Comparison of the Effects on Secretion in Chromaffin and PC12 Cells of Rab3 Family Members and Mutants
EVIDENCE THAT INHIBITORY EFFECTS ARE INDEPENDENT OF DIRECT INTERACTION WITH RABPHILIN3*
- From the ‡Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0632 and the¶Center for Cell Signaling, University of Virginia, Charlottesville, Virginia 22908
Abstract
The Rab class of low molecular weight GTPases has been implicated in the regulation of vesicular trafficking between membrane compartments in eukaryotic cells. The Rab3 family consisting of four highly homologous isoforms is associated with secretory granules and synaptic vesicles. Many different types of experiments indicate that Rab3a is a negative regulator of exocytosis and that its GTP-bound form interacts with Rabphilin3, a possible effector. Overexpression of Rabphilin3 in chromaffin cells enhances secretion. We have investigated the expression, localization, and effects on secretion of the various members of the Rab3 family in bovine chromaffin and PC12 cells. We found that Rab3a, Rab3b, Rab3c, and Rab3d are expressed to varying degrees in PC12 cells and in a fraction enriched in chromaffin granule membranes from the adrenal medulla. Immunocytochemistry revealed that all members of the family when overexpressed in PC12 cells localize to secretory granules. Binding constants for the interaction of the GTP-bound forms of Rab3a, Rab3b, Rab3c, and Rab3d with Rabphilin3 were comparable (K d = 10–20 nm). Overexpression of each of the four members of the Rab3 family inhibited secretion. Mutations in Rab3a were identified that strongly impaired the ability of the GTP-bound form to interact with Rabphilin3. The mutated proteins inhibited secretion similarly to wild type Rab3a. Although Rab3a and Rabphilin3 are located on the same secretory granule or secretory vesicle and interact both in vitro and in situ,it is concluded that the inhibition of secretion by overexpression of Rab3a is unrelated to its ability to interact with Rabphilin3.
Footnotes
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↵* This work was supported by Grants RO1 DK50127 (to R. W. H.) and CA-56300 (to I. G. M.) from the National Institutes of Health, and by a grant from the Student Biomedical Research Program, University of Michigan Medical School (to E. A. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ Current address: Cardiovascular Biology Laboratory, Harvard School of Public Health, Boston, MA 02115.
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↵‖ To whom correspondence should be addressed: Dept. of Pharmacology, University of Michigan Medical School, 1301 MSRB III, Ann Arbor, MI 48109-0632.
- Abbreviations:
- hGH
-
human growth hormone
- GH
-
growth hormone
- HA
-
hemagglutinin
- PMSF
-
phenylmethylsulfonyl fluoride
- GTPγS
-
guanosine 5′-O-(thiotriphosphate)
- DMPP
-
dimethylphenylpiperazinium
- PAGE
-
polyacrylamide gel electrophoresis
- DTT
-
dithiothreitol
- CMV
-
cytomegalovirus
- SNARE
-
solubleN-ethylmaleimide-sensitive factorattachment protein receptor
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- Received January 22, 1999.
- Revision received March 30, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
