Abstract
In the present study, we used the N terminus (amino acids 1 approximately 160) 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 1 approximately 86 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(Delta33-51), 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-DeltaA87), 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.
Highlights
Adenylyl cyclases (ACs)1 are a family of enzymes that produce cyclic AMP from ATP upon extracellular stimu
Double immunostaining of ACVI and Snapin shown in the present study reveals the colocalization of ACVI and Snapin in the brain and in primary hippocampal neurons, supporting the physiological relevance of this interaction
Snapin Interaction with the N-terminal Domain of ACVI— the N terminus is variable among different AC members, it is highly conserved between species
Summary
Materials—Forskolin, cAMP, ATP, prostaglandin E1, thapsigargin, and essential amino acids were obtained from Sigma. The halves of gels that contained proteins larger than 35 kDa were transferred to polyvinylidene difluoride membranes, and the levels of MBP-Snapin and MBP were analyzed by the Western blot analysis using anti-MBP antiserum (New England BioLabs; 1: 10,000 dilution) and anti-SNA-c (1:5,000 dilution), respectively. For triple immunostaining with ACVI, Snapin, and MAP2 in primary hippocampal neurons, cells were blocked with 3% bovine serum albumin for 1 h, stained with a monoclonal anti-MAP2 antibody (1: 1000, Chemicon, Temecula CA) at 4 °C overnight, and incubated in a goat anti-mouse IgG conjugated to Cy5 (1:200, Jackson ImmunoResearch, West Grove, PA) for 2 h.
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