N-glycosylation and residues Asn805 and Asn890 are involved in the functional properties of type VI adenylyl cyclase.
In this study, we demonstrate that type VI adenylyl cyclase (ACVI) is glycosylated in vivo. Treating HEK293 cells expressing ACVI with tunicamycin to block the addition of N-linked oligosaccharide or removing the N-linked oligosaccharide by in vitro peptidyl-N-glycosidase F digestion reduced the molecular mass of ACVI. Furthermore, tunicamycin treatment suppressed the forskolin-stimulated activity of ACVI. Mutation of either one or both potential N-glycosylation sites (Asn(805) and Asn(890), located on extracellular loops 5 and 6, respectively) also reduced the molecular mass of ACVI. Therefore, ACVI was glycosylated at both Asn(805) and Asn(890). Confocal analysis indicated that glycosylation was not required for the delivery of ACVI to the cell surface. Although no significant alterations in K(m) values for ATP or sensitivity to divalent cations were detected, the glycosylation-deficient ACVI mutant N805Q/N890Q-ACVI exhibited much lower forskolin-, Mn(2+)-, and Mg(2+)-stimulated cyclase activities than did wild-type ACVI. By contrast, the Galpha(s)-stimulated cyclase activities of wild-type ACVI and N805Q/N890Q-ACVI were indistinguishable. Furthermore, compared with wild-type ACVI, N805Q/N890Q-ACVI was less sensitive to inhibition mediated by dopamine D2 receptors or by protein kinase C. Collectively, glycosylation of ACVI not only affected its catalytic activity in an activator-dependent manner, but also altered its ability to be regulated by a Galpha(i) protein-coupled receptor or by protein kinase C.
Wu
G C
GC
Institute of Life Sciences, National Defense Medical Center, Taipei 104, Taiwan, Republic of China.
Lai
H L
HL
Lin
Y W
YW
Chu
Y T
YT
Chern
Y
Y
D000262:Adenylyl Cyclases / Q000737:chemistry / Q000378:metabolism*; D001216:Asparagine / Q000378:metabolism*; D002384:Catalysis; D002460:Cell Line; D006031:Glycosylation; D006801:Humans; D007150:Immunohistochemistry; D011493:Protein Kinase C / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism
D011994:Recombinant Proteins; D001216:Asparagine; D011493:Protein Kinase C; D000262:Adenylyl Cyclases