title: Residence of adenylyl cyclase type 8 in caveolae is necessary but not sufficient for regulation by capacitative Ca(2+) entry.
abstract: Ca(2+)-sensitive adenylyl cyclases (ACs) depend on capacitative Ca(2+) entry (CCE) for their regulation. Residence of the endogenous Ca(2+)-inhibitable adenylyl cyclase of C6-2B glioma cells in cholesterol-enriched caveolae is essential for its regulation by CCE (Fagan, K. A., Smith, K. E., and Cooper, D. M. F. (2000) J. Biol. Chem. 275, 26530-26537). In the present study, we established that depletion of cellular cholesterol ablated the regulation by CCE of a Ca(2+)-stimulable adenylyl cyclase, AC8, heterologously expressed in HEK293 cells. We considered the possibility that a calmodulin-binding domain in the N terminus of AC8, which is not required for in vitro regulation by Ca(2+), might play a targeting role. Deletion and mutation of the N terminus did attenuate the enzyme's sensitivity to CCE without altering its in vitro responsiveness to Ca(2+)/calmodulin. Both N terminus-deleted AC8 and wild type AC8 were expressed at the plasma membrane, as shown by imaging analysis of green fluorescence protein-tagged constructs. However, not only wild type AC8 but also the CCE-insensitive mutants occurred in caveolar fractions of the plasma membranes, even though a Ca(2+)-insensitive adenylyl cyclase, AC7, was excluded from caveolae. Finally, the AC8 mutants were no more responsive to nonphysiological elevation of Ca(2+) than the wild type. We conclude that (i) not all adenylyl cyclases reside in caveolae, (ii) the calmodulin-binding domain in the N terminus of AC8 does not play a role in caveolar targeting, (iii) the N terminus does play a role in associating AC8 with factors that confer sensitivity to CCE, and (iv) residence of Ca(2+)-sensitive adenylyl cyclases in caveolae is essential but not sufficient for regulation by CCE.
authors.affiliation: Department of Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
authors.forename: Karen E
authors.initials: KE
authors.lastname: Smith
authors.forename: Chen
authors.initials: C
authors.lastname: Gu
authors.forename: Kent A
authors.initials: KA
authors.lastname: Fagan
authors.forename: Biao
authors.initials: B
authors.lastname: Hu
authors.forename: Dermot M F
authors.initials: DM
authors.lastname: Cooper
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D021941:Caveolae / Q000201:enzymology / Q000502:physiology*; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D016296:Mutagenesis; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D019284:Thapsigargin / Q000494:pharmacology; D014162:Transfection
chemical_list: D002121:Calcium Channel Blockers; D007093:Imidazoles; D011994:Recombinant Proteins; D005576:Colforsin; D015759:Ionomycin; D019284:Thapsigargin; D002784:Cholesterol; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; C118292:adenylyl cyclase 8; C063159:1-(2-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenylethyl)-1H-imidazole; D002118:Calcium