Differential Activity of the G Protein β5γ2 Subunit at Receptors and Effectors
1998; Elsevier BV; Volume: 273; Issue: 51 Linguagem: Inglês
10.1074/jbc.273.51.34429
ISSN1083-351X
AutoresMargaret A. Lindorfer, Chang‐Seon Myung, Yoko Savino, Hiroshi Yasuda, Rimma Khazan, James C. Garrison,
Tópico(s)Ion channel regulation and function
ResumoThe G protein β5 subunit differs substantially in amino acid sequence from the other known β subunits suggesting that βγ dimers containing this protein may play specialized roles in cell signaling. To examine the functional properties of the β5 subunit, recombinant β5γ2 dimers were purified from baculovirus-infected Sf9 insect cells using a strategy based on two affinity tags (hexahistidine and FLAG) engineered into the N terminus of the γ2 subunit (γ2HF). The function of the pure β5γ2HF dimers was examined in three assays: activation of pure phospholipase C-β in lipid vesicles; activation of recombinant, type II adenylyl cyclase expressed in Sf9 cell membranes; and coupling of α subunits to the endothelin B (ETB) and M1 muscarinic receptors. In each case, the efficacy of the β5γ2HF dimer was compared with that of the β1γ2HF dimer, which has demonstrated activity in these assays. The β5γ2HF dimer activated phospholipase C-β with a potency and efficacy similar to that of β1γ2 or β1γ2HF; however, it was markedly less effective than the β1γ2HF or β1γ2 dimer in its ability to activate type II adenylyl cyclase (EC50 of ∼700 nm versus 25 nm). Both the β5γ2HF and the β1γ2HF dimers supported coupling of M1 muscarinic receptors to the Gq α subunit. The ETB receptor coupled effectively to both the Gi and Gq α subunits in the presence of the β1γ2HF dimer. In contrast, the β5γ2HF dimer only supported coupling of the Gq α subunits to the ETB receptor and did not support coupling of the Gi α subunit. These results suggest that the β5γ2HF dimer binds selectively to Gq α subunits and does not activate the same set of effectors as dimers containing the β1subunit. Overall, the data support a specialized role for the β5 subunit in cell signaling. The G protein β5 subunit differs substantially in amino acid sequence from the other known β subunits suggesting that βγ dimers containing this protein may play specialized roles in cell signaling. To examine the functional properties of the β5 subunit, recombinant β5γ2 dimers were purified from baculovirus-infected Sf9 insect cells using a strategy based on two affinity tags (hexahistidine and FLAG) engineered into the N terminus of the γ2 subunit (γ2HF). The function of the pure β5γ2HF dimers was examined in three assays: activation of pure phospholipase C-β in lipid vesicles; activation of recombinant, type II adenylyl cyclase expressed in Sf9 cell membranes; and coupling of α subunits to the endothelin B (ETB) and M1 muscarinic receptors. In each case, the efficacy of the β5γ2HF dimer was compared with that of the β1γ2HF dimer, which has demonstrated activity in these assays. The β5γ2HF dimer activated phospholipase C-β with a potency and efficacy similar to that of β1γ2 or β1γ2HF; however, it was markedly less effective than the β1γ2HF or β1γ2 dimer in its ability to activate type II adenylyl cyclase (EC50 of ∼700 nm versus 25 nm). Both the β5γ2HF and the β1γ2HF dimers supported coupling of M1 muscarinic receptors to the Gq α subunit. The ETB receptor coupled effectively to both the Gi and Gq α subunits in the presence of the β1γ2HF dimer. In contrast, the β5γ2HF dimer only supported coupling of the Gq α subunits to the ETB receptor and did not support coupling of the Gi α subunit. These results suggest that the β5γ2HF dimer binds selectively to Gq α subunits and does not activate the same set of effectors as dimers containing the β1subunit. Overall, the data support a specialized role for the β5 subunit in cell signaling. guanine nucleotide-binding regulatory proteins Spondoptera frugiperda cells (ATCC no. CRL 1711) phenylmethylsulfonyl fluoride 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate polyoxylethylene (10Berstein G. Blank J.L. Jhon D. Exton J.H. Rhee S.G. Ross E.M. Cell. 1992; 70: 411-418Abstract Full Text PDF PubMed Scopus (348) Google Scholar) dodecyl ether 5′-adenylylimidodiphosphate human endothelin B receptor phospholipase C-β l-[benzylic-4,4′-3H]quinuclidinyl benzilate. Complex biochemical mechanisms exist to discriminate, integrate, and modulate a cell's response to the hormones, autacoids, neurotransmitters, and growth factors in their environment. One of the best characterized signal transduction systems is the pathway used by receptors coupled to heterotrimeric G proteins1 (1Strader C.D. Fong T.M. Tota M.R. Underwood D. Dixon R.A.F. Annu. Rev. Biochem. 1994; 63: 101-132Crossref PubMed Scopus (996) Google Scholar, 2Neer E.J. Cell. 1995; 80: 249-257Abstract Full Text PDF PubMed Scopus (1287) Google Scholar, 3Hamm H.E. J. Biol. Chem. 1998; 273: 669-672Abstract Full Text Full Text PDF PubMed Scopus (939) Google Scholar). Current understanding of this signaling pathway shows it to be surprisingly complex with large families of proteins comprising the receptors, G proteins, and effectors (1Strader C.D. Fong T.M. Tota M.R. Underwood D. Dixon R.A.F. Annu. Rev. Biochem. 1994; 63: 101-132Crossref PubMed Scopus (996) Google Scholar, 4Hepler J.R. Gilman A.G. Trends Biochem. Sci. 1992; 17: 383-387Abstract Full Text PDF PubMed Scopus (925) Google Scholar, 5Clapham D.E. Neer E.J. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 167-203Crossref PubMed Scopus (704) Google Scholar) and with most cell types expressing multiple isoforms of each category. Many receptors are known to signal through several G proteins. In turn, many effectors receive signals from different isoforms of both the α and βγ subunits of G proteins (5Clapham D.E. Neer E.J. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 167-203Crossref PubMed Scopus (704) Google Scholar, 6Sunahara R.K. Dessauer C.W. Gilman A.G. Annu. Rev. Pharmacol. Toxicol. 1996; 36: 461-480Crossref PubMed Scopus (742) Google Scholar). Thus, an important issue in cell signaling is identification of the protein-protein interactions underlying the cellular responses to a particular stimulus. It is clear that a cellular response is the summation of many molecular interactions. A primary determinant of the outcome is selective expression of signaling molecules, as occurs in the rod outer segment of the eye where the visual receptor, rhodopsin, and its G protein transducin are the major signaling proteins in the membrane (7Yau K.W. Invest. Ophthalmol. Visual Sci. 1994; 35: 9-32PubMed Google Scholar). In less specialized cells, compartmentation or targeting of the participants in a signaling cascade may determine the specificity observed in vivo (2Neer E.J. Cell. 1995; 80: 249-257Abstract Full Text PDF PubMed Scopus (1287) Google Scholar, 8Neubig R.R. FASEB J. 1994; 8: 939-946Crossref PubMed Scopus (319) Google Scholar). Indeed, several lines of investigation have suggested specific localization of receptors, G proteins, and effectors within the cell that may lead to selective and efficient interactions between members of a signaling cascade (9Gudermann T. Kalkbrenner F. Schultz G. Annu. Rev. Pharm. Toxicol. 1996; 36: 429-459Crossref PubMed Scopus (333) Google Scholar). Moreover, critical steps in the activation of G proteins, such as the rate of exchange of GTP for GDP on the α subunit, are highly regulated processes wherein receptors, effectors, and accessory proteins such as RGS molecules are all involved in determining the kinetics of GTP/GDP exchange (10Berstein G. Blank J.L. Jhon D. Exton J.H. Rhee S.G. Ross E.M. Cell. 1992; 70: 411-418Abstract Full Text PDF PubMed Scopus (348) Google Scholar, 11Dohlman H.G. Thorner J. J. Biol. Chem. 1997; 272: 3871-3874Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar, 12Berman D.M. Gilman A.G. J. Biol. Chem. 1998; 273: 1269-1272Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar). At the G protein-effector level, it has long been recognized that certain α subunits selectively regulate specific effectors (2Neer E.J. Cell. 1995; 80: 249-257Abstract Full Text PDF PubMed Scopus (1287) Google Scholar, 3Hamm H.E. J. Biol. Chem. 1998; 273: 669-672Abstract Full Text Full Text PDF PubMed Scopus (939) Google Scholar, 4Hepler J.R. Gilman A.G. Trends Biochem. Sci. 1992; 17: 383-387Abstract Full Text PDF PubMed Scopus (925) Google Scholar), and recently, defined isoforms of βγ dimers have been reported to differentially activate effectors (13Zhang S. Coso O.A. Lee C. Gutkind J.S. Simonds W.F. J. Biol. Chem. 1996; 271: 33575-33579Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 14Bayewitch M.L. Avidor-Reiss T. Levy R. Pfeuffer T. Nevo I. Simonds W.F. Vogel Z. J. Biol. Chem. 1998; 273: 2273-2276Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Finally, the large diversity in the G protein α and βγ subunits suggests that there are important protein-protein interactions involving the α and βγ subunits of the heterotrimer that determine specificity (4Hepler J.R. Gilman A.G. Trends Biochem. Sci. 1992; 17: 383-387Abstract Full Text PDF PubMed Scopus (925) Google Scholar, 9Gudermann T. Kalkbrenner F. Schultz G. Annu. Rev. Pharm. Toxicol. 1996; 36: 429-459Crossref PubMed Scopus (333) Google Scholar). Indeed, selective interaction of α and βγ isoforms has been observed in vitro between the β5 subunit and α subunits in the Gq family (15Fletcher J.E. Lindorfer M.A. DeFilippo J.M. Yasuda H. Guilmard M. Garrison J.C. J. Biol. Chem. 1998; 273: 636-644Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). As receptors coupled to the αq subunit regulate important functions in most all tissues, this latter finding makes it important to determine the functional properties of βγ dimers containing the β5 subunit. To date, there is no information about the receptors that couple to dimers containing the β5 subunit and release this potentially unique signal. Only limited data are available about the functionality of βγ dimers containing the β5 subunit. However, both the β5 subunit and its splice variant, β5L, have been shown to stimulate PLC-β2 activity when transfected into COS-7 cells with the γ2 subunit (16Watson A.J. Katz A. Simon M.I. J. Biol. Chem. 1994; 269: 22150-22156Abstract Full Text PDF PubMed Google Scholar,17Watson A.J. Aragay A.M. Slepak V.Z. Simon M.I. J. Biol. Chem. 1996; 271: 28154-28160Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Interestingly, the β1γ2 dimer markedly activates the mitogen-activated protein kinase pathway in COS cells, whereas the β5γ2 dimer does not (13Zhang S. Coso O.A. Lee C. Gutkind J.S. Simonds W.F. J. Biol. Chem. 1996; 271: 33575-33579Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Moreover, the β1γ2 dimer stimulates type II adenylyl cyclase and inhibits type I adenylyl cyclase when co-transfected into these cells, but the β5γ2 subunit appears to inhibit both type I and type II cyclase activity (14Bayewitch M.L. Avidor-Reiss T. Levy R. Pfeuffer T. Nevo I. Simonds W.F. Vogel Z. J. Biol. Chem. 1998; 273: 2273-2276Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). These observations suggest that dimers containing the β5 subunit may have different functions in cell signaling from those containing other β subunits. In the experiments reported here, we have taken two approaches toward examining the function of the β5γ2 dimer. Recombinant dimers were purified using two affinity tags (hexahistidine and FLAG) engineered into the N terminus of the γ2subunit (γ2HF). In one approach, we examined the ability of the purified dimer to activate two effectors in vitro,PLC-β and type II adenylyl cyclase. In a second approach, we compared the ability of β5γ2HF and β1γ2HF dimers to support coupling of α subunits with the M1 muscarinic and ETBreceptors. The M1 muscarinic receptor was chosen because it couples selectively to Gq α subunits (18Berstein G. Blank J.L. Smrcka A.V. Higashijima T. Sternweis P.C. Exton J.H. Ross E.M. J. Biol. Chem. 1992; 267: 8081-8088Abstract Full Text PDF PubMed Google Scholar, 19Offermanns S. Wieland T. Homann D. Sandmann J. Bombien E. Spicher K. Schultz G. Jakobs K.H. Mol. Pharmacol. 1994; 45: 890-898PubMed Google Scholar) and the ETB receptor because it couples to both the Giand Gq α subunits (20Aramori I. Nakanishi S. J. Biol. Chem. 1992; 267: 12468-12474Abstract Full Text PDF PubMed Google Scholar, 21Elshourbagy N.A. Korman D.R. Wu H. Sylvester D.R. Lee J.A. Nuthalaganti P. Bergsma D.J. Kumar C.S. Nambi P. J. Biol. Chem. 1993; 268: 3873-3879Abstract Full Text PDF PubMed Google Scholar). The results show that the β5γ2HF dimer is able to fully activate PLC-β but is much less effective at activating type II adenylyl cyclase. The β5γ2HF dimer supports coupling of Gq α subunits to M1 muscarinic and ETB receptors with an efficacy similar to that of the β1γ2HF dimer. However, in keeping with its apparent lower affinity for the Gi α subunit (15Fletcher J.E. Lindorfer M.A. DeFilippo J.M. Yasuda H. Guilmard M. Garrison J.C. J. Biol. Chem. 1998; 273: 636-644Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), the β5γ2HF dimer does not support coupling of the Gi α subunit to the ETB receptor. Overall, the data suggest that the β5γ2dimer couples selectively to αq-linked receptors and may only interact with a limited set of effectors. Thus, in tissues such as brain where it is highly expressed, this dimer may indeed have specialized functions in cellular signaling. A clone encoding the human ETB receptor (GenBankTM accession no. L06623) was the kind gift of Dr. Ponal Nambi at SmithKline Beecham Pharmaceuticals (21Elshourbagy N.A. Korman D.R. Wu H. Sylvester D.R. Lee J.A. Nuthalaganti P. Bergsma D.J. Kumar C.S. Nambi P. J. Biol. Chem. 1993; 268: 3873-3879Abstract Full Text PDF PubMed Google Scholar). The receptor cDNA was excised from pBluescript (Stratagene) with SacI and KpnI, further digested with MspI and subcloned into the pCNTR shuttle vector. The ETB coding sequence was excised with BamHI and subcloned into the pVL1393 baculovirus transfer vector. A recombinant baculovirus encoding the receptor was obtained by co-transfecting the transfer vector with linearized viral DNA into Sf9 cells using the Pharmingen BaculoGold® kit (15Fletcher J.E. Lindorfer M.A. DeFilippo J.M. Yasuda H. Guilmard M. Garrison J.C. J. Biol. Chem. 1998; 273: 636-644Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The recombinant baculoviruses were purified by one round of plaque purification (22Summers M.D. Smith G.E. Tex. Agric. Exp. Stn. Bull. 1987; 1555: 1-56Google Scholar). The construction of recombinant baculoviruses coding for the Gi1, Gq, Gs α subunits, the β1, β1HF, β5, γ2 and γ2HF subunits have been described (15Fletcher J.E. Lindorfer M.A. DeFilippo J.M. Yasuda H. Guilmard M. Garrison J.C. J. Biol. Chem. 1998; 273: 636-644Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 23Graber S.G. Figler R.A. Garrison J.C. J. Biol. Chem. 1992; 267: 1271-1278Abstract Full Text PDF PubMed Google Scholar, 24Graber S.G. Figler R.A. Kalman-Maltese V.K. Robishaw J.D. Garrison J.C. J. Biol. Chem. 1992; 267: 13123-13126Abstract Full Text PDF PubMed Google Scholar, 25Yasuda H. Lindorfer M.A. Myung C.-S. Garrison J.C. J. Biol. Chem. 1998; 273: 21958-21965Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The baculovirus for the M1 muscarinic receptor was the kind gift of Dr. Elliott M. Ross. Sf9 cells were infected with recombinant baculovirus encoding the ETB or M1muscarinic receptor at a multiplicity of infection of 3 and the incubation continued for 48–60 h (26Graber S.G. Figler R.A. Garrison J.C. Methods Enzymol. 1994; 237: 212-226Crossref PubMed Scopus (30) Google Scholar). Washed cell pellets were resuspended in either ETB membrane homogenization buffer (25 mm Hepes, pH 7.5, 1% (w/v) glycerol, 100 mm NaCl, 17 μg/ml PMSF, 20 μg/ml benzamidine, and 2 μg/ml of aprotinin, leupeptin, and pepstatin A) or M1membrane homogenization buffer (20 mm Hepes, pH 8.0, 2 mm MgCl2, 1 mm EDTA, 17 μg/ml PMSF, 2 μg/ml aprotinin, and 10 μg/ml leupeptin) (27Parker E.M. Kameyama K. Higashijima T. Ross E.M. J. Biol. Chem. 1991; 266: 519-527Abstract Full Text PDF PubMed Google Scholar) and stored frozen at −70 °C. All subsequent steps were at 4 °C or on ice. Cell pellets were thawed in ice-cold membrane homogenization buffer and burst by N2 cavitation (600 psi, 20 min). The cell lysate was centrifuged for 10 min at 750 × g. The low speed supernatant was centrifuged for 30 min at 28,000 × g. To reduce the level of endogenous Sf9 G proteins, the pelleted membranes were resuspended in membrane resuspension buffer (50 mm Hepes, pH 7.5, 1 mm EDTA, 3 mmMgSO4) containing 7 m urea and incubated for 30 min (28Shichi H. Somers R.L. J. Biol. Chem. 1978; 253: 7040-7046Abstract Full Text PDF PubMed Google Scholar, 29Debburman S.K. Kunapuli P. Benovic J.L. Hosey M.M. Mol. Pharmacol. 1995; 47: 224-233PubMed Google Scholar, 30Hartman IV, J.L. Northup J.K. J. Biol. Chem. 1996; 271: 22591-22597Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The suspension was then centrifuged at 142,000 ×g for 30 min. The stripped membranes were washed twice with membrane resuspension buffer and resuspended in membrane homogenization buffer at 5–15 mg of protein/ml, frozen, and stored at −70 °C. TheB max and K d values of the receptors were measured before and after urea treatment. The binding parameters for recombinant ETB receptors were determined using 125I-endothelin-1 in a buffer containing 10 mm Hepes, pH 7.4, 5 mm MgCl2, 1 mm EDTA, and 0.1% bovine serum albumin using our published techniques (31Figler R.A. Graber S.G. Lindorfer M.A. Yasuda H. Linden J. Garrison J.C. Mol. Pharmacol. 1996; 50: 1587-1595PubMed Google Scholar) and the conditions described previously (32Elshourbagy N.A. Lee J.A. Korman D.R. Nuthalaganti P. Sylvester D.R. Dilella A.G. Sutiphong J.A. Kumar C.S. Mol. Pharmacol. 1992; 41: 465-473PubMed Google Scholar). Nonspecific binding was determined using 10 μm unlabeled endothelin-1 and varied from 13% at 0.01 nm to 90% at 100 nm endothelin-1. Binding of the antagonist ligand [3H]QNB to the M1 muscarinic receptor was performed as described (33Wong S.K. Parker E.M. Ross E.M. J. Biol. Chem. 1990; 265: 6219-6224Abstract Full Text PDF PubMed Google Scholar) using a concentration range of 0.5–50 nm [3H]QNB. Nonspecific binding was determined using 10 μm atropine and was less than 10% at 50 nm [3H]QNB. TheB max and K d were determined by fitting the data to a rectangular hyperbola using the nonlinear least squares routines in the GraphPad Prizm® software. The binding parameters of the membranes as determined before and after urea treatment were as follows: ETB receptors (beforeB max, 27 pmol/mg membrane protein;K d 0.05 nm; after Bmax, 100 pmol/mg membrane protein; K d 0.1 nm); M1 muscarinic receptors (beforeB max, 2.6 pmol/mg membrane protein;K d 0.15 nm; afterB max, 2 pmol/mg membrane protein;K d 0.4 nm). These data indicate that urea treatment did not greatly change the affinity for ligand. The recombinant Gi1 α subunit and β1γ2 and β1γ2HFdimers were purified from baculovirus-infected Sf9 cells as described previously (26Graber S.G. Figler R.A. Garrison J.C. Methods Enzymol. 1994; 237: 212-226Crossref PubMed Scopus (30) Google Scholar, 34Graber S.G. Lindorfer M.A. Garrison J.C. Methods Neurosci. 1996; 29: 207-226Crossref Scopus (23) Google Scholar). The purification of Gq α was based on the method of Biddlecome et al. (35Biddlecome G.H. Berstein G. Ross E.M. J. Biol. Chem. 1996; 271: 7999-8007Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). The Gq α subunit was expressed in Sf9 cells in combination with the β1HFγ2HF dimer at a multiplicity of infection of 3 each. The cells were harvested 48 h after infection, washed three times with insect phosphate-buffered saline, resuspended in a few milliliters of Q lysis buffer (20 mm Tris, pH 8.0, 10 μm GDP, 17 μg/ml PMSF, and 2 μg/ml pepstatin, leupeptin, and aprotinin), frozen in liquid nitrogen and stored at −70 °C. All extractions and centrifugation steps were performed at 4 °C or on ice. Cell pellets were thawed in half the original culture volume of Q lysis buffer and lysed by nitrogen cavitation (26Graber S.G. Figler R.A. Garrison J.C. Methods Enzymol. 1994; 237: 212-226Crossref PubMed Scopus (30) Google Scholar). The lysate was centrifuged (24,000 ×g, 20 min) and the pellet resuspended in one-fourth the previous volume of Q lysis buffer containing 10 μg/ml DNase and 5 mm MgCl2 with a Dounce homogenizer. After a 15-min incubation period, the suspension was rehomogenized and repelleted. The washed pellet was resuspended in Q extraction buffer (1% cholate, 20 mm Tris, pH 8.0, 100 mm NaCl, 5 mm β-mercaptoethanol, 10 μm GDP, PMSF, and 2 μg/ml pepstatin, leupeptin, and aprotinin) at 5 mg/ml total protein. The suspension was stirred for 1 h and then centrifuged (142,000 × g, 45 min). The cholate extract was frozen and stored at −70 °C. The Q chromatography buffer used to purify the Gq α subunit contained 20 mm Hepes, pH 8.0, 100 mm NaCl, 1 mm MgCl2, 5 mm β-mercaptoethanol, 17 μg/ml PMSF, and 2 μg/ml each leupeptin and aprotinin. The cholate extract (∼100 ml) from 1.5 × 109 Sf9 cells was diluted with 400 ml of loading buffer (Q chromatography buffer containing 0.5% (v/v) Genapol C-100 and 10 μm GDP) and applied to a 15-ml Ni2+-NTA superflow agarose resin column equilibrated in loading buffer. The loaded column was washed sequentially with 75-ml volumes of loading buffer, loading buffer containing 0.5 mNaCl, loading buffer containing 0.5 m NaCl and 10 mm imidazole, loading buffer containing 1 mNaCl, and loading buffer. The column was warmed to room temperature and subsequent wash and elution buffers were applied at room temperature. The column was washed with 75 ml of loading buffer containing 0.2% cholate and 3 μm GTPγS, 75 ml of Q chromatography buffer containing 0.3% cholate and finally eluted with Q chromatography buffer containing 1% cholate, 10 μm GDP, 20 μm AlCl3, 10 mmMgCl2, and 10 mm NaF. Fractions containing the Gq α subunit were pooled and concentrated on ice using an Amicon ultrafiltration apparatus and a PM10 membrane. The concentrated protein was diluted 10-fold with Q chromatography buffer containing 0.4% (w/v) cholate with 1 mm dithiothreitol substituted for 5 mm β-mercaptoethanol and re-concentrated. This procedure was repeated three times. The retentate (∼1 ml) was further concentrated to approximately 100 μl in a spin concentrator (Microcon 10, Amicon). The spin concentrator was pretreated with 0.1% bovine serum albumin in phosphate-buffered saline overnight at room temperature and rinsed twice by centrifugation with deionized water. Approximately 10 μg of highly purified αq was recovered from 1.5 × 109 cells. The β5γ2HF subunit was generated by infecting Sf9 cells with the β5 and γ2HF viruses at a multiplicity of infection of 3 each (23Graber S.G. Figler R.A. Garrison J.C. J. Biol. Chem. 1992; 267: 1271-1278Abstract Full Text PDF PubMed Google Scholar). The washed cell pellet was resuspended in a few milliliters of histag lysis buffer (20 mm Hepes, pH 7.4, 150 mm NaCl, 1 mm β-mercaptoethanol, 3 mm MgCl2, 17 μg/ml PMSF, 20 μg/ml benzamidine, and 2 μg/ml aprotinin, leupeptin, and pepstatin), frozen, and stored at −70 °C. The cell pellet was thawed and lysed by nitrogen cavitation in ice-cold histag lysis buffer (26Graber S.G. Figler R.A. Garrison J.C. Methods Enzymol. 1994; 237: 212-226Crossref PubMed Scopus (30) Google Scholar). The cell lysate was centrifuged (10 min, 750 × g) and membranes were pelleted (100,000 × g, 30 min) from the low speed supernatant. The membrane pellet was resuspended in Genapol extraction buffer (histag lysis buffer containing 0.1% (v/v) Genapol C-100) at a ratio of 1 ml of buffer/20 mg of wet pellet weight and stirred on ice for 1 h. The detergent extract was centrifuged (100,000 ×g, 60 min), the supernatant decanted, frozen in liquid nitrogen and stored at −70 °C. Typically, 50 ml of Genapol extract (prepared from 5 gm of cell pellet) was applied to a 5-ml FLAG M2 column equilibrated in Genapol extraction buffer, washed with 30 ml of Genapol extraction buffer, and eluted with 30 ml of 400 μm FLAG peptide in Genapol extraction buffer. This step was repeated with a second aliquot of Genapol extract. The elution fractions were pooled, the Genapol concentration adjusted to 0.5% (v/v) by addition of histag lysis buffer containing 2% Genapol C-100, and the pool applied to a 2.5-ml Ni2+-NTA agarose column equilibrated in histag-loading buffer (histag lysis buffer containing 0.5% (v/v) Genapol). The column was washed with the following buffers: 12.5 ml of histag loading buffer; 12.5 ml of histag-loading buffer plus 350 mm NaCl; 25 ml of histag-loading buffer plus 350 mm NaCl and 10 mm imidazole; 10 ml of histag-loading buffer plus 1 m NaCl; and 10 ml of histag-loading buffer. The bound β5γ2HF was eluted with 10 ml of histag-loading buffer containing 250 mm imidazole. The eluted dimers were diluted one-fourth with CHAPS dilution buffer (20 mm Hepes, pH 7.5, 1 mm EDTA, 3 mm MgCl2, 0.1% (w/v) CHAPS, 1 mm dithiothreitol), applied to a 1-ml HiTrap Q column (Amersham Pharmacia Biotech) equilibrated in the same buffer, washed with 10 ml of buffer and eluted with CHAPS dilution buffer containing 500 mm NaCl. The highly purified β5γ2HF was stored at −70 °C. Approximately 100 μg of the pure β5γ2HFdimer was obtained from a 10-gm Sf9 cell pellet. The purified β5γ2HF dimer was analyzed by electrospray mass spectrometry to determine the extent of modification of the carboxyl terminus of its γ subunit (36Lindorfer M.A. Sherman N.E. Woodfork K.A. Fletcher J.E. Hunt D.F. Garrison J.C. J. Biol. Chem. 1996; 271: 18582-18587Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The deconvoluted mass spectrum of the γ2HF derived from the dimer indicated that the sample contained two molecular weight species. One species, with a molecular mass of 10,014 Da, is consistent with full processing of the γ2HF subunit: removal of the three carboxyl-terminal amino acids (-AIL), addition of a geranylgeranyl lipid to the carboxyl-terminal cysteine, and addition of a carboxylmethyl group to the carboxyl terminus. The other species, with a molecular mass of 10,024 Da, corresponded to a γ2HFsubunit that had undergone no carboxyl-terminal processing. Both species had molecular weights consistent with the removal of the amino-terminal methionine and acetylation of the resulting amino-terminal alanine. Based on analysis of two different β5γ2HF preparations, approximately half of the γ2HF subunits in the sample had a fully processed carboxyl terminus and were therefore capable of high-affinity interactions with α subunits, receptors, and effectors. Urea-treated Sf9 cell membranes expressing recombinant receptors were washed with reconstitution buffer (25 mm Hepes, pH 7.4, 100 mm NaCl, 5 mmMgCl2, 1 mm EDTA, 0.1% bovine serum albumin), and resuspended at 0.5–1 mg of protein/ml in reconstitution buffer. Typically, 50 μl of membrane suspension containing 2.7 pmol of recombinant endothelin receptors (measured as125I-endothelin binding sites) or 120 fmol of recombinant M1 muscarinic receptors (measured as [3H]QNB binding sites) was mixed with 400 μl of reconstitution buffer containing 1 μm AMP-PNP and incubated on ice for 15 min. The α subunit was diluted into the mix before addition of the βγ dimer, then concentrated GDP was added to give a final concentration of 50 nm. The final concentration of Gα and βγ added to the membranes was 5–14 nm and 5–47 nm, respectively, and the receptor:G protein ratio was approximately 1:2 for the ETB receptor and 1:40 for the M1muscarinic receptor. The final concentration of CHAPS was held to less than 0.006% (w/v), and in the case of the αqexperiments, the final cholate concentration was held to less than 0.001% (w/v). The receptor:G protein mixture was incubated on ice for 45 min followed by a 10 min equilibration at 25 °C. The assay was begun by the addition of [35S]GTPγS to a final concentration of 7 nm. The ligand was added 8 min later, and membrane aliquots removed for filtration through nitrocellulose filters (Millipore, HAWP-025) every 60 s. The ability of βγ dimers to stimulate PLC-β was measured exactly as described (25Yasuda H. Lindorfer M.A. Myung C.-S. Garrison J.C. J. Biol. Chem. 1998; 273: 21958-21965Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The ability of the βγ subunits to activate adenylyl cyclase was measured using Sf9 insect cell membranes overexpressing recombinant, rat type II adenylyl cyclase (37Jacobowitz O. Iyengar R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10630-10634Crossref PubMed Scopus (150) Google Scholar). The assay was performed as described (15Fletcher J.E. Lindorfer M.A. DeFilippo J.M. Yasuda H. Guilmard M. Garrison J.C. J. Biol. Chem. 1998; 273: 636-644Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 25Yasuda H. Lindorfer M.A. Myung C.-S. Garrison J.C. J. Biol. Chem. 1998; 273: 21958-21965Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) except that it was incubated 7 min at 30 °C. Procedures for sodium dodecyl sulfate-polyacrylamide gel electrophoresis, quantitation, and Western blotting were as described previously (15Fletcher J.E. Lindorfer M.A. DeFilippo J.M. Yasuda H. Guilmard M. Garrison J.C. J. Biol. Chem. 1998; 273: 636-644Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The primary antibodies used for immunoblotting were previously described (15Fletcher J.E. Lindorfer M.A. DeFilippo J.M. Yasuda H. Guilmard M. Garrison J.C. J. Biol. Chem. 1998; 273: 636-644Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) with the addition of an anti-G protein β5 subunit antibody (CytoSignal, 1:1000 dilution) and the anti-FLAG M2® antibody (Kodak, 1:1000 dilution). The primary antibodies were detected using donkey anti-rabbit or sheep anti-mouse IgG F(ab′)2 horseradish peroxidase conjugates (Amersham Pharmacia Biotech).
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