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This Article Full Text Full Text (PDF) All Versions of this Article: 297/2/E452    most recent 90585.2008v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Vikman, J. Articles by Eliasson, L. PubMed PubMed Citation Articles by Vikman, J. Articles by Eliasson, L.

Truncation of SNAP-25 reduces the stimulatory action of cAMP on rapid exocytosis in insulin-secreting cells

¹Lund University Diabetes Centre, ²Department of Clinical Sciences Lund, Biomedical Center, Lund; ³Department of Clinical Sciences Malmö, Clinical Research Centre, Malmö, Sweden; and ⁴Department of Medicine, University of Toronto, Toronto, Ontario, Canada

Submitted 11 July 2008 ; accepted in final form 1 June 2009

Synaptosomal protein of 25 kDa (SNAP-25) is important for Ca²⁺-dependent fusion of large dense core vesicles (LDCVs) in insulin-secreting cells. Exocytosis is further enhanced by cAMP-increasing agents such as glucagon-like peptide-1 (GLP-1), and this augmentation includes interaction with both PKA and cAMP-GEFII. To investigate the coupling between SNAP-25- and cAMP-dependent stimulation of insulin exocytosis, we have used capacitance measurements, protein-binding assays, and Western blot analysis. In insulin-secreting INS-1 cells overexpressing wild-type SNAP-25 (SNAP-25_WT), rapid exocytosis was stimulated more than threefold by cAMP, similar to the situation in nontransfected cells. However, cAMP failed to potentiate rapid exocytosis in INS-1 cells overexpressing a truncated form of SNAP-25 (SNAP-25_1-197) or Botulinum neurotoxin A (BoNT/A). Close dissection of the exocytotic response revealed that the inability of cAMP to stimulate exocytosis in the presence of a truncated SNAP-25 was confined to the release of primed LDCVs within the readily releasable pool, especially from the immediately releasable pool, whereas cAMP enhanced mobilization of granules from the reserve pool in both SNAP-25_1-197 (P < 0.01) and SNAP-25_WT (P < 0.05) cells. This was supported by hormone release measurements. Augmentation of the immediately releasable pool by cAMP has been suggested to act through the cAMP-GEFII-dependent, PKA-independent pathway. Indeed, we were able to verify an interaction between SNAP-25 with both cAMP-GEFII and RIM2, two proteins involved in the PKA-independent pathway. Thus we hypothesize that SNAP-25 is a necessary partner in the complex mediating cAMP-enhanced rapid exocytosis in insulin-secreting cells.

synaptosomal protein of 25 kDa; insulin; INS-1; cAMP-GEFII; Epac; capacitance measurements