constructs in COS-7 cells resulted in high levels of membrane expression of the receptor binding site (some 15–25 pmol/mg protein in individual transfections) as measured by the specific binding of the highly selective and high affinity α2-antagonist [3H]RS-79948–197 (23Gillard N.P. Linton C.J. Milligan G. Carr I.C. Patmore L. Brown C.M. Br. J. Pharmacol. 1996; 117: 298PGoogle Scholar). There were no differences in levels of expression of the individual fusion proteins in concurrently performed transfections (data not shown, but see Fig. 5). Immunoblotting membranes and cytosolic fractions derived from these transfections with the Gi1α specific antiserum I1C (24Green A. Johnson J.L. Milligan G. J. Biol. Chem. 1990; 265: 5206-5210Abstract Full Text PDF PubMed Google Scholar) resulted in identification of low levels of the endogenously expressed Gi1α as a 41-kDa polypeptide in the membrane fraction of each of mock-transfected cells and all the specific transfects (Fig. 3). An ∼100-kDa I1C immunoreactive band of the expected size for the fusion proteins was detected only in positively transfected cells (Fig. 3).Figure 3Expression of α2A-adrenoreceptor-Gi1α fusion proteins, immunoblotting studies. Homogenates of mock-transfected cells (lanes 1 and 2) and those expressing wild-type (lanes 3–10) or C442A (lanes 11–18) α2A-adrenoreceptor fusion proteins containing C351G Gi1α (lanes 3, 4, 11, and 12), C3S/C351G Gi1α (lanes 5,6, 13, and 14), G2A/C351G Gi1α (lanes 7, 8, 15, and 16), or G2A/C3S/C351G Gi1α (lanes 9, 10, 17, and 18) were resolved into P2 particulate (even numbered lanes) and cytosolic (odd numbered lanes) preparations, resolved by SDS-polyacrylamide gel electrophoresis, and immunoblotted to detect the presence of Gi1α. In cells expressing each of the fusion proteins, a polypeptide of some 100 kDa was detected.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Membranes of cells expressing the α2A-adrenoreceptor-C351G Gi1α fusion protein were able to support concentration-dependent stimulation of pertussis toxin-insensitive high affinity GTPase activity upon addition of UK14304 (EC50 = 2.3 ± 0.3 × 10−7m) (Fig.4 A). In contrast to the lack of functional interactions between co-expressed but independent α2A-adrenoreceptor and C3S/C351G Gi1α (Fig.1), expression of the fusion protein containing the C3S mutation also resulted in agonist-mediated stimulation of high affinity GTPase activity (EC50 = 4.4 ± 1.1 × 10−7m) (Fig. 4 A). This was also true for the two fusion proteins containing the G2A mutation in the G protein (Fig.4 A) and again EC50 values for UK14304 (G2A/C351G Gi1α = 3.5 ± 0.8 × 10−7m, G2A/C3S/C351G Gi1α = 3.2 ± 0.9 × 10−7m) (mean ± S.E.,n = 3 in each case) were not different. The porcine α2A-adrenoreceptor has a cysteine residue 9 amino acids from the C terminus that has been shown to act as an acceptor for post-translational palmitoylation (27Kennedy M.E. Limbird L.E. J. Biol. Chem. 1993; 268: 8003-8011Abstract Full Text PDF PubMed Google Scholar). Although a C442A mutation of the receptor has been reported not to interfere with G protein activation (27Kennedy M.E. Limbird L.E. J. Biol. Chem. 1993; 268: 8003-8011Abstract Full Text PDF PubMed Google Scholar), we considered whether potential palmitoylation of this cysteine might alter agonist regulation of the fusion proteins as palmitoylation is believed to provide an extra site of membrane anchorage and generate a "fourth intracellular loop" in the receptor (11Milligan G. Parenti M. Magee A.I. Trends Biochem. Sci. 1995; 20: 181-186Abstract Full Text PDF PubMed Scopus (283) Google Scholar). Furthermore, the C-terminal tail of the α2A-adrenoreceptor is only some 20 amino acids long, which is relatively short within the family of G protein-coupled receptors, and we wished to consider if the extra flexibility following removal of the site of palmitoylation might influence regulation of fusion protein activation by agonist. Fusion proteins containing the pertussis toxin-insensitive C351G Gi1α in concert with G2A, C3S, and G2A/C3S mutations were constructed with the C442A α2A-adrenoreceptor (Fig. 2) and expressed in COS-7 cells. Each of these constructs was expressed to a similar level as those containing the wild-type receptor (Fig. 3), and each was able to stimulate pertussis toxin-insensitive high affinity GTPase activity in response to UK14304 to similar extents and with similar EC50 values (2.2–2.8 × 10−7m in individual transfections) as the fusion proteins containing the wild-type receptor sequence (Fig. 4 B). Co-expression of the β1γ2 complex with the wild-type α2A-adrenoreceptor-C351G Gi1α fusion protein resulted in substantially greater maximal stimulation of GTPase activity by UK14304 than achieved without excess β1γ2 (Fig.5 A). This did not reflect increased levels of expression of the fusion protein (Fig.5 B) or alterations in the EC50 for UK14304 (data not shown but see (19Wise A. Carr I.C. Milligan G. Biochem. J. 1997; 325: 17-21Crossref PubMed Scopus (47) Google Scholar)). Additional expression of β1γ2 also increased the maximal GTPase activity in response to UK14304 of each of the G2A/C351G, C3S/C351G, and G2A/C3S/C351G forms of Gi1α constrained in fusion proteins with the wild-type α2A-adrenoreceptor (Fig.5 A), consistent with interaction of the β1γ2 complex with all of the individual fusion proteins. This is despite the known role of the N-terminal region of the G protein α subunit, which is constrained in the fusion constructs, in βγ binding (28Wall M.A. Coleman D.E. Lee E. Iniguez-Lluhi J.A. Posner B.A. Gilman A.G. Sprang S.R. Cell. 1995; 83: 1047-1058Abstract Full Text PDF PubMed Scopus (1001) Google Scholar, 29Lambright D.G. Sondek J. Bohm A. Skiba N.P. Hamm H.E. Sigler P.B. Nature. 1996; 379: 311-319Crossref PubMed Scopus (1041) Google Scholar). Acylation of G protein α subunits plays a key role in the membrane targeting and association of these proteins (3Casey P.J. Science. 1995; 268: 221-225Crossref PubMed Scopus (726) Google Scholar, 4Wedegaertner P.B. Wilson P.T. Bourne H.R. J. Biol. Chem. 1995; 270: 503-506Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar, 11Milligan G. Parenti M. Magee A.I. Trends Biochem. Sci. 1995; 20: 181-186Abstract Full Text PDF PubMed Scopus (283) Google Scholar). Furthermore, prevention of myristoylation of the α subunit of Gi-like G proteins reduces their affinity of interaction with the βγ complex and can render them cytoplasmic (5Mumby S.M. Heuckeroth R.O. Gordon J.I. Gilman A.G. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 728-732Crossref PubMed Scopus (210) Google Scholar, 6Jones T.L.Z. Simonds W.F. Merendino J.J. Brann M.R. Spiegel A.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 568-572Crossref PubMed Scopus (163) Google Scholar). Prevention of palmitoylation also limits membrane association and has been reported to limit or prevent interactions with receptors (7Parenti M. Vigano M.A. Newman C.M.H. Milligan G. Magee A.I. Biochem. J. 1993; 291: 349-353Crossref PubMed Scopus (123) Google Scholar, 9Galbiati F. Guzzi F. Magee A.I. Milligan G. Parenti M. Biochem. J. 1994; 303: 697-700Crossref PubMed Scopus (41) Google Scholar,12Wedegaertner P.B. Chu D.H. Wilson P.T. Levis M.J. Bourne H.R. J. Biol. Chem. 1993; 268: 25001-25008Abstract Full Text PDF PubMed Google Scholar, 14Edgerton M.D. Chabert C. Chollett A. Arkinstall S. FEBS Lett. 1994; 354: 195-199Crossref PubMed Scopus (41) Google Scholar). However, it has been unclear whether poor activation of acylation-defective G protein α subunits by receptors is simply a problem associated with the lack of appropriate targeting and thus proximity to a receptor or is inherently due to the acylation status of the G protein. In the current study, we have taken a highly novel approach to address this question. This has involved the construction of chimeric fusion proteins between the α2A-adrenoreceptor and the α subunit of the G protein Gi1. To do so involved the apparently simplistic expedient of linking the N terminus of the G protein directly to the C terminus of the receptor. The strategy used resulted in a minimal alteration to the sequence of the protein in the region of fusion. Indeed, only the C-terminal amino acid of the receptor was altered (Val to Ala), and the initiator Met of the G protein, which would normally be removed, remained in the sequence of the new protein (see Fig. 2). Only a single previous example has examined the potential for signal transduction following expression of a GPCR fused to its cognate G protein α subunit. In the case of the β2-adrenoreceptor-Gsα chimera, addition of agonist was able to cause activation of adenylyl cyclase following expression of the fusion protein in a cell line that genetically lacks endogenous Gsα (18Bertin B. Friessmuth M. Jockers R. Strosberg A.D. Marullo S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8827-8831Crossref PubMed Scopus (108) Google Scholar). As well as using the wild-type receptor and G protein, fusion chimeras were generated between the wild-type α2A-adrenoreceptor and mutant forms of Gi1α in which the sites that are normally palmitoylated and myristoylated in the G protein were modified. Expression of each of these constructs could be detected in the membrane fraction following transient transfection of each of these in COS-7 cells. This was achieved both in binding assays using the highly selective and high affinity α2-adrenoreceptor antagonist [3H]RS-79948–197 (23Gillard N.P. Linton C.J. Milligan G. Carr I.C. Patmore L. Brown C.M. Br. J. Pharmacol. 1996; 117: 298PGoogle Scholar) to detect the ligand binding site of the receptor (Fig. 5) and by immunoblotting cytosolic and membrane fractions of these cells with the specific Gi1α antiserum I1C (24Green A. Johnson J.L. Milligan G. J. Biol. Chem. 1990; 265: 5206-5210Abstract Full Text PDF PubMed Google Scholar) (Fig. 3). Gi1α is expressed endogenously in COS-7 cells at low levels and could be detected as a 41-kDa polypeptide by this antiserum in membranes from both mock and positively transfected cells. In contrast, membranes of positively transfected but not mock-transfected cells displayed the presence of an I1C reactive polypeptide of some 100 kDa, which corresponds to the chimeric fusion proteins (Fig. 3). Similar levels of the fusion proteins were expressed whether the G protein element of the fusion protein was wild type at the N terminus or contained G2A, C3S, or both mutations (Fig. 3), and none of these immunoreactive proteins were detected in the cytosolic fractions. As with many GPCRs (11Milligan G. Parenti M. Magee A.I. Trends Biochem. Sci. 1995; 20: 181-186Abstract Full Text PDF PubMed Scopus (283) Google Scholar), the α2A-adrenoreceptor is also a target for post-translational palmitoylation, at Cys442 within the C-terminal tail. Because this acylation is proposed to create a "fourth intracellular loop" in the receptor structure and because this receptor has only a short C-terminal tail, we also created chimeric fusion proteins between a C442A mutant of the α2A-adrenoreceptor and the various forms of Gi1α detailed above. Although previous studies have indicated this mutation in the receptor not to interfere with agonist-mediated G protein activation, we wished to assess whether potential greater flexibility provided by a longer "linker" between the seventh transmembrane element of the receptor and the N terminus of the G protein α subunit would affect agonist activation of the G protein. All of the C442A-α2A-adrenoreceptor-Gi1α fusions were also expressed and to similar levels as the versions that included the wild-type receptor (Fig. 3). As an aid to subsequent analysis, we generated all of the fusion proteins using a form of Gi1α in which Cys-351, which is the normal target for pertussis toxin-catalyzed ADP-ribosylation, was substituted by Gly. We have previously demonstrated that C351G Gi1α is not a substrate for pertussis toxin-catalyzed ADP-ribosylation (20Wise A. Watson-Koken M.-A. Rees S. Lee M. Milligan G. Biochem. J. 1997; 321: 721-728Crossref PubMed Scopus (67) Google Scholar). It can, however, be activated by the α2A-adrenoreceptor, with the only clear difference, compared with the wild-type G protein, being that some 10–15-fold higher concentrations of agonist are required to produce the same degree of stimulation (20Wise A. Watson-Koken M.-A. Rees S. Lee M. Milligan G. Biochem. J. 1997; 321: 721-728Crossref PubMed Scopus (67) Google Scholar). This is presumably a reflection of the alteration in conformation of the C terminus of the G protein, an element known to be a key receptor contact site (30Milligan G. Biochem. J. 1988; 255: 1-13Crossref PubMed Scopus (204) Google Scholar). Such mutations, however, allow receptor regulation of the mutated G protein to be studied in isolation following pertussis toxin-treatment of cells to eliminate potential functional contacts between the receptor and the endogenously expressed Gi-family G proteins (20Wise A. Watson-Koken M.-A. Rees S. Lee M. Milligan G. Biochem. J. 1997; 321: 721-728Crossref PubMed Scopus (67) Google Scholar, 31Senogles S.E. J. Biol. Chem. 1994; 269: 23120-23127Abstract Full Text PDF PubMed Google Scholar, 32Hunt T.W. Carroll R.C. Peralta E.G. J. Biol. Chem. 1994; 269: 29565-29570Abstract Full Text PDF PubMed Google Scholar). As such, all of the functional experiments herein were performed following pertussis toxin treatment of the cells for times and with amounts of toxin that cause modification of all of the endogenous Gi-like G proteins (20Wise A. Watson-Koken M.-A. Rees S. Lee M. Milligan G. Biochem. J. 1997; 321: 721-728Crossref PubMed Scopus (67) Google Scholar). All of the expressed chimeric fusion proteins were able to stimulate high affinity pertussis toxin-insensitive GTPase activity in membranes of the transfected COS-7 cells upon addition of the α2-adrenoreceptor agonist UK14304. Agonist stimulation of high affinity GTPase activity is a classical assay to measure both GDP-GTP exchange and then subsequent GTP hydrolysis by a G protein α subunit (25McKenzie F.R. Milligan G. Biochem. J. 1990; 267: 391-398Crossref PubMed Scopus (199) Google Scholar, 26Koski G. Klee W.A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 4185-4189Crossref PubMed Scopus (179) Google Scholar). As such, these results demonstrate both that the G protein α subunit is folded appropriately to exchange guanine nucleotide and to act as an enzyme and that information, presumably mediated via conformational change, can be transmitted from the binding of agonist to the receptor on to the G protein. These data thus further demonstrate the general utility of generating chimeric fusion proteins to examine receptor regulation of G protein function (18Bertin B. Friessmuth M. Jockers R. Strosberg A.D. Marullo S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8827-8831Crossref PubMed Scopus (108) Google Scholar, 19Wise A. Carr I.C. Milligan G. Biochem. J. 1997; 325: 17-21Crossref PubMed Scopus (47) Google Scholar). G2A and G2A/C3S mutants of Gi1α are essentially completely cytosolic (9Galbiati F. Guzzi F. Magee A.I. Milligan G. Parenti M. Biochem. J. 1994; 303: 697-700Crossref PubMed Scopus (41) Google Scholar), and thus it is not surprising that they are not activated by receptor agonists (21Wise, A., Grassie, M. A., Parenti, M., Lee, M., Rees, S., and Milligan, G. (1997) Biochemistry, in pressGoogle Scholar). Physical proximity, as guaranteed from the fusion protein approach, showed that the lack of functional activation of such acylation negative mutants of G proteins by a co-expressed receptor is related to deficiencies in cellular targeting and location rather than an inherent incapacity to produce appropriate protein-protein interactions for signal transmission. As the fusion chimeras containing the G2A and C3S mutants required similar concentrations of UK14304 to cause half-maximal effects, this also argues that acylation is not inherently required to produce effective protein-protein contacts between the receptor and G protein α subunit but rather is to position the G protein appropriately. It was noted, however, that the fusion constructs containing both the G2A and C3S mutations in Gi1α displayed somewhat lower GTPase activity in response to maximally effective concentrations of UK14304 rather than the other constructs (Fig. 4). The reason for this requires further investigation. The C3S mutation of Gi1α results in expression of a protein that is partially cytoplasmic but partially membrane-associated (9Galbiati F. Guzzi F. Magee A.I. Milligan G. Parenti M. Biochem. J. 1994; 303: 697-700Crossref PubMed Scopus (41) Google Scholar). As such, by increasing levels of expression of C3S Gi1α, it is possible to obtain levels of this mutant at the membrane as high or higher than that following expression of wild-type Gi1α (21Wise, A., Grassie, M. A., Parenti, M., Lee, M., Rees, S., and Milligan, G. (1997) Biochemistry, in pressGoogle Scholar). Despite this, the independently expressed α2A-adrenoreceptor is unable to cause significant activation of C3S Gi1α (Fig. 1). The reason for this remains to be resolved as the fusion proteins containing C3S Gi1α were as effectively stimulated by UK14304 as any of the others (Fig. 4). Given the nature of the physical linkage between the receptor and G protein in the fusion proteins, the knowledge that the N terminus of the G protein α subunit plays a central role in interaction with the βγ complex (28Wall M.A. Coleman D.E. Lee E. Iniguez-Lluhi J.A. Posner B.A. Gilman A.G. Sprang S.R. Cell. 1995; 83: 1047-1058Abstract Full Text PDF PubMed Scopus (1001) Google Scholar, 29Lambright D.G. Sondek J. Bohm A. Skiba N.P. Hamm H.E. Sigler P.B. Nature. 1996; 379: 311-319Crossref PubMed Scopus (1041) Google Scholar), the concept that the βγ complex may play a key role in receptor interactions with the α subunit (33Kisselev O. Ermolaeva M. Gautam N. J. Biol. Chem. 1995; 270: 25356-25358Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 34Kisselev O. Pronin A. Ermolaeva M. Gautam N. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9102-9106Crossref PubMed Scopus (95) Google Scholar, 35Taylor J.M. Jacob-Mosier G.G. Lawton R.G. VanDort M. Neubig R.R. J. Biol. Chem. 1996; 271: 3336-3339Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), and the understanding that G protein α subunit acylation is important in interactions with the βγ complex, it was of considerable interest to observe that co-expression of excess β1γ2 along with any of the α2A-adrenoreceptor-Gi1α fusion proteins resulted in greater maximal UK14304 stimulation of GTPase activity (Fig. 5). A trivial explanation for this observation based on higher steady-state levels of expression of the fusion protein in the presence of excess βγ was eliminated by performing 3H-antagonist binding studies (Fig. 5). These results imply interaction of the fusion protein with the βγ complex, but understanding the details of this will require further study. It is of interest in this regard to note, however, that Taylor et al. (36Taylor J.M. Jacob-Mosier G.G. Lawton R.G. Remmers A.E. Neubig R.R. J. Biol. Chem. 1994; 269: 27618-27624Abstract Full Text PDF PubMed Google Scholar) have previously indicated a role for βγ in receptor stimulation of the GTPase activity of the α2A-adrenoreceptor. Overall these studies demonstrate the receptor-G protein fusion approach to be a novel and useful means to study receptor-G protein interactions and indicate a key role for G protein acylation in cellular targeting but not intrinsically in transmission of information between receptors and G proteins. We thank Dr. Lee Limbird, Vanderbilt University, Tennessee, for provision of the wild-type and C442A forms of the porcine α2A-adrenoreceptor.
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