Structure, Mechanism, and Regulation of Mammalian Adenylyl Cyclase
1999; Elsevier BV; Volume: 274; Issue: 12 Linguagem: Inglês
10.1074/jbc.274.12.7599
ISSN1083-351X
Autores Tópico(s)Phosphodiesterase function and regulation
Resumostimulatory G-protein α subunit inhibitory G-protein α subunit G-protein βγ subunit Ca2+/calmodulin-dependent protein kinase The discovery of 3′,5′-cyclic adenosine monophosphate (cAMP) in the late 1950s by Sutherland and co-workers was the pivotal event that led to our current paradigm of hormone signaling through second messengers. Despite the subsequent discovery of many other second messengers, cAMP has never left center stage. The adenylyl cyclases are the family of enzymes that synthesize cAMP (1Sunahara R.K. Dessauer C.W. Gilman A.G. Annu. Rev. Pharmacol. Toxicol. 1996; 36: 461-480Crossref PubMed Scopus (732) Google Scholar, 2Xia Z.G. Storm D.R. Curr. Opin. Neurobiol. 1997; 7: 391-396Crossref PubMed Scopus (122) Google Scholar, 3Hanoune J. Pouille Y. Tzavara E. Shen T.S. Lipskaya L. Miyamoto N. Suzuki Y. Defer N. Mol. Cell. Endocrinol. 1997; 128: 179-194Crossref PubMed Scopus (139) Google Scholar, 4Cooper D.M.F. Adv. Second Messenger Phosphoprotein Res. 1998; : 32Google Scholar, 5Tang W.-J. Hurley J.H. Mol. Pharmacol. 1998; 54: 231-240Crossref PubMed Scopus (160) Google Scholar).Breakthroughs in determining the first structures of the mammalian adenylyl cyclase catalytic core (6Zhang G. Liu Y. Ruoho A.E. Hurley J.H. Nature. 1997; 386: 247-253Crossref PubMed Scopus (323) Google Scholar, 7Tesmer J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (670) Google Scholar) provide a new context for understanding the action of many regulators, both physiological and pharmacological: free metal ions, P-site inhibitors, forskolin, G-proteins, Ca2+/calmodulin, and protein phosphorylation. Understanding the catalytic mechanism of an enzyme is a prerequisite to understanding its regulation. Here I will describe the essentials of catalysis and then consider how these elements are controlled by each of the major regulators.Structure of Adenylyl CyclaseThe nine cloned isoforms of mammalian adenylyl cyclase share a primary structure consisting of two transmembrane regions, M1 and M2, and two cytoplasmic regions, C1 and C2 (8Krupinski J. Coussen F. Bakalyar H. Tang W.-J. Feinstein P.G. Orth K. Slaughter C. Reed R.R. Gilman A.G. Science. 1989; 244: 1558-1564Crossref PubMed Scopus (505) Google Scholar) (Fig.1). The transmembrane regions each contain six predicted membrane-spanning helices. The function of M1 and M2, aside from membrane localization, is unknown despite their topological analogy to transporters. The C1 and C2 regions are subdivided into C1a and C1b; and C2a and C2b. The C1a and C2a are well conserved, homologous to each other, and contain all of the catalytic apparatus (9Tang W.-J. Gilman A.G. Science. 1995; 268: 1769-1772Crossref PubMed Scopus (165) Google Scholar). C1a and C2a domains heterodimerize with each other in solution (10Yan 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, 11Whisnant R.E. Gilman A.G. Dessauer C.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6621-6625Crossref PubMed Scopus (116) Google Scholar). These domains can also form homodimers. Domains derived from different isoforms can form chimeric heterodimers. The C1b region is large (∼15 kDa), variable, and contains several regulatory sites. The C2b is vanishingly short in some isoforms and lacks identified functions; hence C2 and C2a are sometimes referred to interchangeably.The structure of the type II adenylyl cyclase C2 region revealed a homodimer with two C2 monomers in a wreath-like arrangement (6Zhang G. Liu Y. Ruoho A.E. Hurley J.H. Nature. 1997; 386: 247-253Crossref PubMed Scopus (323) Google Scholar). A deep ventral groove runs between the two in the center of the wreath. Two forskolin molecules bind to this groove in the homodimer. The monomer is built around large β sheet that folds back onto itself on the "inside" facing the dimer interface. The "outside" is α-helical. A ∼80-amino acid substructure within the monomer is similar to the palm domains of the DNA polymerase I and reverse transcriptase families (12Artymiuk P.J. Poirrett A.R. Rice D.W. Willett P.A. Nature. 1997; 388: 33-34Crossref PubMed Scopus (63) Google Scholar, 13Bryant S.H. Madej T. Janin J. Liu Y. Ruoho A.E. Zhang G. Hurley J.H. Nature. 1997; 388: 34Crossref Scopus (13) Google Scholar).The type V C1a region and type II C2 region arrange themselves in a heterodimeric wreath that is nearly identical in overall structure to the C2 homodimer, with some critical differences in detail (7Tesmer J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (670) Google Scholar). The active site is at one end of the ventral groove. The single forskolin binding site, as anticipated by equilibrium binding (14Dessauer C.W. Scully T.T. Gilman A.G. J. Biol. Chem. 1997; 272: 22272-22277Crossref PubMed Scopus (108) Google Scholar), is at the other. The active site is formed at the interface by residues contributed by both C1a and C2. Because the active site is shared between the two domains, association of two catalytic domains in the proper orientation is an absolute prerequisite of catalytic activity. The activity of mammalian adenylyl cyclases depends on the heterologous association of C1a and C2. This is not the case for many other related cyclases (15Liu Y. Ruoho A.E. Rao V.D. Hurley J.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13414-13419Crossref PubMed Scopus (244) Google Scholar, 16Hurley J.H. Curr. Opin. Struct. Biol. 1998; 8: 770-777Crossref PubMed Scopus (71) Google Scholar). Mammalian membrane guanylyl cyclases and many microbial homologues of mammalian adenylyl cyclases are active as homodimers. The mammalian C2 homodimer has measurable activity (9Tang W.-J. Gilman A.G. Science. 1995; 268: 1769-1772Crossref PubMed Scopus (165) Google Scholar, 17Zhang G. Liu Y. Qin J. Vo B. Tang W.-J. Ruoho A.E. Hurley J.H. Protein Sci. 1997; 6: 903-908Crossref PubMed Scopus (27) Google Scholar, 18Mitterauer T. Hohenegger M. Tang W.-J. Nanoff C. Freissmuth M. Biochemistry. 1998; 37: 16183-16191Crossref PubMed Scopus (17) Google Scholar), although reduced by many orders of magnitude because of the loss of two catalytic Asp residues relative to the heterodimer.Nucleotide Binding Site and SpecificityThe ATP binding site has been revealed by the structure of the P-site inhibitor complex (7Tesmer J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (670) Google Scholar), molecular modeling (15Liu Y. Ruoho A.E. Rao V.D. Hurley J.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13414-13419Crossref PubMed Scopus (244) Google Scholar), and mutagenic analysis (15Liu Y. Ruoho A.E. Rao V.D. Hurley J.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13414-13419Crossref PubMed Scopus (244) Google Scholar, 19Tang W.-J. Stanzel M. Gilman A.G. Biochemistry. 1995; 34: 14563-14572Crossref PubMed Scopus (109) Google Scholar, 20Yan S.-Z. Huang Z.-H. Shaw R.S. Tang W.-J. J. Biol. Chem. 1997; 272: 12342-12349Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 21Tucker C.L. Hurley J.H. Miller T.R. Hurley J.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5993-5997Crossref PubMed Scopus (185) Google Scholar, 22Sunahara R.K. Beuve A. Tesmer J.J.G. Sprang S.R. Garbers D.L. Gilman A.G. J. Biol. Chem. 1998; 273: 16332-16338Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). Lys-923 1Unless the isoform is explicitly stated, residue numbering is for type I. and Asp-1000 from C2 interact directly with the N-1 and N-6 of the adenine ring. Gln-417 of C1 plays a supporting role by orienting the Lys. Mutation of these three residues destroys the ATPversus GTP nucleotide specificity of adenylyl cyclase, although it does not convert it into a guanylyl cyclase (22Sunahara R.K. Beuve A. Tesmer J.J.G. Sprang S.R. Garbers D.L. Gilman A.G. J. Biol. Chem. 1998; 273: 16332-16338Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). This is because of a main chain carbonyl that hydrogen bonds to the adenine N-6 and disfavors guanine. Guanylyl cyclases can be converted completely into adenylyl cyclases by mutating their guanine binding Glu and Cys to their adenylyl cyclase counterparts, Lys and Asp (21Tucker C.L. Hurley J.H. Miller T.R. Hurley J.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5993-5997Crossref PubMed Scopus (185) Google Scholar, 22Sunahara R.K. Beuve A. Tesmer J.J.G. Sprang S.R. Garbers D.L. Gilman A.G. J. Biol. Chem. 1998; 273: 16332-16338Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar).The ATP binding site is rounded out by hydrophobic residues that pack against the purine ring and by charged interactions with phosphate groups. Hydrophobic contacts are contributed mainly by C2. Charged interactions are formed by Arg (C1) and Lys (C2). The Lys is part of a flexible lid over the active site that is capable of undergoing an order-disorder transition (6Zhang G. Liu Y. Ruoho A.E. Hurley J.H. Nature. 1997; 386: 247-253Crossref PubMed Scopus (323) Google Scholar,7Tesmer J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (670) Google Scholar).Mg2+ Binding SiteThe Mg2+ binding site consists of two mutationally sensitive Asp residues (15Liu Y. Ruoho A.E. Rao V.D. Hurley J.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13414-13419Crossref PubMed Scopus (244) Google Scholar, 19Tang W.-J. Stanzel M. Gilman A.G. Biochemistry. 1995; 34: 14563-14572Crossref PubMed Scopus (109) Google Scholar). The P-site inhibitor complex shows a single Mg2+ ion interacting with both phosphate moieties of pyrophosphate (7Tesmer J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (670) Google Scholar). There is abundant kinetic evidence for a two-ion mechanism (23Pieroni J.P. Harry A. Chen J. Jacobowitz O. Magnusson R.P. Iyengar R. J. Biol. Chem. 1995; 270: 21368-21373Crossref PubMed Scopus (78) Google Scholar, 24Zimmermann G. Zhou D. Taussig R. J. Biol. Chem. 1998; 273: 19650-19655Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). One ion acts kinetically as free Mg2+whereas the other binds as a complex with ATP. An analogous situation holds for the DNA polymerase family. The polymerases carry out the intermolecular attack of a primer 3′-hydroxyl on the α-phosphate of a deoxynucleotide rather than the intramolecular attack of a nucleotide 3′-hydroxyl on its own α-phosphate.Two Mg2+ ions bind to DNA polymerase-primer-template-nucleotide complexes (25Doublie S. Tabor S. Long A.M. Richardson C.C. Ellenberger T. Nature. 1998; 391: 251-258Crossref PubMed Scopus (1099) Google Scholar, 26Kiefer J.R. Mao C. Braman J.C. Beese L.S. Nature. 1998; 391: 304-307Crossref PubMed Scopus (479) Google Scholar, 27Sawaya M.R. Prasad R. Wilson S.H. Kraut J. Pelletier H. Biochemistry. 1997; 36: 11205-11215Crossref PubMed Scopus (572) Google Scholar, 28Steitz T.A. Nature. 1998; 391: 231-232Crossref PubMed Scopus (493) Google Scholar), providing a model for the coordination of two ions in the adenylyl cyclase active site. The polymerase "B" metal ion corresponds to the observed ion in the adenylyl cyclase complex. It binds all three nucleotide phosphates in a tridentate arrangement (25Doublie S. Tabor S. Long A.M. Richardson C.C. Ellenberger T. Nature. 1998; 391: 251-258Crossref PubMed Scopus (1099) Google Scholar, 27Sawaya M.R. Prasad R. Wilson S.H. Kraut J. Pelletier H. Biochemistry. 1997; 36: 11205-11215Crossref PubMed Scopus (572) Google Scholar). These tight interactions leave little doubt that this is the ion that acts kinetically as an ATP complex. The polymerase "A" metal ion is less tightly bound as judged by higher temperature factors and fewer interactions with protein and nucleotide (25Doublie S. Tabor S. Long A.M. Richardson C.C. Ellenberger T. Nature. 1998; 391: 251-258Crossref PubMed Scopus (1099) Google Scholar). It interacts with the 3′-hydroxyl of the primer and the nucleotide α-phosphate. Both metal ions are coordinated by both Asps. This coordination geometry fits the adenylyl cyclase structure consistent with known stereochemistry (29Koch K.W. Eckstein F. Stryer L. J. Biol. Chem. 1990; 265: 9659-9663Abstract Full Text PDF PubMed Google Scholar).Mechanism of Cyclic AMP FormationCyclic AMP formation requires the deprotonation and activation of the ATP 3′-hydroxyl for nucleophilic attack; stabilization of the transition state at the α-phosphate; and stabilization of increased negative charge on the leaving group, pyrophosphate. Metal ion A activates the 3′-hydroxyl, and both metal ions share in transition state stabilization. Asn-1007, Arg-1011, and Lys-1047 approach the phosphate moieties (Fig. 2). Their modeled interactions with the non-bridging α-phosphate oxygens have poor geometry. It seems likely that at least one of these residues stabilizes the leaving group. Their precise positions in the ATP complex and, therefore, their precise roles in catalysis have yet to be determined. The fate of the proton on the 3′-hydroxyl is unknown. It has been suggested that Asp-354 in adenylyl cyclase or its counterpart in DNA polymerase could act as a general base in these reactions (15Liu Y. Ruoho A.E. Rao V.D. Hurley J.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13414-13419Crossref PubMed Scopus (244) Google Scholar,26Kiefer J.R. Mao C. Braman J.C. Beese L.S. Nature. 1998; 391: 304-307Crossref PubMed Scopus (479) Google Scholar). Substrate-assisted catalysis is the other leading possibility suggested for base catalysis (7Tesmer J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (670) Google Scholar, 20Yan S.-Z. Huang Z.-H. Shaw R.S. Tang W.-J. J. Biol. Chem. 1997; 272: 12342-12349Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar).Figure 2Model for the mechanism of adenylyl cyclase. Metal coordination by phosphates and Asp is derived from the T7 DNA polymerase structure (25Doublie S. Tabor S. Long A.M. Richardson C.C. Ellenberger T. Nature. 1998; 391: 251-258Crossref PubMed Scopus (1099) Google Scholar), and the mechanism is adapted from that of Steitz (28Steitz T.A. Nature. 1998; 391: 231-232Crossref PubMed Scopus (493) Google Scholar). C1 residues are green, and C2 residues are red. Numbering is for type I. Interactions regulated by conformational changes are marked. Asp-310 moves about 1 Å between the two conformations, and Asp-354 and Gln-417 move about 2 Å. A hypothetical hydrogen bond between the putative base Asp-354 and the ATP 3′-hydroxyl is shown. Arg-1011 probably interacts with the α-β bridging oxygen of ATP, but its other interactions are less certain.View Large Image Figure ViewerDownload (PPT)A Conformational Change That Controls Domain Orientation and Active Site StructureThe determinants of both nucleotide binding and catalysis are shared between C1 and C2. This insight is central to understanding regulation of adenylyl cyclase catalytic activity. It means that any factor that alters the relative orientation of the C1 and C2 domains can alter the structure of the active site and thereby alter substrate affinity, catalytic velocity, or both.The structure of the catalytic core is known in two conformations: that of the forskolin-bound homodimer (6Zhang G. Liu Y. Ruoho A.E. Hurley J.H. Nature. 1997; 386: 247-253Crossref PubMed Scopus (323) Google Scholar) and that of the forskolin and Gsα2-bound heterodimer (7Tesmer J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (670) Google Scholar). The two structures differ by a 7o rotation of the heterodimer C1 domain relative to the correspondent C2 in the homodimer. The domain rotation brings key catalytic elements from the two domains about 2 Å closer to each other. The structural differences between the two might be caused by differences in interface residues in the two different dimers, by occupancy of one versus two forskolin molecules, or, most probably, by the binding of Gsα. Despite some uncertainties about which of these factors are driving the observed structural change, there is no doubt that the two boughs of the adenylyl cyclase wreath are capable of moving into more or less active conformations.Regulation by Free Metal IonsMammalian adenylyl cyclases are strongly activated by Mn2+ and inhibited by millimolar concentrations of free Ca2+. These effects are unlikely to have any physiological meaning or to reflect distinct binding sites for these ions. Many otherwise unrelated Mg2+-dependent enzymes can be activated by replacing Mg2+ with Mn2+, probably because the latter is nearly the same size but is a stronger Lewis acid. At high concentrations free Ca2+ binds competitively to Mg2+ sites on enzymes but fails to replace it catalytically. A high affinity and possibly physiological inhibition of type V and VI adenylyl cyclase by free Ca2+ has been reported (30Yoshimura M. Cooper D.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6716-6720Crossref PubMed Scopus (201) Google Scholar), and the possibility of a high affinity binding site in the C1b domain of these isoforms has been raised (31Scholich K. Barbier A.J. Mullenix J.B. Patel T.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2915-2920Crossref PubMed Scopus (64) Google Scholar).Regulation by P-site InhibitorsP-site inhibitors are a class of nucleoside inhibitors of adenylyl cyclase so-called because they all contain a purine ring (32Desaubry L. Shoshani I. Johnson R.A. J. Biol. Chem. 1996; 271: 14028-14034Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The most potent lack a 2′-hydroxyl and are polyphosphorylated at the 3′-position (32Desaubry L. Shoshani I. Johnson R.A. J. Biol. Chem. 1996; 271: 14028-14034Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 33Desaubry L. Shoshani I. Johnson R.A. J. Biol. Chem. 1996; 271: 2380-2382Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 34Desaubry L. Johnson R.A. J. Biol. Chem. 1998; 273: 24972-24977Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). P-site inhibition is potentiated when adenylyl cyclase is activated. P-site inhibition is hypersensitive to certain mutations that slightly reduce enzyme activity. P-site inhibitors are non- or uncompetitive with respect to the forward reaction but compete with the product cyclic AMP in the reverse reaction (35Dessauer C.W. Gilman A.G. J. Biol. Chem. 1997; 272: 27787-27795Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). P-site inhibitors are effective against engineered ATP-specific guanylyl cyclases that are in all other ways regulated by different mechanisms than the adenylyl cyclases (22Sunahara R.K. Beuve A. Tesmer J.J.G. Sprang S.R. Garbers D.L. Gilman A.G. J. Biol. Chem. 1998; 273: 16332-16338Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). P-site inhibitors bind to the active site primarily through conserved residues. Different adenylyl cyclases do show some differences in P-site inhibition, and a physiological role for this type of regulation has been postulated (36Johnson R.A. Désaubry L. Bianchi G. Shoshani I. Lyons Jr., E. Taussig R. Watson P.A. Cali J.J. Krupinski J. Pieroni J.P. Iyengar R. J. Biol. Chem. 1997; 272: 8962-8966Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar).Regulation by ForskolinForskolin is a hydrophobic activator of all the mammalian adenylyl cyclases except type IX. It is an extremely powerful activator of some of the synthetic soluble adenylyl cyclase systems, increasing activity by up to 103, although other forms of soluble adenylyl cyclase barely respond. Forskolin binds to the catalytic core at the opposite end of the same ventral cleft that contains the active site (7Tesmer J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (670) Google Scholar, 15Liu Y. Ruoho A.E. Rao V.D. Hurley J.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13414-13419Crossref PubMed Scopus (244) Google Scholar). It activates the enzyme by gluing together the two domains in the core using a combination of hydrophobic and hydrogen bonding interactions that are distributed equally between the two domains (6Zhang G. Liu Y. Ruoho A.E. Hurley J.H. Nature. 1997; 386: 247-253Crossref PubMed Scopus (323) Google Scholar). Type IX adenylyl cyclase is non-responsive to forskolin because of a Ser → Ala and a Leu → Tyr change in the binding pocket. When these changes are reversed by site-directed mutagenesis, the resulting type IX mutant can be activated by forskolin as well as other adenylyl cyclases (37Yan S.-Z. Huang Z.-H. Andrews R.K. Tang W.-J. Mol. Pharmacol. 1998; 53: 182-187Crossref PubMed Scopus (68) Google Scholar).The forskolin binding pocket is a narrow hydrophobic crevice that almost completely buries the forskolin molecule once bound. The pocket residues are absolutely conserved in types I–VIII and differ only subtly in type IX. The presence of a hydrophobic crevice in a protein is highly destabilizing in the absence of bound ligand. It seems improbable that such a destabilizing feature would be so highly conserved if it had no function. This paradox led us to revive the idea that there exists an endogenous forskolin-like small molecule activator of adenylyl cyclase.Regulation by G-protein SubunitsAll mammalian adenylyl cyclases are potently and physiologically activated by the GTP-bound G-protein α-subunit Gsα. This activation is synergistic, not competitive, with respect to forskolin. GTP-Gsα binds to a crevice on the outside of the wreath formed by α2′ and α3′ of C2 and by the N-terminal portion of C1 (7Tesmer J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (670) Google Scholar, 38Yan S.-Z. Huang Z.-H. Rao V.D. Hurley J.H. Tang W.-J. J. Biol. Chem. 1997; 272: 18849-18854Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 39Zimmermann G. Zhou D.M. Taussig R. J. Biol. Chem. 1998; 273: 6968-6975Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). GTP-Gsα is capable of gluing together C1 and C2 as does forskolin, but mutational analysis suggests this cannot be its only function. If the C1 contact is abolished, activation can be partially rescued when forskolin is used to dimerize C1 and C2. Therefore there must be a non-glue role for GTP-Gsα (38Yan S.-Z. Huang Z.-H. Rao V.D. Hurley J.H. Tang W.-J. J. Biol. Chem. 1997; 272: 18849-18854Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). This role is probably to induce a conformational change that allosterically stimulates catalysis. The 7o rotation of C1, which moves the catalytic residues into their proper positions, is probably the result of a torque applied by Gsα as it "pushes" the C1 away from its binding site (7Tesmer J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (670) Google Scholar).Giα selectively inhibits adenylyl cyclase types V and VI. Symmetry and sequence homology arguments led to the suggestion that Giα binds to the adenylyl cyclase catalytic core on a groove pseudosymmetrically related to the Gsα binding groove (7Tesmer J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (670) Google Scholar, 38Yan S.-Z. Huang Z.-H. Rao V.D. Hurley J.H. Tang W.-J. J. Biol. Chem. 1997; 272: 18849-18854Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Mutational analysis confirmed that the groove formed by α2 and α3 of C1 is the primary site for binding of Giα to type V (40Dessauer C.W. Tesmer J.J.G. Sprang S.R. Gilman A.G. J. Biol. Chem. 1998; 273: 25831-25839Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). The inhibitory mechanism postulates a rotation of the C1 in the opposite sense as that induced by Gsα.Gβγ subunits conditionally regulate several adenylyl cyclases. Type II adenylyl cyclase is activated by Gβγ when Gsα is bound. At least part of the Gβγ binding site of type II has been located using peptide competition studies (41Chen J.Q. Devivo M. Dingus J. Harry A. Li J.R. Sui J.L. 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 (235) Google Scholar). The site spans a flexible loop between β3′ and α3′ and the first two-thirds of α3′ (6Zhang G. Liu Y. Ruoho A.E. Hurley J.H. Nature. 1997; 386: 247-253Crossref PubMed Scopus (323) Google Scholar). The Gβγ site is adjacent to, but does not overlap, the Gsα site, consistent with conditional activation.G-protein interactions with non-catalytic regions of adenylyl cyclase seem likely. The α4–β6 region of Gsα was predicted to interact with adenylyl cyclase based on mutagenic analysis (42Itoh H. Gilman A.G. J. Biol. Chem. 1991; 266: 16226-16231Abstract Full Text PDF PubMed Google Scholar, 43Berlot C.H. Bourne H.R. Cell. 1992; 68: 911-922Abstract Full Text PDF PubMed Scopus (160) Google Scholar), but no such contact with the catalytic domain was seen in the crystal structure. Gβγ regulation of the soluble adenylyl cyclase model has not been established, even though the known binding site is located within the type II C2 domain. C1b (44Scholich K. Wittpoth C. Barbier A.J. Mullenix J.B. Patel T.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9602-9607Crossref PubMed Scopus (29) Google Scholar), M1, or M2 might be involved in either of these processes.Regulation by Ca2+/CalmodulinCa2+/calmodulin activates type I adenylyl cyclase by binding to a putative helical region on the C1b (45Vorherr T. Knopfel L. Hofmann F. Mollner S. Pfeuffer T. Carafoli E. Biochemistry. 1993; 32: 6081-6088Crossref PubMed Scopus (139) Google Scholar, 46Wu Z. Wong S.T. Storm D.R. J. Biol. Chem. 1993; 268: 23766-23768Abstract Full Text PDF PubMed Google Scholar). The precise activation mechanism is unknown. If other Ca2+/calmodulin-activated enzymes are a precedent, it is likely that Ca2+/calmodulin binding will disrupt an autoinhibitory interaction between the C1a/C2catalytic core and sequences within the C1b.Regulation by Protein PhosphorylationProtein kinase C activates type II adenylyl cyclase by phosphorylating it on Thr-1057 (47Bol G.F. Gros C. Hulster A. Bosel A. Pfeuffer T. Biochem. Biophys. Res. Commun. 1997; 237: 251-256Crossref PubMed Scopus (24) Google Scholar). This site is within a region known to be required for protein kinase C activation (48Levin L.R. Reed R.R. J. Biol. Chem. 1995; 270: 7573-7579Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). This Thr is at the edge of the "lid," a flexible region that is disordered in the homodimer structure but folds over the top of the active site in the heterodimer-P-site complex. Phosphorylation might enhance the ability of the lid to adopt the correct conformation. CaM kinase II inhibits type III adenylyl cyclase by phosphorylating it at Ser-1076 (49Wei J. Wayman G. Storm D.R. J. Biol. Chem. 1996; 271: 24231-24235Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). This Ser is at the outer lip of the active site, hence its phosphorylation could directly interfere with catalysis. Protein kinase A phosphorylates Ser-674 in the C1b of type VI (50Chen Y. Harry A. Li J. Smit M.J. Bai X. Magnusson R. Pieroni J.P. Weng G. Iyengar R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14100-14104Crossref PubMed Scopus (123) Google Scholar) and appears to regulate a low affinity secondary binding site for Gsα. CaM kinase IV phosphorylates type I adenylyl cyclase in its C1b domain and disables Ca2+/calmodulin activation by interfering with the calmodulin binding site (51Wayman G.A. Wei J. Wong S. Storm D.R. Mol. Cell. Biol. 1996; 16: 6075-6082Crossref PubMed Scopus (67) Google Scholar).Conclusions and PerspectivesA great deal of regulatory complexity has been layered onto the rather simple core structure of adenylyl cyclase. The core consists of two parts. The all important two metal ions bind to one part, C1. The nucleotide binding pocket and other catalytic residues are contributed primarily by the other part, C2. Both parts need to be aligned to carry out catalysis. The most potent activators of the broad range of mammalian adenylyl cyclases, Gsα and forskolin, bind to the domain interface and thereby control domain orientation in a powerful and direct manner. The small molecule forskolin binds on the inside, whereas the large protein activator binds to the outside of the wreath. More specialized regulatory sites have been added to the surface of the catalytic domain (Gβγ, protein kinase C) or appended to it (Ca2+/calmodulin, protein kinase A). The structural work reinforces the concept of mammalian adenylyl cyclases as sophisticated coincidence detectors and provides a new framework for a precise understanding of regulation.Progress in understanding the structure and function of the C1a and C2 regions has not been matched by information on the rest of adenylyl cyclase. Proposed roles for the transmembrane segments M1 and M2 as a transporter or ion channel (8Krupinski J. Coussen F. Bakalyar H. Tang W.-J. Feinstein P.G. Orth K. Slaughter C. Reed R.R. Gilman A.G. Science. 1989; 244: 1558-1564Crossref PubMed Scopus (505) Google Scholar), membrane potential sensor (52Reddy R. Smith D. Wayman G. Wu Z. Villacres E.C. Storm D.R. J. Biol. Chem. 1995; 270: 14340-14346Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 53Cooper D.M.F. Schell M.J. Thorn P. Irvine R.F. J. Biol. Chem. 1998; 273: 27703-27707Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), or Ca2+ channel interaction domain (54Fagan K.A. Mons N. Cooper D.M.F. J. Biol. Chem. 1998; 273: 927-9305Abstract Full Text Full Text PDF Scopus (106) Google
Referência(s)