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J. Biol. Chem., Vol. 276, Issue 38, 35450-35457, September 21, 2001

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N-Glycosylation and Residues Asn⁸⁰⁵ and Asn⁸⁹⁰ Are Involved in the Functional Properties of Type VI Adenylyl Cyclase^*

Gour-Cheng Wu, Hsing-Lin Lai^§, Ya-Wen Lin^§, Yin-Ting Chu^§, and Yijuang Chern^§¶

From the  Institute of Life Sciences, National Defense Medical Center, Taipei 104 and the ^§ Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, Republic of China

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 (Asn⁸⁰⁵ and Asn⁸⁹⁰, located on extracellular loops 5 and 6, respectively) also reduced the molecular mass of ACVI. Therefore, ACVI was glycosylated at both Asn⁸⁰⁵ and Asn⁸⁹⁰. 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-, Mn²⁺-, and Mg²⁺-stimulated cyclase activities than did wild-type ACVI. By contrast, the G_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 G_i protein-coupled receptor or by protein kinase C.

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