A Highly Effective Dominant Negative αs Construct Containing Mutations That Affect Distinct Functions Inhibits Multiple Gs-coupled Receptor Signaling Pathways

To investigate the subcellular organization of receptor-G protein signaling pathways, a robust dominant negative αs mutant containing substitutions that alter distinct functions was produced and tested for its effects on Gs-coupled receptor activity in HEK-293 cells. Mutations in the α3β5 loop region, which increase receptor affinity, decrease receptor-mediated activation, and impair activation of adenylyl cyclase, were combined with G226A, which increases affinity for βγ, and A366S, which decreases affinity for GDP. This triple αs mutant can inhibit signaling to Gs from the luteinizing hormone receptor by 97% and from the calcitonin receptor by 100%. In addition, this αs mutant blocks all signaling from the calcitonin receptor to Gq. These results lead to two conclusions about receptor-G protein signaling. First, individual receptors have access to multiple types of G proteins in HEK-293 cell membranes. Second, different G protein α subunits can compete with each other for binding to the same receptor. This dominant negative αs construct will be useful for determining interrelationships among distinct receptor-G protein interactions in a wide variety of cells and tissues. To investigate the subcellular organization of receptor-G protein signaling pathways, a robust dominant negative αs mutant containing substitutions that alter distinct functions was produced and tested for its effects on Gs-coupled receptor activity in HEK-293 cells. Mutations in the α3β5 loop region, which increase receptor affinity, decrease receptor-mediated activation, and impair activation of adenylyl cyclase, were combined with G226A, which increases affinity for βγ, and A366S, which decreases affinity for GDP. This triple αs mutant can inhibit signaling to Gs from the luteinizing hormone receptor by 97% and from the calcitonin receptor by 100%. In addition, this αs mutant blocks all signaling from the calcitonin receptor to Gq. These results lead to two conclusions about receptor-G protein signaling. First, individual receptors have access to multiple types of G proteins in HEK-293 cell membranes. Second, different G protein α subunits can compete with each other for binding to the same receptor. This dominant negative αs construct will be useful for determining interrelationships among distinct receptor-G protein interactions in a wide variety of cells and tissues. Stimulation of heterotrimeric G proteins by cell surface receptors activates signaling pathways that mediate specific responses to hormones and neurotransmitters. Cells express a wide variety of G protein-coupled receptors as well as numerous G protein α, β, and γ subunits. Many receptors can activate more than one type of G protein, and the G protein subunits can interact with many different types of receptors. The manner in which signaling specificity is maintained in the midst of this vast range of potential interactions is not well understood. This report investigates the interdependence of distinct signaling pathways activated by receptors with broad G protein specificities using a receptor-sequestering dominant negative G protein α subunit. Many potential mechanisms could establish that distinct receptor-G protein interactions will be independent of each other. Among these, one possibility is that specific receptor-G protein complexes localize to separate membrane compartments (1Neubig R.R. FASEB J. 1994; 8: 939-946Crossref PubMed Scopus (318) Google Scholar, 2Neubig R.R. Semin. Neurosci. 1998; 9: 189-197Crossref Scopus (14) Google Scholar). Differential associations with particular proteins or lipids (3Oh P. Schnitzer J.E. Mol. Biol. Cell. 2001; 12: 685-698Crossref PubMed Scopus (346) Google Scholar, 4Moffett S. Brown D.A. Linder M.E. J. Biol. Chem. 2000; 275: 2191-2198Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar) or covalent modifications such as phosphorylation (5Daaka Y. Luttrell L.M. Lefkowitz R.J. Nature. 1997; 390: 88-91Crossref PubMed Scopus (1054) Google Scholar) may result in subpopulations of receptors and G proteins that have restricted access to each other. Although G proteins are often expressed at much higher levels than their receptors are (6Ostrom R.S. Post S.R. Insel P.A. J. Pharmacol. Exp. Ther. 2000; 294: 407-412PubMed Google Scholar), there is evidence that different receptors utilize separate pools of G proteins (7Graeser D. Neubig R.R. Mol. Pharmacol. 1993; 43: 434-443PubMed Google Scholar, 8Simmons M.A. Mather R.J. J. Neurosci. 1991; 11: 2130-2134Crossref PubMed Google Scholar). An alternative potential mechanism for isolating distinct receptor-G protein interactions is that receptors utilize separate regions for binding different G proteins. If this is the case, then multiple types of receptor-G protein interaction can occur simultaneously without affecting each other. Localization of G protein-binding sites have indicated that separate receptor regions may specify interactions with distinct G proteins (9Shyu J.F. Inoue D. Baron R. Horne W.C. J. Biol. Chem. 1996; 271: 31127-31134Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 10Wade S.M. Lim W.K. Lan K.L. Chung D.A. Nanamori M. Neubig R.R. Mol. Pharmacol. 1999; 56: 1005-10013Crossref PubMed Scopus (61) Google Scholar). Dominant negative G protein α subunits can test potential mechanisms for separating the distinct G protein interactions of broad specificity receptors. For instance, if interactions with different G proteins are localized to separate subcellular compartments and/or receptor regions, signaling from a receptor to one type of G protein α subunit will be blocked by a dominant negative version of that α subunit, whereas the other signaling pathways will be unperturbed. Alternatively, if each receptor has access to multiple types of G protein and these different G proteins can compete with each other for receptor binding, then a dominant negative α subunit will block all of the G protein signaling pathways activated by the receptor. Several dominant negative G protein α subunits have been developed previously, but they inhibit G protein signaling incompletely and therefore are not optimal for investigating receptor-G protein signaling pathways. One dominant negative αs mutant contains three substitutions that disrupt different α subunit functions, but it is extremely unstable (11Iiri T. Bell S.M. Baranski T.J. Fujita T. Bourne H.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 499-504Crossref PubMed Scopus (57) Google Scholar), which contributes to its inability to inhibit signaling completely (12Skoglund G. Hussain M.A. Holz G.G. Diabetes. 2000; 49: 1156-1164Crossref PubMed Scopus (103) Google Scholar, 13Vezza R. Rokach J. FitzGerald G.A. Mol. Pharmacol. 2001; 59: 1506-1513Crossref PubMed Scopus (15) Google Scholar). Xanthine-binding mutants of αo, α11, and α16 can inhibit signaling of specific G protein-coupled receptor families, but inhibition of receptor-mediated phospholipase C stimulation is incomplete (14Yu B. Simon M.I. J. Biol. Chem. 1998; 273: 30183-30188Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 15Yu B., Gu, L. Simon M.I. J. Biol. Chem. 2000; 275: 71-76Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). α subunit carboxyl-terminal fragments exhibit dominant negative activity (16Gilchrist A. Vanhauwe J.F., Li, A. Thomas T.O. Voyno-Yasenetskaya T. Hamm H.E. J. Biol. Chem. 2001; 276: 25672-25679Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), but inhibition of Gs signaling is only partial (13Vezza R. Rokach J. FitzGerald G.A. Mol. Pharmacol. 2001; 59: 1506-1513Crossref PubMed Scopus (15) Google Scholar). This report describes the development of a highly effective dominant negative αs mutant that contains substitutions that alter distinct α subunit functions, each of which should stabilize the receptor-bound, nucleotide-free state of Gs. One set of mutations, located in the α3β5 loop region, specifically increases receptor affinity and decreases receptor-mediated activation without affecting nucleotide handling (17Grishina G. Berlot C.H. Mol. Pharmacol. 2000; 57: 1081-1092PubMed Google Scholar) and also disrupts activation of adenylyl cyclase (18Berlot C.H. Bourne H.R. Cell. 1992; 68: 911-922Abstract Full Text PDF PubMed Scopus (160) Google Scholar). G226A increases affinity for βγ (19Miller R.T. Masters S.B. Sullivan K.A. Beiderman B. Bourne H.R. Nature. 1988; 334: 712-715Crossref PubMed Scopus (123) Google Scholar, 20Lee E. Taussig R. Gilman A.G. J. Biol. Chem. 1992; 267: 1212-1218Abstract Full Text PDF PubMed Google Scholar), and A366S decreases affinity for GDP (21Iiri T. Herzmark P. Nakamoto J.M. Van Dop C. Bourne H.R. Nature. 1994; 371: 164-168Crossref PubMed Scopus (243) Google Scholar). Although A366S alone causes αs to be thermolabile, αs(α3β5/G226A/A366S), containing all three sets of mutations, is expressed at close to wild-type levels and blocks signaling from the luteinizing hormone receptor to Gs by up to 97%. The effects of αs(α3β5/G226A/A366S) on signaling by the calcitonin receptor to Gs and Gq are tested in transiently transfected HEK-293 cells. The results demonstrate that this dominant negative αs mutant can block multiple G protein signaling pathways, which indicates that each receptor has access to multiple types of G protein and that these G proteins can compete with each other for receptor binding. αs mutant constructs in the expression vector pcDNAI/Amp (Invitrogen) were generated from the rat αs cDNA (22Jones D.T. Reed R.R. J. Biol. Chem. 1987; 262: 14241-14249Abstract Full Text PDF PubMed Google Scholar) containing the EE epitope (23Grussenmeyer T. Scheidtmann K.H. Hutchinson M.A. Eckhart W. Walter G. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7952-7954Crossref PubMed Scopus (188) Google Scholar), which was generated by mutating αs residuesDYVPSD (residues 189–194) to EYMPTE. Mutations were generated by oligonucleotide-directed in vitro mutagenesis using the Bio-Rad Muta-Gene kit except for those in the α3β5 region, which were produced by subcloning mutagenic oligodeoxynucleotide cassettes. Subcloning and mutagenesis procedures were verified by restriction enzyme analysis and DNA sequencing. HEK-293 cells (ATCC, CRL-1573) (106 per 60-mm dish) were transfected with plasmids as described in the figure legends using 10 μl of LipofectAMINE 2000 Reagent (Invitrogen) according to the manufacturer's instructions. 24 h after transfection, the cells were replated in 24-well plates and labeled with either [3H]adenine or [3H]inositol. After an additional 24 h, intracellular cAMP levels in cells labeled with [3H]adenine and inositol phosphate levels in cells labeled with [3H]inositol were determined as described previously (24Medina R. Grishina G. Meloni E.G. Muth T.R. Berlot C.H. J. Biol. Chem. 1996; 271: 24720-24727Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). cAMP accumulation was measured in the presence of 3-isobutyl-1-methylxanthine and in the presence or absence of agonist as indicated in the figure legends. Inositol phosphate formation was measured in the presence of 5 mm LiCl and in the presence or absence of agonist as indicated in the figure legends. The cells were cultured and assayed at 37 °C. To determine EC50 values for stimulation of cAMP or inositol phosphate formation, the observed activity was fitted to Equation 1,Y=b+(a−b)/(1+(X/c )d)Equation 1 where X is the concentration of agonist; Y is the observed cAMP or inositol phosphate formation; a is the cAMP or inositol phosphate formation observed in the absence of agonist; b is the maximum observed cAMP or inositol phosphate formation; c is the half-maximal effective concentration (EC50) of the agonist; and d is the slope factor. HEK-293 cells (6.25 × 106 per 150-mm dish) were transfected with 0.2 μg/106 cells of plasmid encoding the rat luteinizing hormone receptor in pCIS (25McFarland K.C. Sprengel R. Phillips H.S. Kohler M. Rosemblit N. Nikolics K. Segaloff D.L. Seeburg P.H. Science. 1989; 245: 494-499Crossref PubMed Scopus (800) Google Scholar) or 0.04 μg/106cells of plasmid encoding the rabbit C1a calcitonin receptor in pBKCMV (9Shyu J.F. Inoue D. Baron R. Horne W.C. J. Biol. Chem. 1996; 271: 31127-31134Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) and 2 μg/106 cells of vector (pcDNAI/Amp) using 62.5 μl of LipofectAMINE 2000 Reagent. 24 h after transfection, the cells were replated in 24-well plates. Assays for ligand binding were performed after an additional 24 h. Receptor numbers per cell were determined by fitting data from saturation binding assays using 125I-labeled hCG 1The abbreviations used are: hCGhuman chorionic gonadotropinsCTsalmon calcitonin (26Kosugi S. Mori T. Shenker A. J. Biol. Chem. 1996; 271: 31813-31817Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 27Min L. Ascoli M. Mol. Endocrinol. 2000; 14: 1797-1810Crossref PubMed Scopus (59) Google Scholar) or (9Shyu J.F. Inoue D. Baron R. Horne W.C. J. Biol. Chem. 1996; 271: 31127-31134Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) to Equation 2 where X is the concentration of agonist; Y is the specific is the maximum of binding and is the In each case, the was and the cells were with binding with without containing 1 The was then and with binding containing hCG in the presence or absence of hCG or in the presence or absence of 1 The cells were for 1 h at three with and with The were and in a chorionic calcitonin HEK-293 cells (6.25 × 106 per 150-mm dish) were transfected with of plasmid using R. R. D. J. J. and K. in Scholar). h after transfection, were as described (24Medina R. Grishina G. Meloni E.G. Muth T.R. Berlot C.H. J. Biol. Chem. 1996; 271: 24720-24727Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). of membrane proteins were by to and with a to the EE epitope (23Grussenmeyer T. Scheidtmann K.H. Hutchinson M.A. Eckhart W. Walter G. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7952-7954Crossref PubMed Scopus (188) Google Scholar). The complexes were using an according to the αs in the and the α3β5 loop Gilman A.G. S.R. Science. 1997; PubMed Scopus Google Scholar) with the and results in an αs that affinity for and a to be activated by the receptor (17Grishina G. Berlot C.H. Mol. Pharmacol. 2000; 57: 1081-1092PubMed Google Scholar). the also disrupt activation of adenylyl cyclase by αs (18Berlot C.H. Bourne H.R. Cell. 1992; 68: 911-922Abstract Full Text PDF PubMed Scopus (160) Google Scholar). These that be to Gs-coupled receptors and exhibit dominant negative was transiently expressed in HEK-293 cells that were with plasmid encoding the luteinizing hormone cAMP accumulation in to hCG was by the of more effective dominant negative additional to stabilize the receptor-G protein were αs in with the α3β5 The effects of each of three mutations, G226A, and A366S, to were these three partial dominant negative activity in αs (11Iiri T. Bell S.M. Baranski T.J. Fujita T. Bourne H.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 499-504Crossref PubMed Scopus (57) Google Scholar). G226A in for of αs from βγ (19Miller R.T. Masters S.B. Sullivan K.A. Beiderman B. Bourne H.R. Nature. 1988; 334: 712-715Crossref PubMed Scopus (123) Google Scholar, 20Lee E. Taussig R. Gilman A.G. J. Biol. Chem. 1992; 267: 1212-1218Abstract Full Text PDF PubMed Google Scholar). disrupts a with that may stabilize the activated of αs T. Bourne H.R. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). A366S GDP αs to be activated and to more in the state (21Iiri T. Herzmark P. Nakamoto J.M. Van Dop C. Bourne H.R. Nature. 1994; 371: 164-168Crossref PubMed Scopus (243) Google Scholar). cAMP accumulation in to hCG by G226A and A366S produced dominant negative activity the cAMP to hCG by whereas the by In addition, cAMP with the of A366S on GDP (21Iiri T. Herzmark P. Nakamoto J.M. Van Dop C. Bourne H.R. Nature. 1994; 371: 164-168Crossref PubMed Scopus (243) Google Scholar). In the same dominant negative activity as the two that the dominant negative activity of G226A and A366S, with the α3β5 substitutions in a highly effective dominant negative αs construct that the cAMP accumulation to hCG by 97% increase in dominant negative activity the was to αs(α3β5/G226A/A366S) The dominant negative αs and αs(α3β5/G226A/A366S), the of signaling from the luteinizing hormone receptor to Gs by the EC50 of the cAMP to hCG and by the of cAMP responses to hCG 2 and with and the effects of αs(α3β5/G226A/A366S) on of these were These effects of the G226A, and A366S are with their independent sites and mechanisms of values and inhibition of cAMP responses to hCG in HEK-293 cells the luteinizing hormone receptor in the absence or presence of αs inhibition of inhibition of to were determined in three is the in a were determined in three is the To investigate the α3β5 loop of αs dominant negative the effects of numbers of substitutions in this were the two are located in the and and three and are in the α3β5 on their only the loop are to interact with receptors (17Grishina G. Berlot C.H. Mol. Pharmacol. 2000; 57: 1081-1092PubMed Google Scholar). the effects of substitutions in these two regions that the dominant negative is to the substitutions in the α3β5 than those in the was only effective as a dominant negative than whereas the same and cAMP accumulation as αs the dominant negative of to result from the of a receptor on substitutions of each of the α3β5 loop receptor-mediated activation of αs but not dominant negative activity or whereas the of and were to that of The dominant negative activity produced by simultaneously the three may result from in receptor interaction or may in the loop to interactions among the previously of is its (11Iiri T. Bell S.M. Baranski T.J. Fujita T. Bourne H.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 499-504Crossref PubMed Scopus (57) Google Scholar), to the A366S which increases the of in the nucleotide-free state (21Iiri T. Herzmark P. Nakamoto J.M. Van Dop C. Bourne H.R. Nature. 1994; 371: 164-168Crossref PubMed Scopus (243) Google Scholar). To determine the dominant negative activity of αs(α3β5/G226A/A366S) with is in to a of the α3β5 the expression levels of these αs constructs in of transfected HEK-293 cells were The expression of was to that of whereas that of was much The expression of αs(α3β5/G226A/A366S) was to that of and of αs but was much higher than that of the α3β5 substitutions to the of To investigate dominant negative αs activity the signaling pathways of Gs-coupled receptors that also interact with other G protein signaling of the calcitonin receptor to Gs and Gq was in the presence and absence of αs(α3β5/G226A/A366S) completely blocked cAMP accumulation in to up to at of this was by αs(α3β5/G226A/A366S) the stimulation of cAMP accumulation by by a of 10 previously (9Shyu J.F. Inoue D. Baron R. Horne W.C. J. Biol. Chem. 1996; 271: 31127-31134Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, E. H.E. L. Bourne H.R. Mol. Endocrinol. 1992; PubMed Google Scholar), the calcitonin receptor to Gq than to Gs 5 and In the presence of αs(α3β5/G226A/A366S), inositol phosphate formation in to all of was blocked values of responses to in HEK-293 cells the calcitonin receptor in the absence or presence of not values were determined in three is the inositol not in a EC50 values were determined in three is the inositol The of a dominant negative αs mutant to block signaling from the calcitonin receptor to Gs and Gq to two conclusions calcitonin receptor-G protein signaling. First, each calcitonin receptor has access to αs and in transfected HEK-293 that these α subunits a of receptors. Second, αs can compete with for binding to the calcitonin receptor. substitutions in three different regions of αs results in dominant negative αs activity that can inhibit signaling from Gs-coupled receptors by close to 100%. The substitutions distinct as receptor affinity, receptor-mediated activation, and activation of adenylyl cyclase affinity for βγ or affinity for GDP these to stabilize the nucleotide-free The increases in dominant negative activity that result from these are with their independent sites and mechanisms of Although the nucleotide-free which is by the A366S is αs(α3β5/G226A/A366S) is expressed at a close to that of wild-type This is the receptor affinity by the α3β5 substitutions the which has the receptor affinity A. M. Lefkowitz R.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The α3β5 on their not the nucleotide handling of αs (17Grishina G. Berlot C.H. Mol. Pharmacol. 2000; 57: 1081-1092PubMed Google Scholar). 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