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 (Kd = 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.
Highlights
The Rab class of low molecular weight GTPases has been implicated in the regulation of vesicular trafficking between membrane compartments in eukaryotic 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
Members of the Rab3 Family Are Enriched into Chromaffin Granule Fraction and Are Expressed in PC12 Cells—Antibodies that were specific for each of the Rab3 GTPases demonstrated that Rab3a, Rab3b, Rab3c, and Rab3d are enriched in a chromaffin granule membrane fraction from the adrenal medulla and are expressed to varying degrees in PC12 cells
Summary
The Rab class of low molecular weight GTPases has been implicated in the regulation of vesicular trafficking between membrane compartments in eukaryotic cells. The Rab 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 Rabphilin, a possible effector. We have investigated the expression, localization, and effects on secretion of the various members of the Rab family in bovine chromaffin and PC12 cells. Overexpression of each of the four members of the Rab family inhibited secretion. The inhibition of secretion caused by overexpression of Rab3a is consistent with the enhancement of secretion from hippocampal neurons in Rab3a knockout mice [17] and the enhancement of secretion by injection into chromaffin cells of antisense rab3a oligonucleotide [16]
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