Characterization of small-molecular-mass guanine-nucleotide-binding regulatory proteins in insulin-secreting cells and PC12 cells

Details

Serval ID
serval:BIB_E7DD09E287D6
Type
Article: article from journal or magazin.
Collection
Publications
Title
Characterization of small-molecular-mass guanine-nucleotide-binding regulatory proteins in insulin-secreting cells and PC12 cells
Journal
European Journal of Biochemistry
Author(s)
Regazzi  R., Vallar  L., Ullrich  S., Ravazzola  M., Kikuchi  A., Takai  Y., Wollheim  C. B.
ISSN
0014-2956 (Print)
Publication state
Published
Issued date
09/1992
Volume
208
Number
3
Pages
729-37
Notes
In Vitro
Journal Article
Research Support, Non-U.S. Gov't --- Old month value: Sep 15
Abstract
The distribution of ras-related small-molecular-mass guanine-nucleotide-binding regulatory proteins (SMG) of two insulin-secreting cell lines, RINm5F and HIT-T15, and of a catecholamine-secreting cell line, PC12, have been studied using different techniques. About ten such proteins were detected by [32P]GTP binding after two-dimensional gel electrophoresis and transfer to nitrocellulose membranes. In insulin-secreting cells, rho protein(s) that cannot be detected with the GTP-binding technique were identified by ADP ribosylation with Clostridium botulinum C3 exoenzyme. After subcellular fractionation, SMG displayed specific distributions. The insulin-secreting cell line RINm5F and the catecholamine-secreting cell line PC12 expressed a similar set of these proteins with analogous localization. [32P]GTP binding analysis revealed that at least seven SMG were associated with the secretory granule enriched fraction of RINm5F cells and with the fraction containing dense secretory granules from PC12 cells, proteins of 27 (pI 5.4), 23 (pI 6.8) and 25 kDa (pI 6.7) being the most abundant. These proteins were present in a highly purified granule fraction of a solid rat insulinoma. The 23 kDa (pI 6.8) and 25 kDa (pI 6.7) proteins, but not the protein migrating at 27 kDa (pI 5.4), were detected in the corresponding fraction from HIT-T15 cells. A monoclonal antibody directed against smg25A/rab3A recognized the SMG in secretory granules migrating at 25 kDa (pI 6.7) and 27 kDa (pI 5.4). This antibody also revealed the presence of such protein(s) in homogenates of rat pancreatic islets. During stimulation of insulin secretion of either intact or permeabilized cells, there was no detectable redistribution to the cytosol or to the plasma membrane of the major proteins located on secretory granules. In view of the invariable presence of at least two of the SMG in granules of secretory cells, these proteins are good candidates for regulation of hormone secretion.
Keywords
Adenosine Diphosphate Ribose/metabolism Animals Blotting, Western Electrophoresis, Gel, Two-Dimensional *Exocytosis GTP-Binding Proteins/analysis/*metabolism Guanosine Triphosphate/metabolism Insulin/*secretion Insulinoma/chemistry/metabolism Islets of Langerhans/chemistry/metabolism Nerve Tissue Proteins/metabolism PC12 Cells/*chemistry/metabolism Rats Subcellular Fractions/chemistry Tumor Cells, Cultured rab3 GTP-Binding Proteins
Pubmed
Web of science
Open Access
Yes
Create date
24/01/2008 14:30
Last modification date
20/08/2019 16:10
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