Comparison of the Effects on Secretion in Chromaffin and PC12 Cells of Rab3 Family Members and Mutants

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 GTPbound 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.

A Direct Inhibitory Role for the Rab3-specific Effector, Noc2, in Ca2+-regulated Exocytosis in Neuroendocrine Cells

“…Noc2 binding was specific for Rab3A, as the endosome-specific Rab5B failed to associate in the presence of GTP␥S. The affinity of the interaction (ϳ500 nm) was lower than that reported for Rabphilin binding to Rab3A of 150 nM (43) or 20 nM (33). This may explain the low efficiency of binding in the in vitro assay that we observed.…”

“…This effect is not directly mediated by the interaction with Rab3A, as point mutants in Rabphilin3A having defective Rab3A binding still enhance secretion from an endocrine cell line (31). In a reciprocal study (33), Rab3A mutants defective in Rabphilin3A binding retained the wild type ability to inhibit secretion. Further evidence for a Rab3A-independent effect of Rabphilin 3A comes from studies using Rabphilin 3A knockout mice (34).…”

“…Overexpression of Rabphilin 3A (30) or an N-terminal fragment of RIM stimulates exocytosis (27). The use of mutagenesis and analysis of gene knockouts has ruled out a requirement for Rab3A interaction for these effects and have eliminated Rabphilin 3A or RIM as the inhibitory effectors of Rab3A (31)(32)(33)(34). Another candidate Rab3A-interacting protein (35) that has been suggested to be involved in the inhibition of exocytosis (32) is calmodulin.…”

Rab8b and Its Interacting Partner TRIP8b Are Involved in Regulated Secretion in AtT20 Cells

“…These results suggest that Rab8a is not involved in the regulated secretory pathway that involves the apical or axonal surfaces. Furthermore, studies have demonstrated that overexpression of Rab8a has no effect on the regulated secretion of human growth hormone (37). In contrast, the overexpression of Rab8b had a significant stimulatory effect on the regulated secretion of ACTH that occurs at the tips of the cell processes (13,38).…”

“…The Rab3 family consists of four highly homologous isoforms associated with secretory granules and synaptic vesicles. Overexpression of each of the four members of the Rab3 family inhibited secretion (37). Moreover, knockout of Rab3A increased the quantal release of synaptic vesicles (39).…”