Genetic Selection of Mammalian Adenylyl Cyclases Insensitive to Stimulation by Gsα

Abstract

We describe the development of a genetic system allowing for the isolation of mutant mammalian adenylyl cyclases defective in their responses to G protein subunits, thus allowing for the identification of structural elements within the cyclase that are responsible for the recognition of these regulators. Expression of mammalian type V adenylyl cyclase in a cyclase-deleted yeast strain can conditionally complement the lethal phenotype of this strain. Type V adenylyl cyclase-expressing yeast grow only when the cyclase is activated by coexpression of Gsα or addition of forskolin to the medium; however, growth arrest is observed in the presence of both activators or under basal conditions. Utilizing this genetic system, we have isolated 25 adenylyl cyclase mutants defective in their response to Gsα. Sequence analysis and biochemical characterization of these mutants have identified residues in both cytoplasmic domains of the cyclase that are involved in the specific binding of and regulation by Gsα. We describe the development of a genetic system allowing for the isolation of mutant mammalian adenylyl cyclases defective in their responses to G protein subunits, thus allowing for the identification of structural elements within the cyclase that are responsible for the recognition of these regulators. Expression of mammalian type V adenylyl cyclase in a cyclase-deleted yeast strain can conditionally complement the lethal phenotype of this strain. Type V adenylyl cyclase-expressing yeast grow only when the cyclase is activated by coexpression of Gsα or addition of forskolin to the medium; however, growth arrest is observed in the presence of both activators or under basal conditions. Utilizing this genetic system, we have isolated 25 adenylyl cyclase mutants defective in their response to Gsα. Sequence analysis and biochemical characterization of these mutants have identified residues in both cytoplasmic domains of the cyclase that are involved in the specific binding of and regulation by Gsα. Regulation of intracellular cyclic AMP concentrations is principally controlled at the level of its synthesis, through the hormonal regulation of adenylyl cyclase, the enzyme responsible for the conversion of ATP into cyclic AMP. The adenylyl cyclase system comprises three components: seven transmembrane-spanning receptors for a variety of hormones and neurotransmitters, heterotrimeric G proteins, 1The abbreviations used are: G protein, heterotrimeric guanine nucleotide-binding regulatory protein; Gsα, the α subunit of the G protein that stimulates adenylyl cyclase; Giα, the α subunit of the G protein that inhibits adenylyl cyclase; GTPγS, guanosine 5′-O-(2-thiodiphosphate); PCR, polymerase chain reaction; AC, adenylyl cyclase. and the catalytic entity itself. Currently, nine isoforms of membrane-bound adenylyl cyclases have been identified by molecular genetic approaches, and studies of these enzymes reveal both common and unique regulatory features (1Sunahara R.K. Dessauer C.W. Gilman A.G. Annu. Rev. Pharmacol. Toxicol. 1996; 36: 461-480Crossref PubMed Scopus (744) Google Scholar, 2Taussig R. Zimmermann G. Cooper D.M.F. Adenylyl Cyclases. 32. Lippincott-Raven, New York1998: 81-98Google Scholar). All isoforms tested to date are activated by the GTP-bound form of Gsα and by forskolin; for some of the isoforms, such as the type V, these stimulators synergistically activate the enzyme. All isoforms of adenylyl cyclase are further regulated by additional inputs in an isoform-specific pattern. For example, Giα inhibits the types I, V, and VI isoforms (3Taussig R. Iniguez-Lluhi J. Gilman A.G. Science. 1993; 261: 218-221Crossref PubMed Scopus (324) Google Scholar, 4Chen J. Iyengar R. J. Biol. Chem. 1993; 268: 12253-12256Abstract Full Text PDF PubMed Google Scholar, 5Taussig R. Tang W.J. Hepler J.R. Gilman A.G. J. Biol. Chem. 1994; 269: 6093-6100Abstract Full Text PDF PubMed Google Scholar); βγ subunits can activate (types II, IV, and VII) or inhibit (type I) adenylyl cyclase activity (6Tang W.J. Gilman A.G. Science. 1991; 254: 1500-1503Crossref PubMed Scopus (748) Google Scholar, 7Gao B. Gilman A.G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10178-10182Crossref PubMed Scopus (277) Google Scholar, 8Federman A.D. Conklin B.R. Schrader K.A. Reed R.R. Bourne H.R. Nature. 1992; 356: 159-161Crossref PubMed Scopus (481) Google Scholar, 9Taussig R. Quarmby L.M. Gilman A.G. J. Biol. Chem. 1993; 268: 9-12Abstract Full Text PDF PubMed Google Scholar, 10Yoshimura M. Ikeda H. Tabakoff B. Mol. Pharmacol. 1996; 50: 43-51PubMed Google Scholar). Increases in intracellular calcium concentrations will inhibit the types V and VI isoforms (11Katsushika S. Chen L. Kawabe J.I. Nilakantan R. Halnon N.J. Homcy C.J. Ishikawa Y. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8774-8778Crossref PubMed Scopus (195) Google Scholar, 12Ishikawa Y. Katsushika S. Chen L. Halnon N.J. Kawabe J. Homcy C.J. J. Biol. Chem. 1992; 267: 13553-13557Abstract Full Text PDF PubMed Google Scholar, 13Yoshimura M. Cooper D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6716-6720Crossref PubMed Scopus (204) Google Scholar) while indirectly activating (via a calmodulin-dependent process) types I and VIII (14Tang W.J. Krupinski J. Gilman A.G. J. Biol. Chem. 1991; 266: 8595-8603Abstract Full Text PDF PubMed Google Scholar, 15Cali J.J. Zwaagstra J.C. Mons N. Cooper D. Krupinski J. J. Biol. Chem. 1994; 269: 12190-12195Abstract Full Text PDF PubMed Google Scholar) and inhibiting (by calmodulin kinase) the type III isoform (16Wayman G.A. Impey S. Storm D.R. J. Biol. Chem. 1995; 270: 21480-21486Crossref PubMed Scopus (127) Google Scholar). Structural motifs responsible for the recognition of these regulatory molecules by the adenylyl cyclases are starting to be uncovered. A region of type II adenylyl cyclase (residues 956–982 from the C2 domain) containing a QXXER motif has been shown to be important for the regulation of this enzyme by G protein βγ subunits (17Chen J. DeVivo M. Dingus J. Harry A. Li J. Sui J. Carty D.J. Blank J.L. Exton J.H. Stoffel R.H. Inglese J. Lefkowitz R.J. Logothetis D.E. Hildebrandt J.D. Iyengar R. Science. 1995; 268: 1166-1169Crossref PubMed Scopus (236) Google Scholar). This site has been proposed to interact with the βγ subunit of the G protein, within the amino-terminal 100 residues of β (18Weng G. Li J. Dingus J. Hildebrandt J.D. Weinstein H. Iyengar R. J. Biol. Chem. 1996; 271: 26445-26448Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 19Yan K. Gautam N. J. Biol. Chem. 1997; 272: 2056-2059Crossref PubMed Scopus (45) Google Scholar). Synthetic peptide and mutational approaches have identified sequences located within the first cytoplasmic (C1a) region of type I adenylyl cyclase (residues 495–522) important for calmodulin activation (20Vorherr T. Knopfel L. Hofmann F. Mollner S. Pfeuffer T. Carafoli E. Biochemistry. 1993; 32: 6081-6088Crossref PubMed Scopus (142) Google Scholar, 21Wu Z. Wong S.T. Storm D.R. J. Biol. Chem. 1993; 268: 23766-23768Abstract Full Text PDF PubMed Google Scholar); this sequence has a hydrophobic/basic composition and an aromatic amino acid in its NH2-terminal portion, typical of most calmodulin binding domains, and is therefore likely to function as the calmodulin binding domain in the cyclase. The recently solved crystal structure of a homodimer of the type II adenylyl cyclase second cytoplasmic domain (C2) bound with forskolin has revealed the binding site(s) of this regulator (22Zhang G.Y. Liu Y. Ruoho A.E. Hurley J.H. Nature. 1997; 386: 247-253Crossref PubMed Scopus (326) Google Scholar). Mutational analysis of Gsα has revealed three regions of the protein critical for the ability of Gsα to activate adenylyl cyclase (23Itoh H. Gilman A.G. J. Biol. Chem. 1991; 266: 16226-16231Abstract Full Text PDF PubMed Google Scholar, 24Berlot C.H. Bourne H.R. Cell. 1992; 68: 911-922Abstract Full Text PDF PubMed Scopus (162) Google Scholar); however, at the time that this study was initiated, the Gsα-binding site on the adenylyl cyclase molecule had not been determined. The yeast adenylyl cyclase, encoded by the CYR1 gene, is structurally distinct from and exhibits much different regulation than the mammalian adenylyl cyclases (25Kataoka T. Broek D. Wigler M. Cell. 1985; 43: 493-505Abstract Full Text PDF PubMed Scopus (265) Google Scholar). Yeast strains containing a disruption of the CYR1 gene are not viable but can be propagated in medium containing cAMP, demonstrating the importance of cAMP for growth of the organism (26Ishikawa T. Matsumoto K. Uno I. Methods Enzymol. 1988; 159: 27-42Crossref PubMed Scopus (3) Google Scholar). Based on this requirement of cAMP for growth, we have expressed mammalian adenylyl cyclases in a CYR1-deleted yeast strain to take advantage of the readily manipulatable genetic properties of the yeast for the isolation of mutant mammalian adenylyl cyclases defective in their regulatory responses. In this report, we describe a genetic system utilizing the expression of mammalian type V adenylyl cyclase in yeast and demonstrate its utility in isolating mutant adenylyl cyclases defective in their regulation by Gsα. Genetic and biochemical analyses of these mutants reveal structural information about the Gsα binding site on the cyclase, as well as regions of the molecule important for responses to activating regulators. Yeast strain TC41-1 (27Casperson G.F. Walker N. Bourne H.R. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 5060-5063Crossref PubMed Scopus (63) Google Scholar) (MAT a, leu2–3, leu2–112, ura3–52, his3, his4, cam1, cam2, cam3, cyr1 Δ::URA3) was a generous gift of Warren Heideman (University of Wisconsin, Madison, WI). Strain 12229 is an isogenic derivative of TC41-1 generated by the targeted integration of plasmid pIntTrp1Cup1Gs at the chromosomal TRP1 locus; this plasmid contains the CUP1copper-inducible promoter (region −460 to −30) fused to the coding region of rat Gsα. Plasmid pADHprACVLeu was constructed by ligating the XbaI-HindIII fragment of dog type V (12Ishikawa Y. Katsushika S. Chen L. Halnon N.J. Kawabe J. Homcy C.J. J. Biol. Chem. 1992; 267: 13553-13557Abstract Full Text PDF PubMed Google Scholar) adenylyl cyclase (in pCDNA1) into the XbaI-HindIII sites of pVT100U and replacing the EcoRI-HpaI fragment of pVT100U (containing the URA3 gene) with the LEU2 gene (HpaI fragment) from YEP13. Randomly mutated libraries of plasmid pADHprACVLeu were generated by passage in the mutator bacterial strain XL1 Red (Stratgene). Libraries with different extents of mutagenesis were prepared by isolating and of growth in this strain. Libraries containing in the or C2 domains were constructed by For the and were used and the were with and and into these sites in the pADHprACVLeu yeast expression mutant libraries were constructed by of the fragment with the fragment generated with the was for polymerase and concentrations were as and level of and was for at at at Plasmid libraries were into yeast strain 12229 by yeast were on yeast containing medium with 100 and and at were from yeast and into by as were into 12229 and TC41-1 yeast strains by and of the adenylyl cyclase mutants was as by the In some was an of mutant adenylyl cyclases (by to the were by ligating the fragment from isolated mutant into plasmid mutant adenylyl cyclases to the C2 region were by replacing the fragment the of type V adenylyl in type plasmid pADHprACVLeu with the fragment of the isolated mutant In the fragment only a were into 12229 and TC41-1 yeast strains by and to that the with the growth phenotype of the isolated mutant of was the system as For the of the type type V adenylyl cyclase, the fragment containing the coding region of dog type V was to the to mutant adenylyl cyclases to the region were by ligating the fragment from isolated mutant pADHprACVLeu yeast expression into mutant adenylyl cyclases to the C2 region were by replacing the fragment the of type V adenylyl from with the fragment of the isolated mutant pADHprACVLeu yeast expression for the of and the of have been by and Scholar). containing adenylyl cyclase isoforms were prepared as R. Tang W.J. Gilman A.G. Methods Enzymol. 1994; PubMed Scopus Google Scholar). Gsα and Giα were in and as by E. Gilman A.G. Methods Enzymol. 1994; PubMed Scopus Google Scholar). concentrations were by with PubMed Scopus Google Scholar). The α subunits were activated by with and at for or M. Gilman A.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar); was by Yeast were to and with for by and cAMP was as by Uno I. H. Ishikawa T. J. Scopus Google Scholar). Adenylyl cyclase activity was the by J. Biol. Chem. 261: Full Text PDF PubMed Google Scholar). All were for at in a of 100 containing of protein and a of type and mutant type V adenylyl cyclase were from R. Quarmby L.M. Gilman A.G. J. Biol. Chem. 1993; 268: 9-12Abstract Full Text PDF PubMed Google Scholar). were by to and as R. Tang W.J. Gilman A.G. Methods Enzymol. 1994; PubMed Scopus Google a specific for type adenylyl cyclase T. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar) and a were on a The Gsα for binding was prepared by of Gsα with for at in and in a of The was through a to The Gsα binding was by of type or mutant adenylyl cyclase, of and Gsα as R. Tang W.J. Hepler J.R. Gilman A.G. J. Biol. Chem. 1994; 269: 6093-6100Abstract Full Text PDF PubMed Google Scholar, W.J. M. Gilman A.G. Biochemistry. 1995; PubMed Scopus Google Scholar). were the for the for were from the for that not concentrations of Gsα that activity as much as the of Gsα tested Yeast adenylyl cyclase by the CYR1 is an gene strains a cyclase are but can be propagated in the presence of cAMP in the growth shown in expression of the mammalian type V adenylyl cyclase the of the yeast on a plasmid can conditionally the lethal phenotype of the CYR1-deleted strain The expression have to the different regulatory of the type V adenylyl cyclase on growth of the In the of cyclase TC41-1 to to cAMP by this isoform under basal of of under activating (in the presence of 100 forskolin in the medium or coexpression of these was by an in intracellular cAMP of of protein for In is synergistically activated by Gsα and forskolin in and under these we were to a growth phenotype than growth of this yeast strain of intracellular cAMP of of The observed of the yeast strain to grow under activation of the cyclase by forskolin and Gsα a to adenylyl cyclase mutants defective in their response to Gsα. We that mutants a of that for growth in the presence of this we have constructed libraries generated mutant type V adenylyl cyclases in a yeast expression these libraries into yeast strain 12229 derivative of TC41-1 rat Gsα under the of the and for that grow in the presence of forskolin and were isolated from from nine three libraries in the of mutagenesis were generated for region of and the coding in these genetic were on medium forskolin to growth in the presence of of expressed Gsα by the of in the growth For mutants that to Gsα by of growth in the presence of the mutant cyclases were and into TC41-1 to their to by forskolin and into the yeast to that the growth phenotype with the mutant adenylyl cyclase the growth of isolated mutants defective in responses Gsα. All of these mutants for their ability to grow in the presence of Gsα and forskolin to grow in the presence of Gsα to growth for the type activation of the promoter by of in the however, will Gsα to growth of yeast some of these mutant All but mutant a growth phenotype in the presence of with a to Gsα of yeast these mutants or is at concentrations of forskolin and not at concentrations not mutants was isolated from three from the domain and from the and mutated was isolated from the mutated was isolated from both the mutated and the C2 domain All mutants were isolated from the C2 domain were to sequence analysis to the sites of responsible for the observed and C2 domains of the cyclase containing and were as shown in mutants were from different mutant libraries and and three and and were mutated to different amino to or and to or in mutated are mammalian adenylyl cyclase We the biochemical properties of these mutant type V cyclases to defective regulatory properties of these mutants for the growth observed when are expressed in characterization of the regulatory properties of mutant or type adenylyl cyclase in yeast or was not to of these the mutant adenylyl cyclase were into and in from these were used as a of cyclase for biochemical in of cyclase protein, the type and mutant adenylyl cyclase were of these cyclases on activity were by with an not For of cyclase protein were with to that of the type enzyme and are in the from to of type and in expression or of the mutant of type and mutant adenylyl cyclases in cyclase in of in a shown in both the and mutants have responses to by Gsα with the type well with the growth observed in S. The in the for both of these mutants a for Gsα to the type enzyme for and are and We not the by Gsα was by these we were to for the mutant type V adenylyl We the responses of these mutants to forskolin to these mutants had in their response to this and to the ability of these mutants to forskolin and Gsα of that the of the mutant to forskolin is to type In the presence of Gsα, activation of the type is as an in to forskolin to and an in the of the forskolin response the mutant of these further demonstrating its defective response to Gsα. In is the of the mutant shown in this mutant has a to forskolin not to at the the activation of this mutant by forskolin Gsα that of the type the observed of to that the with the mutant is a regulation by the mutant is defective in its response to Gsα, likely to a ability to Gsα. this we the ability of these mutants to Gsα. shown in type Gsα in the presence of and this binding is by the addition of Gsα. binding of be for the mutant under these with the of this mutant Gsα and to to Gsα in the presence of The by to Gsα, the level of binding the of this mutant for Gsα is to that of the type enzyme not these a for in Gsα binding and a structural for The of the mutants that were isolated in yeast were to the C2 domain of analysis of these cyclase expression in that are defective in to by Gsα. shown in mutants have responses to by with the type enzyme. of these mutants for Gsα are shown in and G of cyclase activity was in the presence of 100 forskolin and in the presence of were to in adenylyl cyclase expression for the for forskolin were from forskolin The with forskolin concentrations from to were with for the of were from the in concentrations of Gsα that activity as much as the of Gsα tested activation of adenylyl cyclase by forskolin and Gsα was by catalytic in the presence and of Gsα. are expressed as the of activity addition of of activity was in the presence of 100 forskolin and in the presence of were to in adenylyl cyclase expression for the for forskolin were from forskolin The with forskolin concentrations from to were with for the of were from the in concentrations of Gsα that activity as much as the of Gsα tested activation of adenylyl cyclase by forskolin and Gsα was by catalytic in the presence and of Gsα. are expressed as the of activity addition of Gsα. in a All mutants tested to by forskolin; in the presence or of from to of of the type and are to that of type Gsα the of the activity for the type the of these mutants to Gsα was further by this are in Based on this the C2 mutants into distinct cyclases containing at activation by forskolin and Gsα ability to be by Gsα The ability of these mutants to Gsα was and is shown in with the activity both mutants can Gsα in the presence of forskolin to a than type some binding of Gsα to can be in the of forskolin not these that this is not involved in with Gsα but is important in integration of regulatory with this is the that these mutants by Giα not The second of C2 mutants and exhibits a ability to to Gsα as by Gsα activation and the ability to synergistically activate the enzyme in the presence of forskolin but regulation by Giα not and All of these mutants have ability to Gsα mutants and therefore residues critical for of the adenylyl cyclase with Gsα by in Gsα binding or the structural of the Gsα binding The mutant properties to this and the mutants a ability to be activated by Gsα, exhibits Gsα and forskolin activation and with this activity is only to Gsα in the presence of The 25 adenylyl cyclase mutants isolated at that are the identified adenylyl cyclase the are to the cytoplasmic regions and of the can be into on their biochemical The and are defective in some of their responses to regulatory The of the second of mutants are to Gsα responses (in Gsα and with forskolin activation for mutants of forskolin the biochemical properties of these mutants are with the of these residues in the Gsα binding Based on the the cytoplasmic domains of mammalian adenylyl cyclase isoforms and the and C2 regions within a cyclase but the mutants identified in this study can be to on the recently solved crystal structure of the (22Zhang G.Y. Liu Y. Ruoho A.E. Hurley J.H. Nature. 1997; 386: 247-253Crossref PubMed Scopus (326) Google Scholar). is to the of the chain of the homodimer located on of this that its chain with and of the C2 domain residues and of the A of this its ability to to the of these the of the cytoplasmic In of the that both Gsα and forskolin activate adenylyl cyclase by the of for C2 Gilman A.G. Dessauer C.W. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar, D. Tang J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google the of be was observed for the to by forskolin or Gsα and a of the ability to synergistically these The second of mutants identified to three regions of the adenylyl cyclase molecule critical for its with Gsα. and at the α and β and at the of α The residues in these regions are The region is in and is in is the amino-terminal most of the C2 is residues to the of All of these are located on the of the adenylyl cyclase protein a binding site for Gsα is and is likely that was isolated as a mutant in genetic this the of the of α All isolated in this study are located in adenylyl is to that the is types but in type and This the biochemical observed for the isolated much of its Gsα binding and activation (in the presence of binding to and by Gsα The that the Gsα binding site that we have by the mutational analysis comprises residues located in both and C2 domains is with the that Gsα the of the cytoplasmic domains for Gilman A.G. Dessauer C.W. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar, D. Tang J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). most of the mutants isolated in this study to residues in the C2 domain is with the that C2 domain can to Gsα in the domain but can be to Gsα in the presence of R.K. Dessauer C.W. Gilman A.G. J. Biol. Chem. 1997; 272: PubMed Scopus Google Scholar). A study utilizing of cyclase residues has a of that Gsα activation of (type I) and C2 (type cyclase to isolated in Hurley J.H. Tang J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). the importance of and C2 residues and in Gsα and further demonstrate the importance of these residues for Gsα binding and their function in the of the membrane-bound enzyme. is Gsα to the cyclase the structure of Gsα is not however, molecular the structure of Giα D.E. E. Gilman A.G. Science. 1994; PubMed Scopus Google Scholar) a of this In the a is by and when is The amino in Gsα in type V adenylyl cyclase, residues to be important for the ability of Gsα to activate adenylyl cyclase; H. Gilman A.G. J. Biol. Chem. 1991; 266: 16226-16231Abstract Full Text PDF PubMed Google and 24Berlot C.H. Bourne H.R. Cell. 1992; 68: 911-922Abstract Full Text PDF PubMed Scopus (162) Google Scholar) α and of the cyclase. We are yeast genetic system to in Gsα that have the ability to the adenylyl cyclase in an to the Gsα and its binding site on adenylyl cyclase. Expression of mammalian type V adenylyl cyclase in a mutant of S. has to take a genetic the structural the regulation of adenylyl cyclase The utility of this genetic system is by the isolation of mutant adenylyl cyclases defective in their regulation by Gsα. The genetic system be readily to the of additional adenylyl cyclase mutants defective in their responses to such as Giα, G protein βγ subunits, or protein thus to the identification of the binding sites for these We for R. for the specific for type adenylyl cyclase, J. for with molecular analysis of the adenylyl cyclase structure and and R. for with analysis and