An Important Functional Role of the N Terminus Domain of Type VI Adenylyl Cyclase in Gαi-mediated Inhibition

Abstract

The mammalian adenylyl cyclase (AC) 1 superfamily consists of nine membrane-bound isoforms. All of these possess three large cytosolic domains (designated N, C1a, and C2 domains; Fig. 1A), which are separated by two sets of six-transmembrane domains (1-3). The C1a and C2 domains among the nine AC members are highly homologous (with 50 -90% similarity in amino acids). In addition, the C1a and C2 domains of each AC share ϳ50% similarity and form the catalytic core complex, which can be stimulated by forskolin or G␣ s proteins (4 -6). Crystallographic analysis of the catalytic complex consisting of the C1a domain, the C2 domain, and the GTP␥S-bound G␣ s protein revealed that forskolin and/or G␣ s stimulate ACs by enhancing the interaction between the C1a and C2 domains and by stabilizing the C1a-C2 catalytic core complex (7).Although most ACs can be activated by G␣ s and forskolin, regulation of each AC isozyme differs. Studies of the morevariable N and C1b domains reveal that these two domains may play important regulatory roles. For example, the C1b domain has been implicated in the regulation of AC isozymes mediated by Ca 2ϩ /calmodulin, calcineurin, or protein kinase A (8 -11). In addition, the C1b domain of ACV and ACVII has been shown to modulate G␣ s -evoked activation by interacting with catalytic core complexes (12, 13). Others and we have shown that another variable region, the N-terminal domain, also significantly contributes to the regulation of AC activity (14, 15). Specifically, the N-terminal domain of ACVI (amino acids 1-160) plays an important role in the protein kinase C (PKC)-mediated inhibition and phosphorylation of ACVI (14). Removal of the first 86 amino acids (aa) of ACVI reduced the inhibitory effect of PKC on ACVI activity without affecting the basic enzymatic properties, including the affinities toward its substrate and two stimuli (forskolin and G␣ s protein (14)). The N-terminal domain of ACVI therefore functions as a regulatory domain. Further biochemical analyses revealed that at least four PKC phosphorylation sites (Ser-10, Ser-568, Ser-674, and Thr-931), located in the three large cytosolic domains of ACVI, significantly contribute to PKC-mediated inhibition of ACVI (16). Intramolecular interactions among the N, C1a/b, and C2 domains of ACVI therefore appear to be important for regulation of ACVI activity.G␣ i -mediated inhibition is a major regulatory feature of the AC superfamily. Among AC members, only ACI, ACV, and ACVI can effectively be inhibited by G␣ i proteins (17, 18). Based on results obtained from an in vitro binding assay, the C1a domain was shown to bind myristoylated G␣ i proteins and form stable complexes (19,20). Mutagenesis of full-length ACV in the ␣2 and ␣3 helices of the C1a domain revealed several residues important for the inhibition by G␣ i proteins. It was postulated that G␣ i may exert its inhibitory effect through binding to the C1a domain at the site just opposite the G␣ sbinding site on the C2 domain and, subsequently, causes reduced interaction between ...

Conditional Stimulation of Type V and VI Adenylyl Cyclases by G Protein βγ Subunits

“…However, it is becoming apparent that other regions of the AC molecule also participate in the regulation of its enzyme activity. For example, the N-terminal residues of ACVI has been shown to play a major role in the regulation of its activity by protein kinase C and G␣ i (22,23) and is also necessary for its interactions with Snapin (24), although the role of this latter interaction remains unknown. Similarly, the N terminus of ACVIII is necessary for modulation of its activity by capacitative Ca 2ϩ entry in intact cells (25).…”

“…The N-terminal segment of the different AC isoforms is also highly variable, and this region may play an important role in type-specific regulation of these enzymes. In ACVI, the N-terminal region is critical for both protein kinase C-mediated and G␣ i -mediated inhibition of the enzyme (22,23) as well as binding to Snapin (24), although the functional significance of this latter interaction remains unknown. Moreover, the N-terminal segment of ACVIII has been shown to play a critical role in regulating its activity by capacitative calcium entry in intact cells (25).…”

Modeling of Gαs and Gαi Regulation of Human Type V and VI Adenylyl Cyclase

“…The N terminus of human AC V also shares almost no homology with that of AC VI. Deletion of the N terminus in AC VI does not alter the regulation by G␣ s or forskolin or the sensitivity to P-site inhibitors (10). The N terminus of AC VI is the site of PKC inhibition of AC VI (22,35), and it has been suggested that the N terminus modulates G␣ i -mediated inhibition of AC VI via binding to the C 1a domain (10).…”

“…Deletion of the N terminus in AC VI does not alter the regulation by G␣ s or forskolin or the sensitivity to P-site inhibitors (10). The N terminus of AC VI is the site of PKC inhibition of AC VI (22,35), and it has been suggested that the N terminus modulates G␣ i -mediated inhibition of AC VI via binding to the C 1a domain (10). It is possible that the N terminus of AC V also plays a role in the regulation of activity and thus may provide the key to species differences for AC V and the differences in regulation of basal and G␣ s -stimulated activities for human AC V and VI.…”

“…For example, the N terminus of rat AC VI interacts with the C 1 domain to modulate G␣ i -mediated inhibition (10) and glycosylation of AC VI may alter regulatory properties of the enzyme (11). Although native and recombinant G␣ s have similar effects on the cytoplasmic domains of AC, activation of full-length AC is less effectively inhibited by G␣ i upon stimulation of native versus recombinant G␣ s (12).…”