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...

a b s t r a c tAdenylyl cyclase (AC) type VI (AC6) is a calcium-inhibitable enzyme which produces cAMP upon stimulation. Herein, we characterized the specific role of AC6 in the kidneys using two AC6-knockout mouse lines. Immunohistochemical staining revealed that AC6 exists in the tubular parts of the nephron and collecting duct. Activities of AC evoked by forskolin or a selective agonist of the V2 vasopressin receptor were lower in the kidneys of AC6-null mice compared to those of wildtype mice. Results of a metabolic cage assay and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) showed for the first time that AC6 plays a critical role in regulating water homeostasis.

3-5-Cyclic AMP (cAMP) is an important second messenger which regulates neurite outgrowth. We demonstrate here that type VI adenylyl cyclase (AC6), an enzyme which catalyzes cAMP synthesis, regulates neurite outgrowth by direct interaction with a binding protein (Snapin) of Snap25 at the N terminus of AC6 (AC6-N). We first showed that AC6 expression increased during postnatal brain development. In primary hippocampal neurons and Neuro2A cells, elevated AC6 expression suppressed neurite outgrowth, whereas the downregulation or genetic removal of AC6 promoted neurite extension. An AC6 variant (AC6-N5) that contains the N terminus of AC5 had no effect, indicating the importance of AC6-N. The downregulation of endogenous Snapin or the overexpression of a Snapin mutant (Snap ⌬33-51 ) that does not bind to AC6, or another Snapin mutant (Snapin S50A ) that does not interact with Snap25, reversed the inhibitory effect of AC6. Pulldown assays and immunoprecipitation-AC assays revealed that the complex formation of AC6, Snapin, and Snap25 is dependent on AC6-N and the phosphorylation of Snapin. The overexpression of Snap25 completely reversed the action of AC6. Collectively, in addition to cAMP production, AC6 plays a complex role in modulating neurite outgrowth by redistributing localization of the SNARE apparatus via its interaction with Snapin.

BackgroundThe type VI adenylyl cyclase (AC6) is a main contributor of cAMP production in the heart. The amino acid (aa) sequence of AC6 is highly homologous to that of another major cardiac adenylyl cyclase, AC5, except for its N-terminus (AC6-N, aa 1–86). Activation of AC6, rather than AC5, produces cardioprotective effects against heart failure, while the underlying mechanism remains to be unveiled. Using an AC6-null (AC6−/−) mouse and a knockin mouse with AC6-N deletion (AC6 ΔN/ΔN), we aimed to investigate the cardioprotective mechanism of AC6 in the heart.MethodsWestern blot analysis and immunofluorescence staining were performed to determine the intracellular distribution of AC6, AC6-ΔN (a truncated AC6 lacking the first 86 amino acids), and STAT3 activation. Activities of AC6 and AC6-ΔN in the heart were assessed by cAMP assay. Apoptosis of cardiomyocytes were evaluated by the TUNEL assay and a propidium iodine-based survival assay. Fibrosis was examined by collagen staining.ResultsImmunofluorescence staining revealed that cardiac AC6 was mainly anchored on the sarcolemmal membranes, while AC6-ΔN was redistributed to the sarcoplasmic reticulum. AC6ΔN/ΔN and AC6−/− mice had more apoptotic myocytes and cardiac remodeling than WT mice in experimental models of isoproterenol (ISO)-induced myocardial injury. Adult cardiomyocytes isolated from AC6ΔN/ΔN or AC6−/− mice survived poorly after exposure to ISO, which produced no effect on WT cardiomyocytes under the condition tested. Importantly, ISO treatment induced cardiac STAT3 phosphorylation/activation in WT mice, but not in AC6ΔN/ΔN and AC6−/− mice. Pharmacological blockage of PKA-, Src-, or STAT3- pathway markedly reduced the survival of WT myocytes in the presence of ISO, but did not affect those of AC6ΔN/ΔN and AC6−/− myocytes, suggesting an important role of AC6 in mediating cardioprotective action through the activation of PKA-Src-STAT3-signaling.ConclusionsCollectively, AC6-N controls the anchorage of cardiac AC6 on the sarcolemmal membrane, which enables the coupling of AC6 with the pro-survival PKA-STAT3 pathway. Our findings may facilitate the development of novel therapies for heart failure.Electronic supplementary materialThe online version of this article (doi:10.1186/s12929-017-0367-3) contains supplementary material, which is available to authorized users.

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