Title: Coordinated Agonist Regulation of Receptor and G Protein Palmitoylation and Functional Rescue of Palmitoylation-deficient Mutants of the G Protein G11α following Fusion to the α1b-Adrenoreceptor
Abstract: Transfection of either the α1b-adrenoreceptor or Gα11 into a fibroblast cell line derived from a Gαq/Gα11 double knockout mouse failed to produce elevation of intracellular [Ca2+] upon the addition of agonist. Co-expression of these two polypeptides, however, produced a significant stimulation. Co-transfection of the α1b-adrenoreceptor with the palmitoylation-resistant C9S,C10S Gα11 also failed to produce a signal, and much reduced and kinetically delayed signals were obtained using either C9S Gα11 or C10S Gα11. Expression of a fusion protein between the α1b-adrenoreceptor and Gα11 allowed [Ca2+]i elevation, and this was also true for a fusion protein between the α1b-adrenoreceptor and C9S,C10S Gα11, since this strategy ensures proximity of the two polypeptides at the cell membrane. For both fusion proteins, co-expression of transducin α, as a β·γ-sequestering agent, fully attenuated the Ca2+signal. Both of these fusion proteins and one in which an acylation-resistant form of the receptor was linked to wild type Gα11 were also targets for agonist-regulated [3H]palmitoylation and bound [35S]guanosine 5′-3-O-(thio)triphosphate (GTPγS) in an agonist concentration-dependent manner. The potency of agonist to stimulate [35S]GTPγS binding was unaffected by the palmitoylation potential of either receptor or G protein. These studies provide clear evidence for coordinated, agonist-mediated regulation of the post-translational acylation of both a receptor and partner G protein and demonstrate the capacity of such fusions to bind and then release β·γ complex upon agonist stimulation whether or not the G protein can be palmitoylated. They also demonstrate that Ca2+ signaling in EF88 cells by such fusion proteins is mediated via release of the G protein β·γ complex. Transfection of either the α1b-adrenoreceptor or Gα11 into a fibroblast cell line derived from a Gαq/Gα11 double knockout mouse failed to produce elevation of intracellular [Ca2+] upon the addition of agonist. Co-expression of these two polypeptides, however, produced a significant stimulation. Co-transfection of the α1b-adrenoreceptor with the palmitoylation-resistant C9S,C10S Gα11 also failed to produce a signal, and much reduced and kinetically delayed signals were obtained using either C9S Gα11 or C10S Gα11. Expression of a fusion protein between the α1b-adrenoreceptor and Gα11 allowed [Ca2+]i elevation, and this was also true for a fusion protein between the α1b-adrenoreceptor and C9S,C10S Gα11, since this strategy ensures proximity of the two polypeptides at the cell membrane. For both fusion proteins, co-expression of transducin α, as a β·γ-sequestering agent, fully attenuated the Ca2+signal. Both of these fusion proteins and one in which an acylation-resistant form of the receptor was linked to wild type Gα11 were also targets for agonist-regulated [3H]palmitoylation and bound [35S]guanosine 5′-3-O-(thio)triphosphate (GTPγS) in an agonist concentration-dependent manner. The potency of agonist to stimulate [35S]GTPγS binding was unaffected by the palmitoylation potential of either receptor or G protein. These studies provide clear evidence for coordinated, agonist-mediated regulation of the post-translational acylation of both a receptor and partner G protein and demonstrate the capacity of such fusions to bind and then release β·γ complex upon agonist stimulation whether or not the G protein can be palmitoylated. They also demonstrate that Ca2+ signaling in EF88 cells by such fusion proteins is mediated via release of the G protein β·γ complex. G protein-coupled receptor Dulbecco's modified Eagle's medium guanosine 5′-3-O-(thio)triphosphate polymerase chain reaction The phosphoinositidase C-linked G proteins Gαq and Gα11 are widely co-expressed (1Strathmann M. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9113-9117Crossref PubMed Scopus (385) Google Scholar, 2Wilkie T.M. Scherle P.A. Strathmann M.P. Slepak V.Z. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10049-10053Crossref PubMed Scopus (256) Google Scholar, 3Milligan G. J. Neurochem. 1993; 61: 845-851Crossref PubMed Scopus (52) Google Scholar, 4Milligan G. Mullaney I. McCallum J.F. Biochim. Biophys. Acta. 1993; 1179: 208-212Crossref PubMed Scopus (37) Google Scholar, 5Johnson G.J. Leis L.A. Dunlop P.C. Biochem. J. 1996; 318: 1023-1031Crossref PubMed Scopus (32) Google Scholar, 6Offermanns S. Toombs C.F. Hu Y.H. Simon M.I. Nature. 1997; 389: 183-186Crossref PubMed Scopus (494) Google Scholar). To gain insight into their function and into potential functions of G protein-coupled receptors (GPCRs)1 in the absence of these G proteins, their genes have been inactivated in mice (6Offermanns S. Toombs C.F. Hu Y.H. Simon M.I. Nature. 1997; 389: 183-186Crossref PubMed Scopus (494) Google Scholar, 7Haley J.E. Delmas P. Offermanns S. Abogadie F.C. Simon M.I. Buckley N.J. Brown D.A. J. Neurosci. 2000; 20: 3973-3979Crossref PubMed Google Scholar, 8Offermanns S. Zhao L.P. Gohla A. Sarosi I. Simon M.I. Wilkie T.M. EMBO J. 1998; 17: 4304-4312Crossref PubMed Scopus (203) Google Scholar, 9Zywietz A. Gohla A. Schmelz M. Schultz G. Offermanns S. J. Biol. Chem. 2001; 276: 3840-3845Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 10Mao J. Yuan H. Xie W. Simon M.I. Wu D. J. Biol. Chem. 1998; 273: 27118-27123Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 11Gohla A. Offermanns S. Wilkie T.M. Schultz G. J. Biol. Chem. 1999; 274: 17901-17907Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 12Klages B. Brandt U. Simon M.I. Schultz G. Offermanns S. J. Cell Biol. 1999; 144: 745-754Crossref PubMed Scopus (305) Google Scholar). Although double Gαq/Gα11 knockout mice are not viable, cells derived from such embryos have been extremely useful, particularly in the elucidation of function of the G12/G13 class of ubiquitously expressed, pertussis-insensitive G proteins (11Gohla A. Offermanns S. Wilkie T.M. Schultz G. J. Biol. Chem. 1999; 274: 17901-17907Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 12Klages B. Brandt U. Simon M.I. Schultz G. Offermanns S. J. Cell Biol. 1999; 144: 745-754Crossref PubMed Scopus (305) Google Scholar). Such cells have also been used to demonstrate that agonist-induced internalization of Gαq/Gα11-coupled GPCRs is not dependent upon the presence of these G proteins (13Yu R. Hinkle P.M. J. Biol. Chem. 1999; 274: 15745-15750Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar).Post-translational palmitoylation close to the N terminus of the α subunits of heterotrimeric G proteins appears to be central to their effective interaction with the plasma membrane and thus their capacity to transduce signals from GPCRs to effectors (14Ross E.M. Curr. Biol. 1995; 5: 107-109Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 15Mumby S.M. Curr. Opin. Cell Biol. 1997; 9: 148-154Crossref PubMed Scopus (238) Google Scholar, 16Dunphy J.T. Linder M.E. Biochim. Biophys. Acta. 1998; 1436: 245-261Crossref PubMed Scopus (315) Google Scholar). Since the thioester bond between the protein and the fatty acid is easily cleaved, there is the potential for dynamic regulation of G protein acylation. This has been best examined for the adenylyl cyclase stimulatory G protein Gs, where activation mediated by GPCRs or cholera toxin has been shown to alter [3H]palmitoylation of the G protein (17Degtyarev M.Y. Spiegel A.M. Jones T.L. J. Biol. Chem. 1993; 268: 23769-23772Abstract Full Text PDF PubMed Google Scholar, 18Linder 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 (292) Google Scholar, 19Wedegaertner P.B. Bourne H.R. Cell. 1994; 77: 1063-1070Abstract Full Text PDF PubMed Scopus (304) Google Scholar). Gα11 possesses two adjacent cysteine residues at positions 9 and 10, which have been established to be sites of acylation (20McCallum J.F. Wise A. Grassie M.A. Magee A.I. Guzzi F. Parenti M. Milligan G. Biochem. J. 1995; 310: 1021-1027Crossref PubMed Scopus (40) Google Scholar, 21Wise A. Parenti M. Milligan G. FEBS Lett. 1997; 407: 257-260Crossref PubMed Scopus (20) Google Scholar). Mutation of these two cysteine residues results in production of a soluble polypeptide. Few reports have provided evidence for regulation of the palmitoylation of Gαq/Gα11, although elevated incorporation of [3H]palmitate into these G proteins by stimulation of the gonadotrophin-releasing hormone receptor has been reported (22Stanislaus D. Janovick J.A. Brothers S. Conn P.M. Mol. Endocrinol. 1997; 11: 738-746PubMed Google Scholar) and by molecularly undefined receptors for 5-hydroxytryptamine in rat brain cortical membranes (23Bhamre S. Wang H.-Y. Friedman E. J. Pharmacol. Exp. Ther. 1998; 286: 1482-1489PubMed Google Scholar).Production of fusion proteins between GPCRs and G protein α subunits has become a popular means to explore many aspects of the detailed interactions between these protein classes (24Milligan G. Trends Pharmacol. Sci. 2000; 21: 24-28Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 25Seifert R. Wenzel-Seifert K. Kobilka B.K. Trends Pharmacol. Sci. 1999; 20: 383-389Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Because the N terminus of the G protein α subunit is fused directly to the C terminus of the GPCR in such constructs, it is often unclear whether this might limit interaction with the G protein β·γ complex. This uncertainty reflects that the N terminus of the α subunit is an important contact interface for β·γ, although key amino acids for this interaction are thought to be located some 15 amino acids away from the site(s) of palmitoylation (26Evanko D.S. Thiyagarajan M.M. Wedegaertner P.B. J. Biol. Chem. 2000; 275: 1327-1336Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar).Herein, we use fusion proteins between the α1b-adrenoreceptor and forms of Gα11 to demonstrate that the fusion proteins are activated and regulate their palmitoylation status in response to agonist. By examining fusion constructs in which either the receptor or the G protein is resistant to palmitoylation, we also demonstrate that the acylation status of both polypeptide partners is dynamically regulated by agonist. Moreover, palmitoylation of Gα11 is not necessary for the binding and release of β·γ complex and further transduction of the signal.MATERIALS AND METHODSA fibroblast cell line (EF88) derived from a combined Gαq/Gα11 double knockout mouse (9Zywietz A. Gohla A. Schmelz M. Schultz G. Offermanns S. J. Biol. Chem. 2001; 276: 3840-3845Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 10Mao J. Yuan H. Xie W. Simon M.I. Wu D. J. Biol. Chem. 1998; 273: 27118-27123Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 13Yu R. Hinkle P.M. J. Biol. Chem. 1999; 274: 15745-15750Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) was the gift of Dr. M. I. Simon (California Institute of Technology, Pasadena, CA).[9,10-3H]Palmitic acid was obtained from Amersham Pharmacia Biotech. [3H]Prazosin and [35S]GTPγS were purchased from PerkinElmer Life Sciences. Dulbecco's modified Eagle's medium (DMEM), newborn calf serum, l-glutamine, and trypsin/EDTA were purchased from Life Technologies, Inc. Fura-2/AM, phenylephrine HCl, phentolamine, HEPES, Bordetella pertussis toxin, and EGTA were purchased from Sigma. CQ (C terminus of Gq) antisera have previously been described (27Mitchell F.M. Buckley N.J. Milligan G. Biochem. J. 1993; 293: 495-499Crossref PubMed Scopus (55) Google Scholar, 28Kim G.-D. Carr I.C. Anderson L.A. Zabavnik J. Eidne K.A. Milligan G. J. Biol. Chem. 1994; 269: 19933-19940Abstract Full Text PDF PubMed Google Scholar).Construction of Fusion ProteinsProduction and subcloning of wild type and palmitoylation-resistant α1b-adrenoreceptor-Gα11 fusion proteins was performed in two separate stages. In the first step, the coding sequence of each form of Gα11 (20McCallum J.F. Wise A. Grassie M.A. Magee A.I. Guzzi F. Parenti M. Milligan G. Biochem. J. 1995; 310: 1021-1027Crossref PubMed Scopus (40) Google Scholar, 21Wise A. Parenti M. Milligan G. FEBS Lett. 1997; 407: 257-260Crossref PubMed Scopus (20) Google Scholar) was modified by PCR amplification using the amino-terminal primer 5′-GAGGACGGTACCACTCTGGAGTCCATG-3′ the initiating Met of Gα11 was removed, and both a KpnI restriction site (underlined) and a two-amino acid spacer (Gly-Asn) were introduced. Using the C-terminal primer 5′-TTGTGCGGCCGCCGGTCACACCAGGTT-3, a NotI restriction site (underlined) was introduced downstream of the stop codon of Gα11. The amplified fragments digested withKpnI and NotI were subcloned into similarly digested pcDNA3 expression vector (Invitrogen). To obtain the various α1b-adrenoreceptor-Gα11 fusion proteins, the coding sequence of the wild type or C365A, C367G hamster α1b-adrenoreceptor was amplified by PCR. Using the amino-terminal primer 5′-GACGGTACCTCTAAAATGAATCCCGAT-3′, aKpnI restriction site (underlined) was introduced upstream of the initiator Met. Using the carboxyl-terminal primer 5′-GTCCCTGGTACCAAAGTGCCCGGGTG-3′, a second KpnI restriction site (underlined) was introduced immediately upstream of the stop codon. Finally, the Gα11 constructs in pcDNA3 were digested with KpnI and ligated together with the PCR product of the α1b-adrenoreceptor amplification also digested with KpnI. The open reading frames thus produced represent the coding sequence of either α1b-adrenoreceptor-Gα11, C365A,C367G α1b-adrenoreceptor-Gα11, or α1b-adrenoreceptor-C9S,C10S Gα11. Each was fully sequenced before its expression and analysis.Transient Transfection of HEK293 CellsHEK293 cells were maintained in DMEM supplemented with 0.292 g/literl-glutamine and 10% (v/v) newborn calf serum at 37 °C in a 5% CO2 humidified atmosphere. Cells were grown to 60–80% confluence before transient transfection in 60-mm dishes. Transfection was performed using LipofectAMINE reagent (Life Technologies) according to the manufacturer's instructions.3H PalmitoylationCells were labeled with 0.5 mCi/ml [9,10-3H]palmitic acid in DMEM supplemented with 0.292 g/liter l-glutamine, 5% (v/v) dialyzed newborn calf serum, and 5 mm pyruvic acid at 37 °C in a 5% CO2 humidified atmosphere. After incubation for the appropriate time in the presence and absence of varying concentrations of phenylephrine, reactions were terminated by the addition of 200 μl of 1% (w/v) SDS. Proteins were denatured by passage through a 25-gauge needle followed by 5-min incubation at 100 °C. After chilling to 4 °C, 800 μl of Kahn solubilization buffer (1% (v/v) Triton X-100, 10 mm EDTA, 100 mmNaH2PO4, 10 mm NaF, 50 mm HEPES (pH 7.2)) was added, and the samples were precleared by incubation for 1 h at 4 °C with 100 μl of Pansorbin (Calbiochem). The precleared supernatants were then incubated for 16 h at 4 °C with protein-A-Sepharose and 10 μl of antiserum CQ (27Mitchell F.M. Buckley N.J. Milligan G. Biochem. J. 1993; 293: 495-499Crossref PubMed Scopus (55) Google Scholar, 28Kim G.-D. Carr I.C. Anderson L.A. Zabavnik J. Eidne K.A. Milligan G. J. Biol. Chem. 1994; 269: 19933-19940Abstract Full Text PDF PubMed Google Scholar). Immune complexes were isolated by centrifugation, washed three times with Kahn immunoprecipitation buffer (1% (v/v) Triton X-100, 100 mm NaCl, 100 mmNaF, 50 mm NaH2PO4, 50 mm HEPES (pH 7.2) plus 0.5% SDS), and eluted from the protein A-Sepharose by the addition of electrophoresis buffer containing 20 mm dithiothreitol and heating to 80 °C for 3 min. Analysis was by SDS-polyacrylamide gel electrophoresis, using 10% (w/v) polyacrylamide resolving gels and by autoradiography.[35S]GTPγS Binding[35S]GTPγS binding experiments were initiated by the addition of 10 μg of membranes to an assay buffer (20 mm HEPES (pH 7.4), 3 mm MgCl2, 100 mm NaCl, 1 μm guanosine 5′-diphosphate, 0.2 mm ascorbic acid, 50 nCi of [35S]GTPγS) containing the indicated concentrations of phenylephrine. Nonspecific binding was determined in the same conditions but in the presence of 100 μm GTPγS. Reactions were incubated for 15 min at 30 °C and were terminated by the addition of 0.5 ml of ice-cold buffer, containing 20 mm HEPES (pH 7.4), 3 mmMgCl2 and 100 mm NaCl. The samples were centrifuged at 16,000 × g for 15 min at 4 °C, and the resulting pellets were resuspended in solubilization buffer (100 mm Tris, 200 mm NaCl, 1 mm EDTA, 1.25% Nonidet P-40) plus 0.2% sodium dodecylsulfate. Samples were precleared with Pansorbin (Calbiochem), followed by immunoprecipitation with CQ antiserum. Finally, the immunocomplexes were washed twice with solubilization buffer, and bound [35S]GTPγS was estimated by liquid-scintillation spectrometry.[3H]Prazosin Binding StudiesBinding assays were initiated by the addition of 3 μg of cell membranes to an assay buffer (50 mm Tris-HCl, 100 mm NaCl, 3 mm MgCl2, pH 7.4) containing [3H] prazosin (0.05–10 nm in saturation assays and 0.5 nm for competition assays) in the absence or presence of increasing concentrations of phenylephrine (200-μl final volume). Nonspecific binding was determined in the presence of 100 μm phentolamine. Reactions were incubated for 30 min at 30 °C, and bound ligand was separated from free by vacuum filtration through GF/B filters. The filters were washed twice with assay buffer, and bound ligand was estimated by liquid scintillation spectrometry.[Ca2+] ImagingA fibroblast cell line, (EF88), derived from the embryos of mice in which the α subunits of both Gq and G11 had been knocked out by targeted gene disruption (9Zywietz A. Gohla A. Schmelz M. Schultz G. Offermanns S. J. Biol. Chem. 2001; 276: 3840-3845Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 10Mao J. Yuan H. Xie W. Simon M.I. Wu D. J. Biol. Chem. 1998; 273: 27118-27123Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 13Yu R. Hinkle P.M. J. Biol. Chem. 1999; 274: 15745-15750Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) were grown in DMEM supplemented with 10% (v/v) heat-inactivated fetal bovine serum andl-glutamine (1 mm) in a 95% air and 5% CO2 atmosphere at 37 °C. For transfection experiments, a portion of the cells harvested during trypsinization were plated onto glass coverslips (22-mm diameter, grade 0 thickness), and after a 24-h growth period, cells were transfected using LipofectAMINE (Life Technologies) according to the manufacturer's instructions. After 3 h, cells were washed twice with OPTI-MEM-1 and then cultured in DMEM growth medium for a further 24 h. In some experiments, after an initial 24-h transfection/growth period, the transfected cells were treated with pertussis toxin (25 ng/ml, 24 h).Measurement of [Ca2+]iTransfected cells growing on coverslips were loaded with the Ca2+-sensitive dye Fura-2 by incubation (15–20 min, 37 °C) in physiological control saline solution, 130 mm NaCl, 5 mm KCl, 1 mm CaCl2, 1 mm MgCl2, 20 mm HEPES, 10 mmd-glucose, pH adjusted to 7.4 using NaOH) containing the dye's membrane-permeant acetoxymethylester form (1.0 μm). A rise in [Ca2+]i causes a corresponding rise in the Fura-2 fluorescence ratio recorded from cells loaded with this dye, which allows receptor-mediated changes in [Ca2+]i to be monitored using standard, microspectrofluorimetric techniques (29Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 265: 3440-3450Abstract Full Text PDF Google Scholar). An Optoscan monochromator (Cairn Research, Faversham, Kent, UK) was used to alternate the excitation wavelength between 340 and 380 nm (band pass of 10 nm) and to control the excitation frequency. Fura-2 fluorescence emission at 510 nm was monitored either by a low noise COHU CCD camera or a photomultiplier tube with a bialkali photocathode. Images acquired with the CCD camera were stored and analyzed digitally under the control of Meta Fluor imaging software (Universal Imaging Corp., West Chester, PA).Agonist-evoked [Ca2+]i responses were quantified by peak height (i.e. difference between the base-line resting ratio level and that attained at the peak response). Responses were pooled and are expressed as the mean ± S.E. of at least five experiments, with vertical lines (see Fig.3) representing S.E. Statistical significance of any difference between means was determined using Student's t test. Differences in the magnitude of [Ca2+]i responses evoked by phenylephrine in untreated and pertussis toxin-treated cells were evaluated by Student's paired t test.RESULTSCells of a fibroblast-derived line (EF88) from the embryo of a combined Gαq/Gα11 double knockout mouse were grown on glass coverslips. These were transiently transfected with either the hamster α1b-adrenoreceptor or the mouse G protein Gα11. Co-transfection with enhanced green fluorescent protein allowed identification of positively transfected cells. Following loading of the cells with the Ca2+indicator Fura-2/AM (1 μm, 15 min, 37 °C), single cell Ca2+ imaging was performed in the absence of external Ca2+. In both cases, the addition of the α1-adrenoreceptor-selective agonist phenylephrine (10 μm) failed to alter basal intracellular [Ca2+] ([Ca2+]i) (Fig.1, A and B). However, co-expression of both the α1b-adrenoreceptor and Gα11 resulted in a robust and rapid elevation of [Ca2+]i (Fig. 1, C and D). We have previously demonstrated that Gα11 can be post-translationally acylated on both Cys9 and Cys10 (20McCallum J.F. Wise A. Grassie M.A. Magee A.I. Guzzi F. Parenti M. Milligan G. Biochem. J. 1995; 310: 1021-1027Crossref PubMed Scopus (40) Google Scholar) and that mutation of both of these amino acids to Ser prevents membrane association of the G protein (20McCallum J.F. Wise A. Grassie M.A. Magee A.I. Guzzi F. Parenti M. Milligan G. Biochem. J. 1995; 310: 1021-1027Crossref PubMed Scopus (40) Google Scholar, 21Wise A. Parenti M. Milligan G. FEBS Lett. 1997; 407: 257-260Crossref PubMed Scopus (20) Google Scholar). Co-transfection of EF88 cells with the α1b-adrenoreceptor and C9S,C10S Gα11 thus also failed to result in a phenylephrine-mediated elevation of [Ca2+]i (Fig.2). Equivalent studies with either C9S Gα11 or C10S Gα11 did produce an agonist-dependent rise in [Ca2+]i(Fig. 2), but the magnitude of the response was substantially less than with wild type Gα11 and was kinetically much slower. In both of these regards, C10S Gα11 performed more poorly than C9S Gα11 (Fig. 2).Figure 1Co-expression of the α1b-adrenoreceptor and the G protein Gα11 is required to elevate [Ca2+] levels in EF88 cells. EF88 cells were transiently transfected with either the hamster α1b-adrenoreceptor (A) or the mouse G protein Gα11 (B) or co-transfected with the α1b-adrenoreceptor and Gα11 (C). Green fluorescent protein was co-expressed as a marker for positively transfected cells. Cells were loaded with Fura-2/AM and [Ca2+]i levels imaged before and during exposure of the cells to phenylephrine (Phe; 10 μm). Representative images of basal and peak [Ca2+]i are displayed for two cells co-expressing α1b-adrenoreceptor and Gα11 (D).Warmer colors represent higher [Ca2+].View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 2Palmitoylation potential of Gα11 determines functional interactions with a co-expressed α1b-adrenoreceptor. EF88 cells were transiently transfected with the hamster α1b-adrenoreceptor and each of the following: wild type Gα11 (1), C9S Gα11(2), C10S Gα11 (3), and C9S,C10S Gα11 (4). Positively transfected cells were identified by co-expression of green fluorescent protein. The capacity of phenylephrine (3 μm) to elevate [Ca2+]i levels was then imaged. Data are the traces from six individual cells for each set of transfections.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To potentially overcome these deficits, fusion proteins were constructed between the α1b-adrenoreceptor and forms of Gα11. The G protein sequence was attached directly to the C-terminal tail of the receptor cDNA from which the stop codon was eliminated. This allows production of single open reading frames containing the features of both polypeptides (24Milligan G. Trends Pharmacol. Sci. 2000; 21: 24-28Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 25Seifert R. Wenzel-Seifert K. Kobilka B.K. Trends Pharmacol. Sci. 1999; 20: 383-389Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Expression in EF88 cells of the chimeric polypeptide containing the wild type sequences of both receptor and G protein resulted in an elevation of [Ca2+]i upon the addition of phenylephrine (Fig.3 A), although the kinetics of the response were markedly slower than for the isolated but co-transfected receptor and G protein. Now the same was true when a fusion protein between the α1b-adrenoreceptor and C9S,C10S Gα11 was used (Fig. 3 B). This was also the case when a C365A,C367G α1b-adrenoreceptor-Gα11 fusion protein was expressed (data not shown). Elevation of [Ca2+]iin response to activation of a GPCR can proceed from activation of members of the phosphoinositidase C family by either α subunits of the Gq/G11 family or β·γ complexes (Fig.4 A). To ascertain if the signal from the fusion proteins derived from the receptor-attached G protein α subunit, EF88 cells were co-transfected with the α1b-adrenoreceptor-Gα11 fusion protein and transducin α. Transducin α is used regularly as a β·γ-sequestering agent, and in this situation the effect of phenylephrine was fully attenuated (Fig. 3 A). The N-terminal region of Gα subunits is an important binding interface for β·γ (30Wall 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 (1004) Google Scholar, 31Lambright D.G. Sondek J. Bohm A. Skiba N.P. Hamm H.E. Sigler P.B. Nature. 1996; 379: 311-319Crossref PubMed Scopus (1044) Google Scholar). However, transducin α also fully attenuated the phenylephrine signal from the α1b-adrenoreceptor-C9S,C10S Gα11 fusion protein (Fig. 3 B).Figure 4Elevation of [Ca2+]i by the α1b-adrenoreceptor-Gα11fusion protein is not via activation of Gi family G proteins. A, receptor-mediated elevation of [Ca2+]i may proceed via either G protein α subunits or the β·γ complex. The β·γ-mediated elevation of [Ca2+]i (Fig. 3) could potentially derive from release of β·γ from the α1b-adrenoreceptor-Gα11 fusion protein or from pertussis toxin (Ptx)-sensitive, Gi family G proteins expressed endogenously by EF88 cells. B, membranes from either EF88 (2) or HEK 293 (1) cells were immunoblotted to detect the presence of Gα11/Gαq (upper panel) or Gαi (lower panel). C, EF88 cells expressing the α1b-adrenoreceptor-Gα11 fusion protein were treated with pertussis toxin (25 ng/ml) or with vehicle for 24 h. The capacity of phenylephrine to elevate [Ca2+]iwas then measured.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Elevation of [Ca2+]i could also potentially arise from β·γ complex released by interaction of the fusion proteins with members of the pertussis toxin-sensitive Gi family (Fig. 4 A), which, unlike Gαq and Gα11, are expressed in EF88 cells (Fig. 4 B). Expression of receptors in heterologous systems can result in a reduction in specificity of G protein coupling (32Selbie L.A. Hill S.J. Trends Pharmacol. Sci. 1998; 19: 87-93Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). To eliminate this possibility, experiments were repeated following sustained treatment with pertussis toxin of EF88 cells that had been transfected to express the α1b-adrenoreceptor-Gα11 fusion protein. This did not alter the phenylephrine-mediated elevation of [Ca2+]i (Fig. 4 C). The combined data of Figs. 3 and 4 demonstrate that the α1b-adrenoreceptor-Gα11 fusion protein both binds endogenous β·γ and is able to release it upon agonist occupancy and that the palmitoylation status and potential of Gα11 does not limit either its binding or release of β·γ complex.To directly explore palmitoylation of the fusion proteins and its regulation, α1b-adrenoreceptor-Gα11 was expressed transiently in HEK293 cells. Both these and mock-transfected cells were labeled with [3H]palmitate for 2 h, and the samples were immunoprecipitated with an antiserum (CQ) that identifies the C-terminal 10 amino acids shared by Gαqand Gα11 (27Mitchell F.M. Buckley N.J. Milligan G. Biochem. J. 1993; 293: 495-499Crossref PubMed Scopus (55) Google Scholar, 28Kim G.-D. Carr I.C. Anderson L.A. Zabavnik J. Eidne K.A. Milligan G. J. Biol. Chem. 1994; 269: 19933-19940Abstract Full Text PDF PubMed Google Scholar). Following SDS-polyacrylamide gel electrophoresis and autoradiography, a band of some 42 kDa was observed in both mock-transfected and positively transfected cells (Fig.5). This corresponds to a mixture of Gαq and Gα11, which are co-expressed by HEK293 cells and not resolved by the gel conditions employed. A 100-kDa [3H]palmitoylated polypeptide corresponding to the α1b-adrenoreceptor-Gα11 fusion protein was also observed but only in the positively transfected cells (Fig. 5). When the time course of [3H]palmitoylation of the fusion protein was monitored in the presence and absence of phenylephrine, the rate, but not the maximal extent, of [3H]palmitoylation was markedly enhanced by the agonist (Fig.6). This effect was specific for agonist, since the presence of phentolamine, an antagonist/inverse agonist at the α1b-adreno