Key points are not available for this paper at this time.
Forskolin- and Gsα-stimulated adenylyl cyclase activity is observed after mixture of two independently-synthesized ∼25-kDa cytosolic fragments derived from mammalian adenylyl cyclases (native Mr ∼ 120,000). The C1a domain from type V adenylyl cyclase (VC1) and the C2 domain from type II adenylyl cyclase (IIC2) can both be expressed in large quantities and purified to homogeneity. When mixed, their maximally stimulated specific activity, 150 μmol/min/mg protein, substantially exceeds values observed previously with the intact enzyme. A soluble, high-affinity complex containing one molecule each of VC1, IIC2, and guanosine 5′-O-(3-thiotriphosphate) (GTPγS)-Gsα is responsible for the observed enzymatic activity and can be isolated. In addition, GTPγS-Gsαinteracts with homodimers of IIC2 to form a heterodimeric complex (one molecule each of Gsα and IIC2) but not detectably with homodimers of VC1. Nevertheless, Gsα can be cross-linked to VC1 in the activated heterotrimeric complex of VC1, IIC2, and Gsα, indicating its proximity to both components of the enzyme that are required for efficient catalysis. These results and those in the accompanying report (Dessauer, C. W., Scully, T. T., and Gilman, A. G. (1997) J. Biol. Chem. 272, 22272–22277) suggest that activators of adenylyl cyclase facilitate formation of a single, high-activity catalytic site at the interface between C1 and C2. Forskolin- and Gsα-stimulated adenylyl cyclase activity is observed after mixture of two independently-synthesized ∼25-kDa cytosolic fragments derived from mammalian adenylyl cyclases (native Mr ∼ 120,000). The C1a domain from type V adenylyl cyclase (VC1) and the C2 domain from type II adenylyl cyclase (IIC2) can both be expressed in large quantities and purified to homogeneity. When mixed, their maximally stimulated specific activity, 150 μmol/min/mg protein, substantially exceeds values observed previously with the intact enzyme. A soluble, high-affinity complex containing one molecule each of VC1, IIC2, and guanosine 5′-O-(3-thiotriphosphate) (GTPγS)-Gsα is responsible for the observed enzymatic activity and can be isolated. In addition, GTPγS-Gsαinteracts with homodimers of IIC2 to form a heterodimeric complex (one molecule each of Gsα and IIC2) but not detectably with homodimers of VC1. Nevertheless, Gsα can be cross-linked to VC1 in the activated heterotrimeric complex of VC1, IIC2, and Gsα, indicating its proximity to both components of the enzyme that are required for efficient catalysis. These results and those in the accompanying report (Dessauer, C. W., Scully, T. T., and Gilman, A. G. (1997) J. Biol. Chem. 272, 22272–22277) suggest that activators of adenylyl cyclase facilitate formation of a single, high-activity catalytic site at the interface between C1 and C2. Eleven distinct isoforms of mammalian adenylyl cyclase have been identified to date, and the regulatory properties of several of these proteins have been characterized extensively (1Taussig R. Gilman A.G. J. Biol. Chem. 1995; 270: 1-4Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar, 2Sunahara R.K. Dessauer C.W. Gilman A.G. Annu. Rev. Pharm. Toxicol. 1996; 36: 461-480Crossref PubMed Scopus (732) Google Scholar). Although there is remarkable variation in the responsiveness of these enzymes to inhibitory effects of the Giα 1The abbreviations used are: Gsα, the α subunit of the G protein that stimulates adenylyl cyclase; Giα, the α subunit of the G protein that inhibits adenylyl cyclase; G protein, heterotrimeric guanine nucleotide-binding protein; GTPγS, guanosine 5′-O-(3-thiotriphosphate); PAGE, polyacrylamide gel electrophoresis.1The abbreviations used are: Gsα, the α subunit of the G protein that stimulates adenylyl cyclase; Giα, the α subunit of the G protein that inhibits adenylyl cyclase; G protein, heterotrimeric guanine nucleotide-binding protein; GTPγS, guanosine 5′-O-(3-thiotriphosphate); PAGE, polyacrylamide gel electrophoresis. proteins and to such agents as G protein βγ subunits and Ca2+, the catalytic activity of all of the known isoforms is stimulated by the α subunit of Gs and, presumably nonphysiologically, by the diterpene forskolin. The adenylyl cyclases share a unique structure for an enzyme, resembling transporters such as the P-glycoproteins topographically. They are intrinsic membrane proteins by virtue of their two large hydrophobic domains, each of which is hypothesized to contain six membrane-spanning helices. The first of these hydrophobic regions follows a short amino-terminal sequence and precedes a roughly 40-kDa cytoplasmic domain (C1). The second hydrophobic region separates C1 from a second cytosolic domain (C2) of comparable size. Each of the two cytosolic domains includes a sequence of 200–250 amino acid residues that is typically 50% similar to its consort, 50–90% similar to the corresponding domains of other adenylyl cyclase isoforms, and 20–25% similar to the catalytic domains of membrane-bound and cytosolic guanylyl cyclases.Detailed biochemical characterization of adenylyl cyclase is impaired by the insolubility, instability, and sparsity of the native enzyme, as well as our incapacity to express necessary amounts of the protein in heterologous systems. To overcome these hurdles we have synthesized (inEscherichia coli) portions of the two cytosolic domains of adenylyl cyclase in the absence of the remainder of the protein, first as a 55-kDa chimeric fusion protein containing the C1adomain of type I adenylyl cyclase linked to the C2 domain of the type II enzyme (3Tang W.-J. Gilman A.G. Science. 1995; 268: 1769-1772Crossref PubMed Scopus (165) Google Scholar, 4Dessauer C.W. Gilman A.G. J. Biol. Chem. 1996; 271: 16967-16974Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). The specific activity of this engineered enzyme is remarkably high, it is soluble in the absence of detergent, and it is adequately stable. Importantly, it is activated synergistically by Gsα and forskolin and inhibited by so-called P-site inhibitors and the G protein βγ subunit complex, providing ample justification to pursue investigation of this and similar artificial entities. To overcome the remaining hurdle of a relatively low level of accumulation of the chimera in bacterial cytosol, we and others prepared the two cytosolic domains of adenylyl cyclase as distinct entities and found that enzymatic activity with similar regulatory properties can be reconstituted by simple mixture of the two roughly 25-kDa proteins (5Whisnant R.E. Gilman A.G. Dessauer C.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6621-6625Crossref PubMed Scopus (116) Google Scholar, 6Yan S.-Z. Hahn D. Huang Z.-H. Tang W.-J. J. Biol. Chem. 1996; 271: 10941-10945Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Large amounts (50–100 mg or more) of the C2 domain of type II adenylyl cyclase can be prepared readily, but similar results are difficult to achieve with the C1a domain of the type I enzyme. We have now extended this approach by utilizing a fragment of the C1 domain of type V adenylyl cyclase, which can be expressed in reasonable (but not exuberant) quantities, and we have characterized the interactions of the two cyclase fragments with each other and with Gsα.DISCUSSIONWe have expressed and purified a fragment of the C1domain of type V adenylyl cyclase, consisting of amino acid residues 364–591 and including hexa-histidine and Flag tags at the amino and carboxyl termini, respectively. Although this protein itself has no adenylyl cyclase activity, catalysis of cyclic AMP synthesis is restored by simple mixture of VC1 with an appropriate fragment from the second cytosolic domain of the enzyme, such as IIC2. This interaction is similar to that described previously between IC1 and IIC2 (5Whisnant R.E. Gilman A.G. Dessauer C.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6621-6625Crossref PubMed Scopus (116) Google Scholar, 6Yan S.-Z. Hahn D. Huang Z.-H. Tang W.-J. J. Biol. Chem. 1996; 271: 10941-10945Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar); the major advantages are the yield of VC1, which exceeds that of IC1 by 20-fold, and the apparent homogeneity of the product. The adenylyl cyclase activity of the VC1-IIC2 mixture is stimulated markedly by either Gsα or forskolin, and these two regulators activate the enzyme synergistically when present simultaneously. These are characteristics of native type II and type V adenylyl cyclases.A notable difference between this reconstituted adenylyl cyclase and the native enzymes is the maximal stimulated activity, which typically exceeds 100 μmol/min/mg in the case of the mixture of VC1and IIC2; values of 10 μmol/min/mg typify purified preparations of native enzymes (12Smigel M.D. J. Biol. Chem. 1986; 261: 1976-1982Abstract Full Text PDF PubMed Google Scholar, 13Pfeuffer E. Mollner S. Pfeuffer T. EMBO J. 1985; 4: 3675-3679Crossref PubMed Scopus (71) Google Scholar, 14Taussig R. Quarmby L.M. Gilman A.G. J. Biol. Chem. 1993; 268: 9-12Abstract Full Text PDF PubMed Google Scholar). Although the source of this discrepancy is not known, we suspect that the values observed with the VC1/IIC2 mixture may indeed approximate a trueVmax for mammalian adenylyl cyclase. Several factors may cause underestimation of maximal activity when dealing with a native adenylyl cyclase. Overexpression of these enzymes in Sf9 cells is plagued by production of nonfunctional protein; detergents are necessary to maintain solubility of the native proteins but may alter estimates of specific activity; lengthy purification schemes may cause denaturation of these labile entities. Alternatively, it is possible that inhibitory domains have been removed from the soluble constructs described above or that the membrane spanning domains of the enzymes do not permit optimal orientation of the interacting cytosolic segments. Another notable difference between native adenylyl cyclases and the engineered soluble enzymes (whether or not the cytosolic domains are linked covalently) is the relatively low (reduced 20–50-fold) apparent affinities of the soluble enzymes for Gsα. It is possible that the transmembrane spans, the loops that connect them, and/or residues immediately surrounding the remnants of the C1 and C2 domains in the constructs utilized may contribute to the binding site for Gsα. Nevertheless, the essential features of activation of adenylyl cyclase by the G protein α subunit are retained.We demonstrate herein that the C1 and C2domains of adenylyl cyclase interact to form a catalytically active adenylyl cyclase and that the apparent affinity of C1 for C2 is enhanced in the presence of Gsα and/or forskolin. and all demonstrate that Gsα with the C2 domain of adenylyl cyclase in a and that this interaction is by interactions between VC1 and Gsα not by gel or these two proteins be cross-linked by in a We demonstrate that Gsα, and C2 form a relatively high-affinity complex with a Although this is not the in the sequence of the C1 and C2 domains of adenylyl cyclase the of binding for two of Gsα. Several adenylyl cyclase isoforms are inhibited by Although of for the of adenylyl cyclase activity by Giα is not Gsα and is not with the α subunit R. Tang W.-J. Gilman A.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar). We that there is a distinct binding site for Giα adenylyl Giα may well be found to interact with of C1 and C2 is difficult to The may be an of protein and have no with to the membrane-bound native enzyme. we have previously of native adenylyl cyclases in the of of of the cytosolic domains W.-J. Gilman A.G. 1995; PubMed Scopus Google of and proteins to interact with their has been and may be the in no such interaction has been previously in a containing such purified The Gsα, VC1, and IIC2 proteins utilized herein are all expressed in their purification to a of are such as of to in the presence of Nevertheless, the apparent affinity of for adenylyl cyclase is that of The activity of a G protein α subunit of the by the affinity of for its the affinity of the G protein βγ subunit complex for is the of the of α with and it is this interaction that of either α or βγ to and of the interactions of the two cytosolic domains of adenylyl cyclase with each other and with their regulators are Although this has been for a has been by the and of quantities of G adenylyl cyclase domains may now be prepared readily, as can a high-affinity complex of Gsα with the components of the cyclase. We that all can now be utilized to the complex regulatory features of these Eleven distinct isoforms of mammalian adenylyl cyclase have been identified to date, and the regulatory properties of several of these proteins have been characterized extensively (1Taussig R. Gilman A.G. J. Biol. Chem. 1995; 270: 1-4Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar, 2Sunahara R.K. Dessauer C.W. Gilman A.G. Annu. Rev. Pharm. Toxicol. 1996; 36: 461-480Crossref PubMed Scopus (732) Google Scholar). Although there is remarkable variation in the responsiveness of these enzymes to inhibitory effects of the Giα 1The abbreviations used are: Gsα, the α subunit of the G protein that stimulates adenylyl cyclase; Giα, the α subunit of the G protein that inhibits adenylyl cyclase; G protein, heterotrimeric guanine nucleotide-binding protein; GTPγS, guanosine 5′-O-(3-thiotriphosphate); PAGE, polyacrylamide gel electrophoresis.1The abbreviations used are: Gsα, the α subunit of the G protein that stimulates adenylyl cyclase; Giα, the α subunit of the G protein that inhibits adenylyl cyclase; G protein, heterotrimeric guanine nucleotide-binding protein; GTPγS, guanosine 5′-O-(3-thiotriphosphate); PAGE, polyacrylamide gel electrophoresis. proteins and to such agents as G protein βγ subunits and Ca2+, the catalytic activity of all of the known isoforms is stimulated by the α subunit of Gs and, presumably nonphysiologically, by the diterpene forskolin. The adenylyl cyclases share a unique structure for an enzyme, resembling transporters such as the P-glycoproteins topographically. They are intrinsic membrane proteins by virtue of their two large hydrophobic domains, each of which is hypothesized to contain six membrane-spanning helices. The first of these hydrophobic regions follows a short amino-terminal sequence and precedes a roughly 40-kDa cytoplasmic domain (C1). The second hydrophobic region separates C1 from a second cytosolic domain (C2) of comparable size. Each of the two cytosolic domains includes a sequence of 200–250 amino acid residues that is typically 50% similar to its consort, 50–90% similar to the corresponding domains of other adenylyl cyclase isoforms, and 20–25% similar to the catalytic domains of membrane-bound and cytosolic guanylyl biochemical characterization of adenylyl cyclase is impaired by the insolubility, instability, and sparsity of the native enzyme, as well as our incapacity to express necessary amounts of the protein in heterologous systems. To overcome these hurdles we have synthesized (inEscherichia coli) portions of the two cytosolic domains of adenylyl cyclase in the absence of the remainder of the protein, first as a 55-kDa chimeric fusion protein containing the C1adomain of type I adenylyl cyclase linked to the C2 domain of the type II enzyme (3Tang W.-J. Gilman A.G. Science. 1995; 268: 1769-1772Crossref PubMed Scopus (165) Google Scholar, 4Dessauer C.W. Gilman A.G. J. Biol. Chem. 1996; 271: 16967-16974Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). The specific activity of this engineered enzyme is remarkably high, it is soluble in the absence of detergent, and it is adequately stable. Importantly, it is activated synergistically by Gsα and forskolin and inhibited by so-called P-site inhibitors and the G protein βγ subunit complex, providing ample justification to pursue investigation of this and similar artificial entities. To overcome the remaining hurdle of a relatively low level of accumulation of the chimera in bacterial cytosol, we and others prepared the two cytosolic domains of adenylyl cyclase as distinct entities and found that enzymatic activity with similar regulatory properties can be reconstituted by simple mixture of the two roughly 25-kDa proteins (5Whisnant R.E. Gilman A.G. Dessauer C.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6621-6625Crossref PubMed Scopus (116) Google Scholar, 6Yan S.-Z. Hahn D. Huang Z.-H. Tang W.-J. J. Biol. Chem. 1996; 271: 10941-10945Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Large amounts (50–100 mg or more) of the C2 domain of type II adenylyl cyclase can be prepared readily, but similar results are difficult to achieve with the C1a domain of the type I enzyme. We have now extended this approach by utilizing a fragment of the C1 domain of type V adenylyl cyclase, which can be expressed in reasonable (but not exuberant) quantities, and we have characterized the interactions of the two cyclase fragments with each other and with Gsα. have expressed and purified a fragment of the C1domain of type V adenylyl cyclase, consisting of amino acid residues 364–591 and including hexa-histidine and Flag tags at the amino and carboxyl termini, respectively. Although this protein itself has no adenylyl cyclase activity, catalysis of cyclic AMP synthesis is restored by simple mixture of VC1 with an appropriate fragment from the second cytosolic domain of the enzyme, such as IIC2. This interaction is similar to that described previously between IC1 and IIC2 (5Whisnant R.E. Gilman A.G. Dessauer C.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6621-6625Crossref PubMed Scopus (116) Google Scholar, 6Yan S.-Z. Hahn D. Huang Z.-H. Tang W.-J. J. Biol. Chem. 1996; 271: 10941-10945Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar); the major advantages are the yield of VC1, which exceeds that of IC1 by 20-fold, and the apparent homogeneity of the product. The adenylyl cyclase activity of the VC1-IIC2 mixture is stimulated markedly by either Gsα or forskolin, and these two regulators activate the enzyme synergistically when present simultaneously. These are characteristics of native type II and type V adenylyl cyclases.A notable difference between this reconstituted adenylyl cyclase and the native enzymes is the maximal stimulated activity, which typically exceeds 100 μmol/min/mg in the case of the mixture of VC1and IIC2; values of 10 μmol/min/mg typify purified preparations of native enzymes (12Smigel M.D. J. Biol. Chem. 1986; 261: 1976-1982Abstract Full Text PDF PubMed Google Scholar, 13Pfeuffer E. Mollner S. Pfeuffer T. EMBO J. 1985; 4: 3675-3679Crossref PubMed Scopus (71) Google Scholar, 14Taussig R. Quarmby L.M. Gilman A.G. J. Biol. Chem. 1993; 268: 9-12Abstract Full Text PDF PubMed Google Scholar). Although the source of this discrepancy is not known, we suspect that the values observed with the VC1/IIC2 mixture may indeed approximate a trueVmax for mammalian adenylyl cyclase. Several factors may cause underestimation of maximal activity when dealing with a native adenylyl cyclase. Overexpression of these enzymes in Sf9 cells is plagued by production of nonfunctional protein; detergents are necessary to maintain solubility of the native proteins but may alter estimates of specific activity; lengthy purification schemes may cause denaturation of these labile entities. Alternatively, it is possible that inhibitory domains have been removed from the soluble constructs described above or that the membrane spanning domains of the enzymes do not permit optimal orientation of the interacting cytosolic segments. Another notable difference between native adenylyl cyclases and the engineered soluble enzymes (whether or not the cytosolic domains are linked covalently) is the relatively low (reduced 20–50-fold) apparent affinities of the soluble enzymes for Gsα. It is possible that the transmembrane spans, the loops that connect them, and/or residues immediately surrounding the remnants of the C1 and C2 domains in the constructs utilized may contribute to the binding site for Gsα. Nevertheless, the essential features of activation of adenylyl cyclase by the G protein α subunit are retained.We demonstrate herein that the C1 and C2domains of adenylyl cyclase interact to form a catalytically active adenylyl cyclase and that the apparent affinity of C1 for C2 is enhanced in the presence of Gsα and/or forskolin. and all demonstrate that Gsα with the C2 domain of adenylyl cyclase in a and that this interaction is by interactions between VC1 and Gsα not by gel or these two proteins be cross-linked by in a We demonstrate that Gsα, and C2 form a relatively high-affinity complex with a Although this is not the in the sequence of the C1 and C2 domains of adenylyl cyclase the of binding for two of Gsα. Several adenylyl cyclase isoforms are inhibited by Although of for the of adenylyl cyclase activity by Giα is not Gsα and is not with the α subunit R. Tang W.-J. Gilman A.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar). We that there is a distinct binding site for Giα adenylyl Giα may well be found to interact with of C1 and C2 is difficult to The may be an of protein and have no with to the membrane-bound native enzyme. we have previously of native adenylyl cyclases in the of of of the cytosolic domains W.-J. Gilman A.G. 1995; PubMed Scopus Google of and proteins to interact with their has been and may be the in no such interaction has been previously in a containing such purified The Gsα, VC1, and IIC2 proteins utilized herein are all expressed in their purification to a of are such as of to in the presence of Nevertheless, the apparent affinity of for adenylyl cyclase is that of The activity of a G protein α subunit of the by the affinity of for its the affinity of the G protein βγ subunit complex for is the of the of α with and it is this interaction that of either α or βγ to and of the interactions of the two cytosolic domains of adenylyl cyclase with each other and with their regulators are Although this has been for a has been by the and of quantities of G adenylyl cyclase domains may now be prepared readily, as can a high-affinity complex of Gsα with the components of the cyclase. We that all can now be utilized to the complex regulatory features of these We have expressed and purified a fragment of the C1domain of type V adenylyl cyclase, consisting of amino acid residues 364–591 and including hexa-histidine and Flag tags at the amino and carboxyl termini, respectively. Although this protein itself has no adenylyl cyclase activity, catalysis of cyclic AMP synthesis is restored by simple mixture of VC1 with an appropriate fragment from the second cytosolic domain of the enzyme, such as IIC2. This interaction is similar to that described previously between IC1 and IIC2 (5Whisnant R.E. Gilman A.G. Dessauer C.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6621-6625Crossref PubMed Scopus (116) Google Scholar, 6Yan S.-Z. Hahn D. Huang Z.-H. Tang W.-J. J. Biol. Chem. 1996; 271: 10941-10945Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar); the major advantages are the yield of VC1, which exceeds that of IC1 by 20-fold, and the apparent homogeneity of the product. The adenylyl cyclase activity of the VC1-IIC2 mixture is stimulated markedly by either Gsα or forskolin, and these two regulators activate the enzyme synergistically when present simultaneously. These are characteristics of native type II and type V adenylyl A notable difference between this reconstituted adenylyl cyclase and the native enzymes is the maximal stimulated activity, which typically exceeds 100 μmol/min/mg in the case of the mixture of VC1and IIC2; values of 10 μmol/min/mg typify purified preparations of native enzymes (12Smigel M.D. J. Biol. Chem. 1986; 261: 1976-1982Abstract Full Text PDF PubMed Google Scholar, 13Pfeuffer E. Mollner S. Pfeuffer T. EMBO J. 1985; 4: 3675-3679Crossref PubMed Scopus (71) Google Scholar, 14Taussig R. Quarmby L.M. Gilman A.G. J. Biol. Chem. 1993; 268: 9-12Abstract Full Text PDF PubMed Google Scholar). Although the source of this discrepancy is not known, we suspect that the values observed with the VC1/IIC2 mixture may indeed approximate a trueVmax for mammalian adenylyl cyclase. Several factors may cause underestimation of maximal activity when dealing with a native adenylyl cyclase. Overexpression of these enzymes in Sf9 cells is plagued by production of nonfunctional protein; detergents are necessary to maintain solubility of the native proteins but may alter estimates of specific activity; lengthy purification schemes may cause denaturation of these labile entities. Alternatively, it is possible that inhibitory domains have been removed from the soluble constructs described above or that the membrane spanning domains of the enzymes do not permit optimal orientation of the interacting cytosolic segments. Another notable difference between native adenylyl cyclases and the engineered soluble enzymes (whether or not the cytosolic domains are linked covalently) is the relatively low (reduced 20–50-fold) apparent affinities of the soluble enzymes for Gsα. It is possible that the transmembrane spans, the loops that connect them, and/or residues immediately surrounding the remnants of the C1 and C2 domains in the constructs utilized may contribute to the binding site for Gsα. Nevertheless, the essential features of activation of adenylyl cyclase by the G protein α subunit are We demonstrate herein that the C1 and C2domains of adenylyl cyclase interact to form a catalytically active adenylyl cyclase and that the apparent affinity of C1 for C2 is enhanced in the presence of Gsα and/or forskolin. and all demonstrate that Gsα with the C2 domain of adenylyl cyclase in a and that this interaction is by interactions between VC1 and Gsα not by gel or these two proteins be cross-linked by in a We demonstrate that Gsα, and C2 form a relatively high-affinity complex with a Although this is not the in the sequence of the C1 and C2 domains of adenylyl cyclase the of binding for two of Gsα. Several adenylyl cyclase isoforms are inhibited by Although of for the of adenylyl cyclase activity by Giα is not Gsα and is not with the α subunit R. Tang W.-J. Gilman A.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar). We that there is a distinct binding site for Giα adenylyl Giα may well be found to interact with of C1 and C2 is difficult to The may be an of protein and have no with to the membrane-bound native enzyme. we have previously of native adenylyl cyclases in the of of of the cytosolic domains W.-J. Gilman A.G. 1995; PubMed Scopus Google Scholar). The of and proteins to interact with their has been and may be the in no such interaction has been previously in a containing such purified The Gsα, VC1, and IIC2 proteins utilized herein are all expressed in their purification to a of are such as of to in the presence of Nevertheless, the apparent affinity of for adenylyl cyclase is that of The activity of a G protein α subunit of the by the affinity of for its the affinity of the G protein βγ subunit complex for is the of the of α with and it is this interaction that of either α or βγ to and of the interactions of the two cytosolic domains of adenylyl cyclase with each other and with their regulators are Although this has been for a has been by the and of quantities of G adenylyl cyclase domains may now be prepared readily, as can a high-affinity complex of Gsα with the components of the cyclase. We that all can now be utilized to the complex regulatory features of these We and for and for providing purified type Gsα.
Sunahara et al. (Fri,) studied this question.