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W2045593899 abstract "Ca2+ stimulation of adenylyl cyclase type VIII (ACVIII) occurs through loosely bound calmodulin. However, where calmodulin binds in ACVIII and how the binding activates this cyclase have not yet been investigated. We have located two putative calmodulin-binding sites in ACVIII. One site is located at the N terminus as revealed by overlay assays; the other is located at the C terminus, as indicated by mutagenesis studies. Both of these calmodulin-binding sites were confirmed by synthetic peptide studies. The N-terminal site has the typical motif of a Ca2+-dependent calmodulin-binding domain, which is defined by a characteristic pattern of hydrophobic amino acids, basic and aromatic amino acids, and a tendency to form amphipathic α-helix structures. Functional, mutagenesis studies suggest that this binding makes a minor contribution to the Ca2+ stimulation of ACVIII activity, although it might be involved in calmodulin trapping by ACVIII. The primary structure of the C-terminal site resembles another calmodulin-binding motif, the so-called IQ motif, which is commonly Ca2+-independent. Mutagenesis and functional assays indicate that this latter site is a calcium-dependent calmodulin-binding site, which is largely responsible for the Ca2+ stimulation of ACVIII. Removal of this latter calmodulin-binding region from ACVIII results in a hyperactivated enzyme state and a loss of Ca2+ sensitivity. Thus, Ca2+/calmodulin regulation of ACVIII may be through a disinhibitory mechanism, as is the case for a number of other targets of Ca2+/calmodulin. Ca2+ stimulation of adenylyl cyclase type VIII (ACVIII) occurs through loosely bound calmodulin. However, where calmodulin binds in ACVIII and how the binding activates this cyclase have not yet been investigated. We have located two putative calmodulin-binding sites in ACVIII. One site is located at the N terminus as revealed by overlay assays; the other is located at the C terminus, as indicated by mutagenesis studies. Both of these calmodulin-binding sites were confirmed by synthetic peptide studies. The N-terminal site has the typical motif of a Ca2+-dependent calmodulin-binding domain, which is defined by a characteristic pattern of hydrophobic amino acids, basic and aromatic amino acids, and a tendency to form amphipathic α-helix structures. Functional, mutagenesis studies suggest that this binding makes a minor contribution to the Ca2+ stimulation of ACVIII activity, although it might be involved in calmodulin trapping by ACVIII. The primary structure of the C-terminal site resembles another calmodulin-binding motif, the so-called IQ motif, which is commonly Ca2+-independent. Mutagenesis and functional assays indicate that this latter site is a calcium-dependent calmodulin-binding site, which is largely responsible for the Ca2+ stimulation of ACVIII. Removal of this latter calmodulin-binding region from ACVIII results in a hyperactivated enzyme state and a loss of Ca2+ sensitivity. Thus, Ca2+/calmodulin regulation of ACVIII may be through a disinhibitory mechanism, as is the case for a number of other targets of Ca2+/calmodulin. adenylyl cyclase, type I adenylyl cyclase, type VIII polymerase chain reaction antibody Mammalian adenylyl cyclases are a diverse group of variously regulated signaling molecules. Details are emerging on some of the molecular features conferring catalytic and regulatory properties on these enzymes. All of the nine cloned adenylyl cyclases are large (1080–1248 amino acids) polypeptides that are proposed to comprise two cassettes of six transmembrane-spanning domains, each cassette being followed by a large cytoplasmic domain (1Sunahara R.K. Dessauer C.W. Gilman A.G. Annu. Rev. Pharmacol. Toxicol. 1996; 36: 461-480Crossref PubMed Scopus (732) Google Scholar, 2Cooper D.M.F. Mons N. Karpen J.W. Nature. 1995; 374: 421-424Crossref PubMed Scopus (553) Google Scholar). The transmembrane domains are not highly conserved among adenylyl cyclases. However, parts of two of the cytoplasmic domains (termed C1a and C2a) are highly conserved, and, when expressed separately, they can combine to display basic catalytic activity (3Tang W.-J. Krupinski J. Gilman A.G. J. Biol. Chem. 1991; 266: 8595-8603Abstract Full Text PDF PubMed Google Scholar, 4Tang W.-J. Gilman A.G. Science. 1995; 268: 1769-1772Crossref PubMed Scopus (165) Google Scholar, 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, 7Sunahara R.K. Dessauer C.W. Whisnant R.E. Kleuss C. Gilman A.G. J. Biol. Chem. 1997; 272: 22265-22271Crossref PubMed Scopus (159) Google Scholar, 8Dessauer C.W. Scully T.T. Gilman A.G. J. Biol. Chem. 1997; 272: 22272-22277Crossref PubMed Scopus (108) Google Scholar). These molecules have been crystallized, and the combination of C1a and C2a, each consisting of a three layer α/β sandwich, forms an active catalytic core to generate cAMP from ATP (9Zhang G. Liu Y. Ruoho A.E. Hurley J.H. Nature. 1997; 386: 247-253Crossref PubMed Scopus (323) Google Scholar, 10Tesmer J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (670) Google Scholar). The other major cytoplasmic domains of mammalian adenylyl cyclases, the N terminus, the C1b region, and the C2b region, are not conserved at all and are speculated to reflect regulatory features of specific adenylyl cyclases (1Sunahara R.K. Dessauer C.W. Gilman A.G. Annu. Rev. Pharmacol. Toxicol. 1996; 36: 461-480Crossref PubMed Scopus (732) Google Scholar, 11Krupinski J. Coussen F. Bakalyar H.A. 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, 12Cooper D.M.F. Mons N. Fagan K.A. Cell. Signalling. 1994; 6: 823-840Crossref PubMed Scopus (61) Google Scholar, 13Krupinski J. Cali J.J. Adv. Second Messengers Phosphoprotein Res. 1998; 32: 53-79Crossref PubMed Google Scholar).Ca2+ elicits a prominent stimulation of ACI1 and ACVIII, which is mediated by loosely bound calmodulin (3Tang W.-J. Krupinski J. Gilman A.G. J. Biol. Chem. 1991; 266: 8595-8603Abstract Full Text PDF PubMed Google Scholar, 11Krupinski J. Coussen F. Bakalyar H.A. 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, 14Cali J.J. Zwaagstra J.C. Mons N. Cooper D.M.F. Krupinski J. J. Biol. Chem. 1994; 269: 12190-12195Abstract Full Text PDF PubMed Google Scholar). Although the likely calmodulin-binding domain on ACI has been localized to the C1b region (15Levin L.R. Reed R.R. J. Biol. Chem. 1995; 270: 7573-7579Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 16Vorherr T. Knopfel L. Hofmann F. Mollner S. Pfeuffer T. Carafoli E. Biochemistry. 1993; 32: 6081-6088Crossref PubMed Scopus (139) Google Scholar), the corresponding regulatory domain has not been identified on ACVIII. Indeed, ACVIII does not possess analogous calmodulin-binding sites in the C1b region. In the case of ACI, peptides corresponding to putative calmodulin-binding domains were used to identify a site in the C1b region as the likely site of calmodulin binding (16Vorherr T. Knopfel L. Hofmann F. Mollner S. Pfeuffer T. Carafoli E. Biochemistry. 1993; 32: 6081-6088Crossref PubMed Scopus (139) Google Scholar). Mutagenesis studies strongly supported this assignment (15Levin L.R. Reed R.R. J. Biol. Chem. 1995; 270: 7573-7579Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 17Wu Z. Wong S.T. Storm D.R. J. Biol. Chem. 1993; 268: 23766-23768Abstract Full Text PDF PubMed Google Scholar). However, no information is yet available on the possible domains that mediate the Ca2+/calmodulin responsiveness of ACVIII. Given that ACI and ACVIII do not share similar C1b domains (they are only ∼40% similar at the amino acid level, compared with 80% similarity in the C1a region) and also that their regulation by Ca2+/calmodulin shows distinct properties, it might not be unexpected if different motifs and/or locations were involved.Identifying calmodulin-binding sites on proteins still mainly depends on experimentation, although some predictive criteria are available to guide experiments. For instance, many known Ca2+-dependent calmodulin-binding proteins possess a region that is often characterized by an amphipathic helix consisting of approximately 20 amino acid residues (18Rhoads A.R. Friedberg F. FASEB J. 1997; 11: 331-340Crossref PubMed Scopus (734) Google Scholar). In these regions, basic amino acids are interspersed among hydrophobic residues, and aromatic amino acids normally appear near either end (16Vorherr T. Knopfel L. Hofmann F. Mollner S. Pfeuffer T. Carafoli E. Biochemistry. 1993; 32: 6081-6088Crossref PubMed Scopus (139) Google Scholar, 18Rhoads A.R. Friedberg F. FASEB J. 1997; 11: 331-340Crossref PubMed Scopus (734) Google Scholar, 19Deiss L.P. Feinstein E. Berissi H. Cohen O. Kimchi A. Genes Dev. 1995; 9: 15-30Crossref PubMed Scopus (530) Google Scholar). However, sequence analysis based on these criteria does not always identify calmodulin-binding regions, and indeed, regions of proteins that bind to calmodulin sometimes do not fit these criteria. Another calmodulin-binding motif is the so-called “IQ motif,” consensus sequence IQXXXRGXXXR, which often (18Rhoads A.R. Friedberg F. FASEB J. 1997; 11: 331-340Crossref PubMed Scopus (734) Google Scholar) but not always (22Munshi H.G. Burks D.J. Joyal J.L. White M.F. Sacks D.B. Biochemistry. 1996; 35: 15883-15889Crossref PubMed Scopus (48) Google Scholar, 23Weitz D. Zoche M. Muller F. Beyermann M. Korschen H.G. Kaupp U.B. Koch K.-W. EMBO J. 1998; 17: 2273-2284Crossref PubMed Scopus (93) Google Scholar) binds calmodulin in a Ca2+-independent manner.The present studies used a combination of calmodulin overlay assays, mutagenesis, and peptide inhibition studies to locate the calmodulin regulatory domains on ACVIII. Surprisingly, a primary site was located in the C2b region, while an ancillary site that appeared to play a minor autoinhibitory role was located in the N terminus.DISCUSSIONThis study has explored the calmodulin-binding sites on ACVIII. The related Ca2+-stimulable ACI binds calmodulin in the C1b region of the molecule. However, ACVIII and ACI are very dissimilar (only 40% homologous) in this region. Therefore, it might not have been unexpected that different sites would mediate the Ca2+ stimulation of ACVIII. Calmodulin overlay assays revealed one putative Ca2+-dependent calmodulin-binding site in the N terminus of ACVIII (Fig. 1). However, without this region the enzyme was still sensitive to Ca2+(Figs. 2 and 3). On the other hand, using mutagenesis and functional assays, only those mutants lacking the C2b region, such as C2Δ1184–1248 and NC2Δ1–106, Δ1184–1248, could not be stimulated by Ca2+either in vivo or in vitro (Figs. 2 and 3). This suggests that the C-terminal region is responsible for the Ca2+/calmodulin stimulation of ACVIII. Moreover, the high basal activities of C2Δ1184–1248 and NC2Δ1–106, Δ1184–1248 suggests the removal of autoinhibitory domains (the C2b region playing the major role), which suppress the activity of wild type ACVIII. The binding of Ca2+/calmodulin to the autoinhibitory domain apparently relieves the inhibitory binding and activates the enzyme. Such a disinhibitory mechanism is employed in a number of Ca2+/calmodulin-activated enzymes, such as Ca2+-regulated nitric-oxide synthase (44Salerno J.C. Harris D.E. Irizarry K. Patel B. Morales A.J. Smith S.M. Martasek P. Roman L.J. Masters B.S. Jones C.L. Weissman B.A. Lane P. Liu Q. Gross S.S. J. Biol. Chem. 1997; 272: 29769-29777Crossref PubMed Scopus (210) Google Scholar), Ca2+/calmodulin-dependent protein kinases (45Zhi G. Abdullah S.M. Stull J.T. J. Biol. Chem. 1998; 273: 8951-8957Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 46Tokumitsu H. Wayman G.A. Muramatsu M. Soderling T.R. Biochemistry. 1997; 36: 12823-12827Crossref PubMed Scopus (54) Google Scholar, 47Yokokura H. Picciotto M.R. Nairn A.C. Hidaka H. J. Biol. Chem. 1995; 270: 23851-23859Crossref PubMed Scopus (61) Google Scholar), a Na+/H+ exchanger (48Wakabayashi S. Ikeda T. Iwamoto T. Pouyssegur J. Shigekawa M. Biochemistry. 1997; 36: 12854-12861Crossref PubMed Scopus (94) Google Scholar), and calcineurin (49Hashimoto Y. Perrino B.A. Soderling T.R. J. Biol. Chem. 1990; 265: 1924-1927Abstract Full Text PDF PubMed Google Scholar). It appears as though most of the C2b region might participate as the inhibitory binding domain, since the two synthetic peptides 8Ccam (25 residues) and 8CT (20 residues) from the C2b region did not inhibit ACVIII activity in the absence of Ca2+ and calmodulin (Fig. 6 C). This disinhibition mechanism of Ca2+/calmodulin stimulation is different from that of forskolin, which is thought to stabilize the C1/C2 heterodimer to activate the adenylyl cyclase activity (9Zhang G. Liu Y. Ruoho A.E. Hurley J.H. Nature. 1997; 386: 247-253Crossref PubMed Scopus (323) Google Scholar, 10Tesmer J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (670) Google Scholar). The latter suggestion is supported by the fact that forskolin and Ca2+/calmodulin synergistically stimulate ACVIII.The putative calmodulin-binding site in the C terminus has the signature sequence of an IQ motif, which generally reflects Ca2+-independent calmodulin binding. It is possible that the binding of calmodulin is Ca2+-independent and that Ca2+ binding changes the conformation of bound calmodulin to activate the enzyme. Alternatively, the binding of calmodulin may be Ca2-dependent as is the case with the β-subunit of rod photoreceptor cyclic nucleotide-gated channel (23Weitz D. Zoche M. Muller F. Beyermann M. Korschen H.G. Kaupp U.B. Koch K.-W. EMBO J. 1998; 17: 2273-2284Crossref PubMed Scopus (93) Google Scholar). It is of some interest that the peptide synthesized from this region (8Ccam) is only 5 times less effective than 8CamkII, which is a conventional calmodulin-binding peptide. This observation underscores how much we still need to learn about the molecular characteristics of calmodulin-binding sequences.The putative calmodulin-binding site for the N terminus of ACVIII is a conventional Ca2+-dependent calmodulin-binding site, which is reinforced by the results of overlay assays. The fact that the double deletion NC2Δ1–106, Δ1184–1248 has higher activity in vivo (Fig. 2) and can be inhibited by a lower concentration of Ca2+ (Fig. 5 A) than C2Δ1184–1248 suggests that this site contributes to the Ca2+ stimulation of ACVIII, although this contribution must be minor.The fact that there is apparently more residual calmodulin in ACVIII wild type membrane preparations than in those of NΔ1–106(Fig. 5 B) might suggest a role of the N terminus of ACVIII as a Ca2+-independent calmodulin trap, notwithstanding the apparently conflicting evidence of the Ca2+-dependent manner of the N-terminal calmodulin-binding site from overlay assays.Unlike the two regulatory domains (the N terminus and the C2b region) discussed above, the C1b region does not have a free end, which suggests that the disruptions on this region could more easily change the activity of adenylyl cyclases. However, continuous deletions in the C1b region of ACVIII could not eliminate the Ca2+stimulation of ACVIII (Fig. 2 A), while, by contrast, a point mutation in this region of ACI abolished its Ca2+sensitivity (17Wu Z. Wong S.T. Storm D.R. J. Biol. Chem. 1993; 268: 23766-23768Abstract Full Text PDF PubMed Google Scholar). The different calmodulin-binding sites on ACVIII and ACI are underlined by some differences in their regulation by Ca2+/calmodulin; for instance, ACI is more sensitive to lower concentrations of Ca2+ than is ACVIII, and ACVIII is more stimulable by Ca2+/calmodulin than ACI (25Fagan K.A. Mahey R. Cooper D.M.F. J. Biol. Chem. 1996; 271: 12438-12444Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). The calmodulin-binding site in ACI is rich in basic amino acids (net charge is +7), and the binding is Ca2+-dependent (15Levin L.R. Reed R.R. J. Biol. Chem. 1995; 270: 7573-7579Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar,16Vorherr T. Knopfel L. Hofmann F. Mollner S. Pfeuffer T. Carafoli E. Biochemistry. 1993; 32: 6081-6088Crossref PubMed Scopus (139) Google Scholar). Since no hyperactivity was observed by mutating the C1b region on ACI (15Levin L.R. Reed R.R. J. Biol. Chem. 1995; 270: 7573-7579Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 17Wu Z. Wong S.T. Storm D.R. J. Biol. Chem. 1993; 268: 23766-23768Abstract Full Text PDF PubMed Google Scholar) and the movement of the C1b region is likely to be more restrained than those of the N terminus and C terminus, the mechanism of Ca2+/calmodulin regulation of ACI might be to stabilize the C1/C2 heterodimer, as has been proposed for forskolin and Gsαa (9Zhang G. Liu Y. Ruoho A.E. Hurley J.H. Nature. 1997; 386: 247-253Crossref PubMed Scopus (323) Google Scholar, 10Tesmer J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (670) Google Scholar), unlike the disinhibitory mechanism we have proposed for ACVIII.In conclusion, two calmodulin-binding sites exist on ACVIII, one (at the C terminus) is of profound regulatory significance, whereas the other (at the N terminus) plays a more minor role. Whether these two domains of ACVIII physically interact to share the same molecule of calmodulin remains to be determined in future studies. Given that the C1a and C2a regions clearly interact for catalytic activity (4Tang W.-J. Gilman A.G. Science. 1995; 268: 1769-1772Crossref PubMed Scopus (165) Google Scholar, 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, 7Sunahara R.K. Dessauer C.W. Whisnant R.E. Kleuss C. Gilman A.G. J. Biol. Chem. 1997; 272: 22265-22271Crossref PubMed Scopus (159) Google Scholar,9Zhang G. Liu Y. Ruoho A.E. Hurley J.H. Nature. 1997; 386: 247-253Crossref PubMed Scopus (323) Google Scholar, 10Tesmer J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (670) Google Scholar, 35Tang W.-J. Stanzel M. Gilman A.G. Biochemistry. 1995; 34: 14563-14572Crossref PubMed Scopus (109) Google Scholar), the tantalizing possibility that adenylyl cyclase could adopt a transporter-like structure (11Krupinski J. Coussen F. Bakalyar H.A. 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, 50Cooper D.M.F. Karpen J.W. Fagan K.A. Mons N.E. Adv. Second Messengers Phosphoprotein Res. 1998; 32: 23-51Crossref PubMed Scopus (74) Google Scholar) would be greatly strengthened by interactions between the N and C termini. Mammalian adenylyl cyclases are a diverse group of variously regulated signaling molecules. Details are emerging on some of the molecular features conferring catalytic and regulatory properties on these enzymes. All of the nine cloned adenylyl cyclases are large (1080–1248 amino acids) polypeptides that are proposed to comprise two cassettes of six transmembrane-spanning domains, each cassette being followed by a large cytoplasmic domain (1Sunahara R.K. Dessauer C.W. Gilman A.G. Annu. Rev. Pharmacol. Toxicol. 1996; 36: 461-480Crossref PubMed Scopus (732) Google Scholar, 2Cooper D.M.F. Mons N. Karpen J.W. Nature. 1995; 374: 421-424Crossref PubMed Scopus (553) Google Scholar). The transmembrane domains are not highly conserved among adenylyl cyclases. However, parts of two of the cytoplasmic domains (termed C1a and C2a) are highly conserved, and, when expressed separately, they can combine to display basic catalytic activity (3Tang W.-J. Krupinski J. Gilman A.G. J. Biol. Chem. 1991; 266: 8595-8603Abstract Full Text PDF PubMed Google Scholar, 4Tang W.-J. Gilman A.G. Science. 1995; 268: 1769-1772Crossref PubMed Scopus (165) Google Scholar, 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, 7Sunahara R.K. Dessauer C.W. Whisnant R.E. Kleuss C. Gilman A.G. J. Biol. Chem. 1997; 272: 22265-22271Crossref PubMed Scopus (159) Google Scholar, 8Dessauer C.W. Scully T.T. Gilman A.G. J. Biol. Chem. 1997; 272: 22272-22277Crossref PubMed Scopus (108) Google Scholar). These molecules have been crystallized, and the combination of C1a and C2a, each consisting of a three layer α/β sandwich, forms an active catalytic core to generate cAMP from ATP (9Zhang G. Liu Y. Ruoho A.E. Hurley J.H. Nature. 1997; 386: 247-253Crossref PubMed Scopus (323) Google Scholar, 10Tesmer J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (670) Google Scholar). The other major cytoplasmic domains of mammalian adenylyl cyclases, the N terminus, the C1b region, and the C2b region, are not conserved at all and are speculated to reflect regulatory features of specific adenylyl cyclases (1Sunahara R.K. Dessauer C.W. Gilman A.G. Annu. Rev. Pharmacol. Toxicol. 1996; 36: 461-480Crossref PubMed Scopus (732) Google Scholar, 11Krupinski J. Coussen F. Bakalyar H.A. 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, 12Cooper D.M.F. Mons N. Fagan K.A. Cell. Signalling. 1994; 6: 823-840Crossref PubMed Scopus (61) Google Scholar, 13Krupinski J. Cali J.J. Adv. Second Messengers Phosphoprotein Res. 1998; 32: 53-79Crossref PubMed Google Scholar). Ca2+ elicits a prominent stimulation of ACI1 and ACVIII, which is mediated by loosely bound calmodulin (3Tang W.-J. Krupinski J. Gilman A.G. J. Biol. Chem. 1991; 266: 8595-8603Abstract Full Text PDF PubMed Google Scholar, 11Krupinski J. Coussen F. Bakalyar H.A. 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, 14Cali J.J. Zwaagstra J.C. Mons N. Cooper D.M.F. Krupinski J. J. Biol. Chem. 1994; 269: 12190-12195Abstract Full Text PDF PubMed Google Scholar). Although the likely calmodulin-binding domain on ACI has been localized to the C1b region (15Levin L.R. Reed R.R. J. Biol. Chem. 1995; 270: 7573-7579Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 16Vorherr T. Knopfel L. Hofmann F. Mollner S. Pfeuffer T. Carafoli E. Biochemistry. 1993; 32: 6081-6088Crossref PubMed Scopus (139) Google Scholar), the corresponding regulatory domain has not been identified on ACVIII. Indeed, ACVIII does not possess analogous calmodulin-binding sites in the C1b region. In the case of ACI, peptides corresponding to putative calmodulin-binding domains were used to identify a site in the C1b region as the likely site of calmodulin binding (16Vorherr T. Knopfel L. Hofmann F. Mollner S. Pfeuffer T. Carafoli E. Biochemistry. 1993; 32: 6081-6088Crossref PubMed Scopus (139) Google Scholar). Mutagenesis studies strongly supported this assignment (15Levin L.R. Reed R.R. J. Biol. Chem. 1995; 270: 7573-7579Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 17Wu Z. Wong S.T. Storm D.R. J. Biol. Chem. 1993; 268: 23766-23768Abstract Full Text PDF PubMed Google Scholar). However, no information is yet available on the possible domains that mediate the Ca2+/calmodulin responsiveness of ACVIII. Given that ACI and ACVIII do not share similar C1b domains (they are only ∼40% similar at the amino acid level, compared with 80% similarity in the C1a region) and also that their regulation by Ca2+/calmodulin shows distinct properties, it might not be unexpected if different motifs and/or locations were involved. Identifying calmodulin-binding sites on proteins still mainly depends on experimentation, although some predictive criteria are available to guide experiments. For instance, many known Ca2+-dependent calmodulin-binding proteins possess a region that is often characterized by an amphipathic helix consisting of approximately 20 amino acid residues (18Rhoads A.R. Friedberg F. FASEB J. 1997; 11: 331-340Crossref PubMed Scopus (734) Google Scholar). In these regions, basic amino acids are interspersed among hydrophobic residues, and aromatic amino acids normally appear near either end (16Vorherr T. Knopfel L. Hofmann F. Mollner S. Pfeuffer T. Carafoli E. Biochemistry. 1993; 32: 6081-6088Crossref PubMed Scopus (139) Google Scholar, 18Rhoads A.R. Friedberg F. FASEB J. 1997; 11: 331-340Crossref PubMed Scopus (734) Google Scholar, 19Deiss L.P. Feinstein E. Berissi H. Cohen O. Kimchi A. Genes Dev. 1995; 9: 15-30Crossref PubMed Scopus (530) Google Scholar). However, sequence analysis based on these criteria does not always identify calmodulin-binding regions, and indeed, regions of proteins that bind to calmodulin sometimes do not fit these criteria. Another calmodulin-binding motif is the so-called “IQ motif,” consensus sequence IQXXXRGXXXR, which often (18Rhoads A.R. Friedberg F. FASEB J. 1997; 11: 331-340Crossref PubMed Scopus (734) Google Scholar) but not always (22Munshi H.G. Burks D.J. Joyal J.L. White M.F. Sacks D.B. Biochemistry. 1996; 35: 15883-15889Crossref PubMed Scopus (48) Google Scholar, 23Weitz D. Zoche M. Muller F. Beyermann M. Korschen H.G. Kaupp U.B. Koch K.-W. EMBO J. 1998; 17: 2273-2284Crossref PubMed Scopus (93) Google Scholar) binds calmodulin in a Ca2+-independent manner. The present studies used a combination of calmodulin overlay assays, mutagenesis, and peptide inhibition studies to locate the calmodulin regulatory domains on ACVIII. Surprisingly, a primary site was located in the C2b region, while an ancillary site that appeared to play a minor autoinhibitory role was located in the N terminus. DISCUSSIONThis study has explored the calmodulin-binding sites on ACVIII. The related Ca2+-stimulable ACI binds calmodulin in the C1b region of the molecule. However, ACVIII and ACI are very dissimilar (only 40% homologous) in this region. Therefore, it might not have been unexpected that different sites would mediate the Ca2+ stimulation of ACVIII. Calmodulin overlay assays revealed one putative Ca2+-dependent calmodulin-binding site in the N terminus of ACVIII (Fig. 1). However, without this region the enzyme was still sensitive to Ca2+(Figs. 2 and 3). On the other hand, using mutagenesis and functional assays, only those mutants lacking the C2b region, such as C2Δ1184–1248 and NC2Δ1–106, Δ1184–1248, could not be stimulated by Ca2+either in vivo or in vitro (Figs. 2 and 3). This suggests that the C-terminal region is responsible for the Ca2+/calmodulin stimulation of ACVIII. Moreover, the high basal activities of C2Δ1184–1248 and NC2Δ1–106, Δ1184–1248 suggests the removal of autoinhibitory domains (the C2b region playing the major role), which suppress the activity of wild type ACVIII. The binding of Ca2+/calmodulin to the autoinhibitory domain apparently relieves the inhibitory binding and activates the enzyme. Such a disinhibitory mechanism is employed in a number of Ca2+/calmodulin-activated enzymes, such as Ca2+-regulated nitric-oxide synthase (44Salerno J.C. Harris D.E. Irizarry K. Patel B. Morales A.J. Smith S.M. Martasek P. Roman L.J. Masters B.S. Jones C.L. Weissman B.A. Lane P. Liu Q. Gross S.S. J. Biol. Chem. 1997; 272: 29769-29777Crossref PubMed Scopus (210) Google Scholar), Ca2+/calmodulin-dependent protein kinases (45Zhi G. Abdullah S.M. Stull J.T. J. Biol. Chem. 1998; 273: 8951-8957Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 46Tokumitsu H. Wayman G.A. Muramatsu M. Soderling T.R. Biochemistry. 1997; 36: 12823-12827Crossref PubMed Scopus (54) Google Scholar, 47Yokokura H. Picciotto M.R. Nairn A.C. Hidaka H. J. Biol. Chem. 1995; 270: 23851-23859Crossref PubMed Scopus (61) Google Scholar), a Na+/H+ exchanger (48Wakabayashi S. Ikeda T. Iwamoto T. Pouyssegur J. Shigekawa M. Biochemistry. 1997; 36: 12854-12861Crossref PubMed Scopus (94) Google Scholar), and calcineurin (49Hashimoto Y. Perrino B.A. Soderling T.R. J. Biol. Chem. 1990; 265: 1924-1927Abstract Full Text PDF PubMed Google Scholar). It appears as though most of the C2b region might participate as the inhibitory binding domain, since the two synthetic peptides 8Ccam (25 residues) and 8CT (20 residues) from the C2b region did not inhibit ACVIII activity in the absence of Ca2+ and calmodulin (Fig. 6 C). This disinhibition mechanism of Ca2+/calmodulin stimulation is different from that of forskolin, which is thought to stabilize the C1/C2 heterodimer to activate the adenylyl cyclase activity (9Zhang G. Liu Y. Ruoho A.E. Hurley J.H. Nature. 1997; 386: 247-253Crossref PubMed Scopus (323) Google Scholar, 10Tesmer J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (670) Google Scholar). The latter suggestion is supported by the fact that forskolin and Ca2+/calmodulin synergistically stimulate ACVIII.The putative calmodulin-binding site in the C terminus has the signature sequence of an IQ motif, which generally reflects Ca2+-independent calmodulin binding. It is possible that the binding of calmodulin is Ca2+-independent and that Ca2+ binding changes the conformation of bound calmodulin to activate the enzyme. Alternatively, the binding of calmodulin may be Ca2-dependent as is the case with the β-subunit of rod photoreceptor cyclic nucleotide-gated channel (23Weitz D. Zoche M. Muller F. Beyermann M. Korschen H.G. Kaupp U.B. Koch K.-W. EMBO J. 1998; 17: 2273-2284Crossref PubMed Scopus (93) Google Scholar). It is of some interest that the peptide synthesized from this region (8Ccam) is only 5 times less effective than 8CamkII, which is a conventional calmodulin-binding peptide. This observation underscores how much we still need to learn about the molecular characteristics of calmodulin-binding sequences.The putative calmodulin-binding site for the N terminus of ACVIII is a conventional Ca2+-dependent calmodulin-binding site, which is reinforced by the results of overlay assays. The fact that the double deletion NC2Δ1–106, Δ1184–1248 has higher activity in vivo (Fig. 2) and can be inhibited by a lower concentration of Ca2+ (Fig. 5 A) than C2Δ1184–1248 suggests that this site contributes to the Ca2+ stimulation of ACVIII, although this contribution must be minor.The fact that there is apparently more residual calmodulin in ACVIII wild type membrane preparations than in those of NΔ1–106(Fig. 5 B) might suggest a role of the N terminus of ACVIII as a Ca2+-independent calmodulin trap, notwithstanding the apparently conflicting evidence of the Ca2+-dependent manner of the N-terminal calmodulin-binding site from overlay assays.Unlike the two regulatory domains (the N terminus and the C2b region) discussed above, the C1b region does not have a free end, which suggests that the disruptions on this region could more easily change the activity of adenylyl cyclases. However, continuous deletions in the C1b region of ACVIII could not eliminate the Ca2+stimulation of ACVIII (Fig. 2 A), while, by contrast, a point mutation in this region of ACI abolished its Ca2+sensitivity (17Wu Z. Wong S.T. Storm D.R. J. Biol. Chem. 1993; 268: 23766-23768Abstract Full Text PDF PubMed Google Scholar). The different calmodulin-binding sites on ACVIII and ACI are underlined by some differences in their regulation by Ca2+/calmodulin; for instance, ACI is more sensitive to lower concentrations of Ca2+ than is ACVIII, and ACVIII is more stimulable by Ca2+/calmodulin than ACI (25Fagan K.A. Mahey R. Cooper D.M.F. J. Biol. Chem. 1996; 271: 12438-12444Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). The calmodulin-binding site in ACI is rich in basic amino acids (net charge is +7), and the binding is Ca2+-dependent (15Levin L.R. Reed R.R. J. Biol. Chem. 1995; 270: 7573-7579Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar,16Vorherr T. Knopfel L. Hofmann F. Mollner S. Pfeuffer T. Carafoli E. Biochemistry. 1993; 32: 6081-6088Crossref PubMed Scopus (139) Google Scholar). Since no hyperactivity was observed by mutating the C1b region on ACI (15Levin L.R. Reed R.R. J. Biol. Chem. 1995; 270: 7573-7579Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 17Wu Z. Wong S.T. Storm D.R. J. Biol. Chem. 1993; 268: 23766-23768Abstract Full Text PDF PubMed Google Scholar) and the movement of the C1b region is likely to be more restrained than those of the N terminus and C terminus, the mechanism of Ca2+/calmodulin regulation of ACI might be to stabilize the C1/C2 heterodimer, as has been proposed for forskolin and Gsαa (9Zhang G. Liu Y. Ruoho A.E. Hurley J.H. Nature. 1997; 386: 247-253Crossref PubMed Scopus (323) Google Scholar, 10Tesmer J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (670) Google Scholar), unlike the disinhibitory mechanism we have proposed for ACVIII.In conclusion, two calmodulin-binding sites exist on ACVIII, one (at the C terminus) is of profound regulatory significance, whereas the other (at the N terminus) plays a more minor role. Whether these two domains of ACVIII physically interact to share the same molecule of calmodulin remains to be determined in future studies. Given that the C1a and C2a regions clearly interact for catalytic activity (4Tang W.-J. Gilman A.G. Science. 1995; 268: 1769-1772Crossref PubMed Scopus (165) Google Scholar, 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, 7Sunahara R.K. Dessauer C.W. Whisnant R.E. Kleuss C. Gilman A.G. J. Biol. Chem. 1997; 272: 22265-22271Crossref PubMed Scopus (159) Google Scholar,9Zhang G. Liu Y. Ruoho A.E. Hurley J.H. Nature. 1997; 386: 247-253Crossref PubMed Scopus (323) Google Scholar, 10Tesmer J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (670) Google Scholar, 35Tang W.-J. Stanzel M. Gilman A.G. Biochemistry. 1995; 34: 14563-14572Crossref PubMed Scopus (109) Google Scholar), the tantalizing possibility that adenylyl cyclase could adopt a transporter-like structure (11Krupinski J. Coussen F. Bakalyar H.A. 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, 50Cooper D.M.F. Karpen J.W. Fagan K.A. Mons N.E. Adv. Second Messengers Phosphoprotein Res. 1998; 32: 23-51Crossref PubMed Scopus (74) Google Scholar) would be greatly strengthened by interactions between the N and C termini. This study has explored the calmodulin-binding sites on ACVIII. The related Ca2+-stimulable ACI binds calmodulin in the C1b region of the molecule. However, ACVIII and ACI are very dissimilar (only 40% homologous) in this region. Therefore, it might not have been unexpected that different sites would mediate the Ca2+ stimulation of ACVIII. Calmodulin overlay assays revealed one putative Ca2+-dependent calmodulin-binding site in the N terminus of ACVIII (Fig. 1). However, without this region the enzyme was still sensitive to Ca2+(Figs. 2 and 3). On the other hand, using mutagenesis and functional assays, only those mutants lacking the C2b region, such as C2Δ1184–1248 and NC2Δ1–106, Δ1184–1248, could not be stimulated by Ca2+either in vivo or in vitro (Figs. 2 and 3). This suggests that the C-terminal region is responsible for the Ca2+/calmodulin stimulation of ACVIII. Moreover, the high basal activities of C2Δ1184–1248 and NC2Δ1–106, Δ1184–1248 suggests the removal of autoinhibitory domains (the C2b region playing the major role), which suppress the activity of wild type ACVIII. The binding of Ca2+/calmodulin to the autoinhibitory domain apparently relieves the inhibitory binding and activates the enzyme. Such a disinhibitory mechanism is employed in a number of Ca2+/calmodulin-activated enzymes, such as Ca2+-regulated nitric-oxide synthase (44Salerno J.C. Harris D.E. Irizarry K. Patel B. Morales A.J. Smith S.M. Martasek P. Roman L.J. Masters B.S. Jones C.L. Weissman B.A. Lane P. Liu Q. Gross S.S. J. Biol. Chem. 1997; 272: 29769-29777Crossref PubMed Scopus (210) Google Scholar), Ca2+/calmodulin-dependent protein kinases (45Zhi G. Abdullah S.M. Stull J.T. J. Biol. Chem. 1998; 273: 8951-8957Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 46Tokumitsu H. Wayman G.A. Muramatsu M. Soderling T.R. Biochemistry. 1997; 36: 12823-12827Crossref PubMed Scopus (54) Google Scholar, 47Yokokura H. Picciotto M.R. Nairn A.C. Hidaka H. J. Biol. Chem. 1995; 270: 23851-23859Crossref PubMed Scopus (61) Google Scholar), a Na+/H+ exchanger (48Wakabayashi S. Ikeda T. Iwamoto T. Pouyssegur J. Shigekawa M. Biochemistry. 1997; 36: 12854-12861Crossref PubMed Scopus (94) Google Scholar), and calcineurin (49Hashimoto Y. Perrino B.A. Soderling T.R. J. Biol. Chem. 1990; 265: 1924-1927Abstract Full Text PDF PubMed Google Scholar). It appears as though most of the C2b region might participate as the inhibitory binding domain, since the two synthetic peptides 8Ccam (25 residues) and 8CT (20 residues) from the C2b region did not inhibit ACVIII activity in the absence of Ca2+ and calmodulin (Fig. 6 C). This disinhibition mechanism of Ca2+/calmodulin stimulation is different from that of forskolin, which is thought to stabilize the C1/C2 heterodimer to activate the adenylyl cyclase activity (9Zhang G. Liu Y. Ruoho A.E. Hurley J.H. Nature. 1997; 386: 247-253Crossref PubMed Scopus (323) Google Scholar, 10Tesmer J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (670) Google Scholar). The latter suggestion is supported by the fact that forskolin and Ca2+/calmodulin synergistically stimulate ACVIII. The putative calmodulin-binding site in the C terminus has the signature sequence of an IQ motif, which generally reflects Ca2+-independent calmodulin binding. It is possible that the binding of calmodulin is Ca2+-independent and that Ca2+ binding changes the conformation of bound calmodulin to activate the enzyme. Alternatively, the binding of calmodulin may be Ca2-dependent as is the case with the β-subunit of rod photoreceptor cyclic nucleotide-gated channel (23Weitz D. Zoche M. Muller F. Beyermann M. Korschen H.G. Kaupp U.B. Koch K.-W. EMBO J. 1998; 17: 2273-2284Crossref PubMed Scopus (93) Google Scholar). It is of some interest that the peptide synthesized from this region (8Ccam) is only 5 times less effective than 8CamkII, which is a conventional calmodulin-binding peptide. This observation underscores how much we still need to learn about the molecular characteristics of calmodulin-binding sequences. The putative calmodulin-binding site for the N terminus of ACVIII is a conventional Ca2+-dependent calmodulin-binding site, which is reinforced by the results of overlay assays. The fact that the double deletion NC2Δ1–106, Δ1184–1248 has higher activity in vivo (Fig. 2) and can be inhibited by a lower concentration of Ca2+ (Fig. 5 A) than C2Δ1184–1248 suggests that this site contributes to the Ca2+ stimulation of ACVIII, although this contribution must be minor. The fact that there is apparently more residual calmodulin in ACVIII wild type membrane preparations than in those of NΔ1–106(Fig. 5 B) might suggest a role of the N terminus of ACVIII as a Ca2+-independent calmodulin trap, notwithstanding the apparently conflicting evidence of the Ca2+-dependent manner of the N-terminal calmodulin-binding site from overlay assays. Unlike the two regulatory domains (the N terminus and the C2b region) discussed above, the C1b region does not have a free end, which suggests that the disruptions on this region could more easily change the activity of adenylyl cyclases. However, continuous deletions in the C1b region of ACVIII could not eliminate the Ca2+stimulation of ACVIII (Fig. 2 A), while, by contrast, a point mutation in this region of ACI abolished its Ca2+sensitivity (17Wu Z. Wong S.T. Storm D.R. J. Biol. Chem. 1993; 268: 23766-23768Abstract Full Text PDF PubMed Google Scholar). The different calmodulin-binding sites on ACVIII and ACI are underlined by some differences in their regulation by Ca2+/calmodulin; for instance, ACI is more sensitive to lower concentrations of Ca2+ than is ACVIII, and ACVIII is more stimulable by Ca2+/calmodulin than ACI (25Fagan K.A. Mahey R. Cooper D.M.F. J. Biol. Chem. 1996; 271: 12438-12444Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). The calmodulin-binding site in ACI is rich in basic amino acids (net charge is +7), and the binding is Ca2+-dependent (15Levin L.R. Reed R.R. J. Biol. Chem. 1995; 270: 7573-7579Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar,16Vorherr T. Knopfel L. Hofmann F. Mollner S. Pfeuffer T. Carafoli E. Biochemistry. 1993; 32: 6081-6088Crossref PubMed Scopus (139) Google Scholar). Since no hyperactivity was observed by mutating the C1b region on ACI (15Levin L.R. Reed R.R. J. Biol. Chem. 1995; 270: 7573-7579Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 17Wu Z. Wong S.T. Storm D.R. J. Biol. Chem. 1993; 268: 23766-23768Abstract Full Text PDF PubMed Google Scholar) and the movement of the C1b region is likely to be more restrained than those of the N terminus and C terminus, the mechanism of Ca2+/calmodulin regulation of ACI might be to stabilize the C1/C2 heterodimer, as has been proposed for forskolin and Gsαa (9Zhang G. Liu Y. Ruoho A.E. Hurley J.H. Nature. 1997; 386: 247-253Crossref PubMed Scopus (323) Google Scholar, 10Tesmer J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (670) Google Scholar), unlike the disinhibitory mechanism we have proposed for ACVIII. In conclusion, two calmodulin-binding sites exist on ACVIII, one (at the C terminus) is of profound regulatory significance, whereas the other (at the N terminus) plays a more minor role. Whether these two domains of ACVIII physically interact to share the same molecule of calmodulin remains to be determined in future studies. Given that the C1a and C2a regions clearly interact for catalytic activity (4Tang W.-J. Gilman A.G. Science. 1995; 268: 1769-1772Crossref PubMed Scopus (165) Google Scholar, 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, 7Sunahara R.K. Dessauer C.W. Whisnant R.E. Kleuss C. Gilman A.G. J. Biol. Chem. 1997; 272: 22265-22271Crossref PubMed Scopus (159) Google Scholar,9Zhang G. Liu Y. Ruoho A.E. Hurley J.H. Nature. 1997; 386: 247-253Crossref PubMed Scopus (323) Google Scholar, 10Tesmer J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (670) Google Scholar, 35Tang W.-J. Stanzel M. Gilman A.G. Biochemistry. 1995; 34: 14563-14572Crossref PubMed Scopus (109) Google Scholar), the tantalizing possibility that adenylyl cyclase could adopt a transporter-like structure (11Krupinski J. Coussen F. Bakalyar H.A. 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, 50Cooper D.M.F. Karpen J.W. Fagan K.A. Mons N.E. Adv. Second Messengers Phosphoprotein Res. 1998; 32: 23-51Crossref PubMed Scopus (74) Google Scholar) would be greatly strengthened by interactions between the N and C termini. We thank Drs. J. J. Cali and J. Krupinski for the cDNAs of three isoforms of ACVIII and two antibodies, Ab VIII-A 1229–1248 and Ab VIII-A 666–682, and Drs. J. W. Karpen, K. A. Fagan, and M. Yoshimura for comments on the manuscript." @default.
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W2045593899 title "Calmodulin-binding Sites on Adenylyl Cyclase Type VIII" @default.
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