Expression and Functional Role of Syntaxin 1/HPC-1 in Pancreatic Cells

doi:10.1074/jbc.271.2.1160

Expression and Functional Role of Syntaxin 1/HPC-1 in Pancreatic Cells

SYNTAXIN 1A, BUT NOT 1B, PLAYS A NEGATIVE ROLE IN REGULATORY INSULIN RELEASE PATHWAY (*)

From the (1)Departments of Biochemistry and
(2)Physiology and
(3)Clinical Pathology and
(4)Internal Medicine (III) and
(5)Neurosurgery, Kyorin University School of Medicine, Mitaka, Tokyo 181, Japan

^§ To whom correspondence should be addressed:
Dept. of Biochemistry, Kyorin University School of Medicine, Shinkawa 6-20-2, Mitaka, Tokyo 181, Japan.
Fax: 81-422-41-6865.

Abstract

Syntaxin 1/HPC-1 is an integral membrane protein, which is thought to be implicated in the regulation of synaptic neurotransmitter release. We investigated syntaxin 1 expression in pancreatic β cells and the functional role of syntaxin 1 in the insulin release mechanism. Expression of syntaxin 1A, but not 1B, was detected in mouse isolated islets by the reverse transcriptase-polymerase chain reaction procedure. An immunoprecipitation study of metabolically labeled islets with an anti-rat syntaxin 1/HPC-1 antibody demonstrated syntaxin 1A protein with an apparent molecular mass of 35 kDa. Immunohistochemistry of the mouse pancreas demonstrated that syntaxin 1/HPC-1 was present in the plasma membranes of the islets of Langerhans. In order to determine the functional role of syntaxin 1 in pancreatic β-cells, rat syntaxin 1A or 1B was overexpressed in mouse βTC3 cells using the transient transfection procedure. Transfection of βTC3 cells with either syntaxin 1 resulted in approximately 7-fold increases in their immunodetectable protein levels. Glucose-stimulated insulin release by syntaxin 1A-overexpressing cells was suppressed to about 50% of the level in control cells, whereas insulin release by syntaxin 1B-overexpressing and control cells did not differ. Next, we established stable βTC3 cell lines that overexpressed syntaxin 1A and used them to evaluate the effect of syntaxin 1A on the regulatory insulin release pathway. Two insulin secretogogues, 4-β-phorbol 12-myristate 13-acetate or forskolin, increased insulin release by untransfected βTC3 cells markedly, but their effects were diminished in syntaxin 1A-overexpressing βTC3 cells. Glucose-unstimulated insulin release and the proinsulin biosynthetic rate were not affected by syntaxin 1A overexpression, indicating a specific role of syntaxin 1A in the regulatory insulin release pathway. Finally, in vitro binding assays showed that syntaxin 1A binds to insulin secretory granules, indicating an inhibitory role of syntaxin 1A in insulin exocytosis via its interaction with vesicular proteins. These results demonstrate that syntaxin 1A is expressed in the islets of Langerhans and functions as a negative regulator in the regulatory insulin release pathway.

Footnotes

↵* This study was supported in part by a grant-in-aid for Japan Private School Promotion Foundation (to S. N.) and by the Ciba-Geigy Foundation (Japan) for the Promotion of Science (to K. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

PCR

polymerase chain reaction

DMEM

Dulbecco's minimal essential medium

PAGE

polyacrylamide gel electrophoresis

SSC

standard saline citrate

VAMP

vesicle-associated membrane protein

SNAP-25

synaptosomal-associated protein of 25 kDa

NSF

N-ethylmaleimide-sensitive fusion protein

SNAP

soluble NSF attachment protein

IRI

insulin radioimmunoassay

FBS

fetal bovine serum

bp

base pair(s)

PBS

phosphate-buffered saline

MBP

maltose-binding protein.
↵²Y. Kushima, T. Fujiwara, and K. Akagawa, unpublished data.
³S. Nagamatsu, Y. Nakamichi, and H. Sawa, unpublished data.
- Received August 10, 1995.
- Revision received October 18, 1995.