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

Vikman, Jenny LU ; Svensson, Hjalmar; Huang, Ya-Chi; Kang, Youhou; Andersson, Sofia A LU ; Gaisano, Herbert and Eliasson, Lena LU (2009) In American Journal of Physiology: Endocrinology and Metabolism 297. p.452-461
Abstract
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 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-25WT) rapid exocytosis was stimulated >3-fold by cAMP, similar to the situation in non-transfected cells. However, cAMP failed to potentiate rapid exocytosis in INS-1 cells overexpressing a truncated form of SNAP-25 (SNAP-251-197)... (More)
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 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-25WT) rapid exocytosis was stimulated >3-fold by cAMP, similar to the situation in non-transfected cells. However, cAMP failed to potentiate rapid exocytosis in INS-1 cells overexpressing a truncated form of SNAP-25 (SNAP-251-197) or Botulinum neurotoxin A (BoNT/A). Close dissection of the exocytotic response revealed that the inability of cAMP to stimulate exocytosis in presence of a truncated SNAP-25 was confined to the release of primed LDCVs within the Readily Releasable Pool (RRP), especially from the Immediately Releasable Pool (IRP), whereas cAMP enhanced mobilization of granules from the Reserve Pool (RP) in both SNAP-251-197 (P<0.01) and SNAP-25WT (P<0.05) cells. This was supported by hormone release measurements. Augmentation of IRP 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. Key words: cAMP, SNAP-25, insulin, INS-1. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
American Journal of Physiology: Endocrinology and Metabolism
volume
297
pages
452 - 461
publisher
American Physiological Society
external identifiers
  • wos:000268252700020
  • pmid:19509185
  • scopus:68049096966
ISSN
1522-1555
DOI
10.1152/ajpendo.90585.2008
language
English
LU publication?
yes
id
0076a826-aa52-46ed-8f88-90437fb3cbf3 (old id 1434357)
alternative location
http://www.ncbi.nlm.nih.gov/pubmed/19509185?dopt=Abstract
date added to LUP
2009-07-03 14:40:24
date last changed
2017-10-01 05:03:13
@article{0076a826-aa52-46ed-8f88-90437fb3cbf3,
  abstract     = {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 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-25WT) rapid exocytosis was stimulated &gt;3-fold by cAMP, similar to the situation in non-transfected cells. However, cAMP failed to potentiate rapid exocytosis in INS-1 cells overexpressing a truncated form of SNAP-25 (SNAP-251-197) or Botulinum neurotoxin A (BoNT/A). Close dissection of the exocytotic response revealed that the inability of cAMP to stimulate exocytosis in presence of a truncated SNAP-25 was confined to the release of primed LDCVs within the Readily Releasable Pool (RRP), especially from the Immediately Releasable Pool (IRP), whereas cAMP enhanced mobilization of granules from the Reserve Pool (RP) in both SNAP-251-197 (P&lt;0.01) and SNAP-25WT (P&lt;0.05) cells. This was supported by hormone release measurements. Augmentation of IRP 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. Key words: cAMP, SNAP-25, insulin, INS-1.},
  author       = {Vikman, Jenny and Svensson, Hjalmar and Huang, Ya-Chi and Kang, Youhou and Andersson, Sofia A and Gaisano, Herbert and Eliasson, Lena},
  issn         = {1522-1555},
  language     = {eng},
  pages        = {452--461},
  publisher    = {American Physiological Society},
  series       = {American Journal of Physiology: Endocrinology and Metabolism},
  title        = {Truncation of SNAP-25 reduces the stimulatory action of cAMP on rapid exocytosis in insulin-secreting cells.},
  url          = {http://dx.doi.org/10.1152/ajpendo.90585.2008},
  volume       = {297},
  year         = {2009},
}