Regulation of Type VI Adenylyl Cyclase by Snapin, a SNAP25-binding Protein

Abstract

In the present study, we used the N terminus (amino acids 1ϳ160) of type VI adenylyl cyclase (ACVI) as bait to screen a mouse brain cDNA library and identified Snapin as a novel ACVI-interacting molecule. Snapin is a binding protein of SNAP25, a component of the SNARE complex. Co-immunoprecipitation analyses confirmed the interaction between Snapin and full-length ACVI. Mutational analysis revealed that the interaction domains of ACVI and Snapin were located within amino acids 1ϳ86 of ACVI and 33-51 of Snapin, respectively. Co-localization of ACVI and Snapin was observed in primary hippocampal neurons. Moreover, expression of Snapin specifically eliminated protein kinase C (PKC)-mediated suppression of ACVI, but not that of cAMP-dependent protein kinase (PKA) or calcium. Mutation of the potential PKC and PKA phosphorylation sites of Snapin did not affect the ability of Snapin to reverse the PKC inhibitory effect on ACVI. Phosphorylation of Snapin by PKC or PKA therefore might not be crucial for Snapin action on ACVI. In contrast, Snapin ⌬33-51 , which harbors an internal deletion of amino acids 33-51 did not affect PKC-mediated inhibition of ACVI, supporting that amino acids 33-51 of Snapin comprises the ACVI-interacting region. Consistently, Snapin exerted no effect on PKC-mediated inhibition of an ACVI mutant (ACVI-⌬A87), which lacked the Snapin-interacting region (amino acids 1-86). Snapin thus reverses its action via direct interaction with the N terminus of ACVI. Collectively, we demonstrate herein that in addition to its association with the SNARE complex, Snapin also functions as a regulator of an important cAMP synthesis enzyme in the brain. Adenylyl cyclases (ACs)1 are a family of enzymes that produce cyclic AMP (cAMP) from ATP upon extracellular stimulation. To date, at least 9 membrane-bound ACs have been isolated and characterized (1). These enzymes are capable of integrating positive and negative signals that act directly through stimulation of G protein-coupled receptors (GPCRs) or indirectly via intracellular signaling molecules in isozyme-specific patterns. In addition, the regulatory properties and expression patterns of different AC isoforms greatly diverge and may account for the distinctive cell-and tissue-specific responsiveness of ACs. Recently, several different proteins, including RGS2 and the protein associated with Myc (PAM), have been shown to interact and modulate activity of different AC isozymes (2, 3), adding additional dimensions to the isozymespecific regulation of the AC superfamily.Except for the newly identified soluble AC, all membranebound AC members share a primary structure consisting of 12 transmembrane regions and 3 large cytoplasmic domains (N, C1a/b, and C2). The C1a and C2 domains, which form the catalytic core complex, are highly conserved and are homologous to each other. The N-terminal domains of ACs, in contrast, are variable among ACs, and have been demonstrated to play mostly regulatory roles (4, 5). Among the AC isozymes, ACVI is of particular interest, because...

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).…”

Adenylyl cyclase AC8 directly controls its micro-environment by recruiting the actin cytoskeleton in a cholesterol-rich milieu

“…Organization of the immediate downstream targets of cAMP, namely protein kinase A (PKA), exchange protein activated by cAMP (EPAC) and cyclic nucleotide-gated (CNG) channels, offers an additional level of control. However, recent studies on the binding by adenylyl cyclases (ACs) of various regulatory proteins such as PKA, A-kinase anchoring proteins (AKAPs) (Bauman et al, 2006;Efendiev et al, 2010;Willoughby et al, 2010b), protein phosphatase 2A (PP2A) , snapin (Chou et al, 2004) and phosphodiesterases (PDEs) (Halls and Cooper, 2010) indicate that ACs can to a large extent dictate their micro-environmental milieu. Consequently, the placement and compartmentalization of ACs might provide a primary level of organization of cAMP signalling cascades.…”

Distinct pools of cAMP centre on different isoforms of adenylyl cyclase in pituitary-derived GH3B6 cells

“…Experimental evidence is accumulating for the existence of cAMP microdomains in which cAMP targets are discretely and selectively regulated in a manner that bears little relation to changes in gross cellular levels of cAMP (Bacskai et al, 1993;Hempel et al, 1996;Nikolaev et al, 2006;Rich et al, 2001;Zaccolo and Pozzan, 2002). Recent studies suggest that adenylyl cyclases (ACs), the originators of cAMP, can actually orchestrate their own microenvironment by recruiting a variety of signalling and scaffolding molecules (Bauman et al, 2006;Chou et al, 2004;Crossthwaite et al, 2006;Dupre et al, 2007;Piggott et al, 2008). Thus, the nine differently regulated isoforms of AC (Sunahara and Taussig, 2002) can also contribute significantly to the diversity of cellular cAMP microdomains.…”