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

Abstract

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 G␣ s -stimulated cyclase activities of wildtype 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 proteincoupled receptor or by protein kinase C. Adenylyl cyclases (ACs)1 produce cAMP in response to extracellular stimulation. To date, at least nine mammalian membrane-bound ACs have been isolated and characterized (1). All membrane-bound ACs contain two hydrophobic regions that comprise six transmembrane helices and three large cytoplasmic domains (N, C1a/b, and C2) (see Fig. 1). The basic catalytic unit is very conserved among ACs and is composed of the C1a and C2 domains (2-4). The ␣-subunit of the G s protein (G␣ s ), which activates all membrane-bound ACs, at least in vitro, was shown to interact with the catalytic complex of ACs at the C2 domain (5, 6). On the contrary, the ␣-subunit of the G i protein (G␣ i ) only effectively suppresses some of these membranebound AC isozymes (i.e. type I, V, and VI ACs) (7). The G␣ i protein was reported to interact with the C1 domain of type V AC (3). The N-terminal domains of ACs are highly variable and were shown to play modulating roles (9, 10). A recent study by Chen et al. (11) suggests that the two tandem six-transmembrane segments are critical for the targeting and functional assembly of ACs. The regulatory roles of the transmembrane domains and of the six extracellular loops of ACs remain largely uncharacterized.Members of the AC superfamily are tightly controlled by various signals and therefore contribute significantly to the complexity and fine-tuning of cellular signaling mechanisms (12). We are particularly interested in type VI AC (ACVI) because we (9, 13) and others (14, 15) have shown that ACVI can be regulated...

Particulate and soluble adenylyl cyclases participate in the sperm acrosome reaction

“…4B) with different relative mobility (Mr ± standard error: AC1, 157 ± 0.38; AC2, 113 ± 1.14; AC5/6, 250 ± 0 and AC9, 104 ± 1.31; n = 8-17 SDS-PAGEs). Since most mammalian AC enzymes are glycosylated [33], the apparent Mr could be higher than the real MW. We frequently observed protein degradation in the head membrane fractions (see anti-AC1 in Fig.…”

N-Glycan Remodeling on Glucagon Receptor Is an Effector of Nutrient Sensing by the Hexosamine Biosynthesis Pathway

“…Dimerization of the GLP-1R promotes coupling with G protein-coupled receptors and sensitivity to ligand (53). All nine mammalian adenylyl cyclases share conserved N-glycosylation sites in extracellular loops 5 and 6 (54). N-Glycosylation of adenylyl cyclase 8 is required to target the enzyme into lipid raft domains (55), and Golgi modifications may play a role in efficient coupling of this adenylyl cyclase with Gcgr.…”

Role of adenylyl cyclase 6 in the development of lithium-induced nephrogenic diabetes insipidus

“…Therefore, to isolate a potential role of AC6 in kidney and CD PCs, the main "target" cell for Li + effects (24), we generated a condition- al mouse model with AC6 deletion controlled by AQP2-dependent Cre recombinase expression (25). After several attempts with antibodies from various commercial sources, we identified an antibody that, in cortex homogenates from WT mice, detected AC6 as a smear centered at 150 kDa (26,27). No signal was detectable in AC6 -/-mice, demonstrating for the first time to our knowledge specificity of an AC6 antibody in Western blots (Figure 2A).…”