Insights into the residence in lipid rafts of adenylyl cyclase AC8 and its regulation by capacitative calcium entry.
Adenylyl cyclases (ACs) are a family of critically important signaling molecules that are regulated by multiple pathways. Adenylyl cyclase 8 (AC8) is a Ca(2+) stimulated isoform that displays a selective regulation by capacitative Ca(2+) entry (CCE), the process whereby the entry of Ca(2+) into cells is triggered by the emptying of intracellular stores. This selectivity was believed to be achieved through the localization of AC8 in lipid raft microdomains, along with components of the CCE apparatus. In the present study, we show that an intact leucine zipper motif is required for the efficient N-linked glycosylation of AC8, and that this N-linked glycosylation is important to target AC8 into lipid rafts. Disruption of the leucine zipper by site-directed mutagenesis results in the elimination of N-glycosylated forms and their exclusion from lipid rafts. Mutants of AC8 that cannot be N-glycosylated are not demonstrably associated with rafts, although they can still be regulated by CCE; however, raft integrity is required for the regulation of these mutants. These findings suggest that raft localized proteins in addition to AC8 are needed to mediate its regulation by CCE.
Pagano
Mario
M
Dept. of Pharmacology, Univ. of Cambridge, Cambridge, CB2 1PD, United Kingdom.
Clynes
Michael A
MA
Masada
Nanako
N
Ciruela
Antonio
A
Ayling
Laura-Jo
LJ
Wachten
Sebastian
S
Cooper
Dermot M F
DM
D000262:Adenylyl Cyclases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002460:Cell Line; D006031:Glycosylation; D006801:Humans; D007527:Isoenzymes; D016350:Leucine Zippers; D021962:Membrane Microdomains / Q000187:drug effects / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021381:Protein Transport; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology
D007527:Isoenzymes; D014415:Tunicamycin; D000262:Adenylyl Cyclases; C118292:adenylyl cyclase 8; D002118:Calcium