Importance of the Rab3a-GTP binding domain for the intracellular stability and function of Rabphilin3a in secretion.
We had previously demonstrated that Rab3a-GTP inhibits and the Rab3a-binding protein Rabphilin3a enhances secretion in bovine chromaffin cells. In this study, we investigated the role of Rab3a-GTP binding in the intracellular expression and the function of Rabphilin3a in regulated exocytosis in bovine chromaffin cells. Using transient transfections, we found that a minimal domain, Rp(51-190), that inhibits secretion coincides with a minimal domain that effectively binds Rab3a-GTP and allows intracellular stability of the construct. This domain includes a cysteine-rich, Zn2+-binding domain whose integrity is also required for Rab3a-GTP binding and the ability to inhibit secretion. A Rabphilin3a mutant, containing both C2 domains but defective in Rab3a-GTP, and wild-type Rabphilin3a both localized to chromaffin granules and stimulated secretion similarly despite lessened intracellular expression of the mutant protein. The data are consistent with a sequence of events in which a Rab3a-GTP x Rabphilin3a complex forms on the secretory granule as a precursor in a pathway that enhances secretion. The complex dissociates (perhaps because of GTP hydrolysis) to permit the enhancement of secretion by Rabphilin3a.
Chung
S H
SH
Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109-0632, U.S.A.
Stabila
P
P
Macara
I G
IG
Holz
R W
RW
D000818:Animals; D001665:Binding Sites / Q000502:physiology; D001692:Biological Transport / Q000502:physiology; D002417:Cattle; D019439:Chromaffin Cells / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D003594:Cytoplasmic Granules / Q000737:chemistry / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D015870:Gene Expression / Q000502:physiology; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D016296:Mutagenesis / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D015032:Zinc / Q000378:metabolism; D020692:rab3 GTP-Binding Proteins
D009419:Nerve Tissue Proteins; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins; D015032:Zinc