Truncation of SNAP-25 reduces the stimulatory action of cAMP on rapid exocytosis in insulin-secreting cells.
Synaptosomal protein of 25 kDa (SNAP-25) is important for Ca(2+)-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.
Vikman
Jenny
J
Department of Clinical Sciences Lund, Biomedical Center, Lund University Diabetes Centre, Lund, Sweden.
Svensson
Hjalmar
H
Huang
Ya-Chi
YC
Kang
Youhou
Y
Andersson
Sofia A
SA
Gaisano
Herbert Y
HY
Eliasson
Lena
L
D000818:Animals; D019274:Botulinum Toxins, Type A / Q000494:pharmacology; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000494:pharmacology*; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics; D004351:Drug Resistance / Q000187:drug effects / Q000235:genetics; D005089:Exocytosis / Q000187:drug effects* / Q000235:genetics*; D017353:Gene Deletion; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006801:Humans; D050417:Insulin-Secreting Cells / Q000187:drug effects* / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D050825:Synaptosomal-Associated Protein 25 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D013997:Time Factors
D020662:Guanine Nucleotide Exchange Factors; D009419:Nerve Tissue Proteins; C531571:Rapgef4 protein, rat; C484282:Rims2 protein, rat; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D000242:Cyclic AMP; D019274:Botulinum Toxins, Type A