Subcellular distribution and function of Rab3A, B, C, and D isoforms in insulin-secreting cells.
Insulin-secreting cells express four GTPases of the Rab3 family. After separation of extracts of INS-1 cells on a sucrose density gradient, the bulk of the A, B, and C isoforms was recovered in the fractions enriched in insulin-containing secretory granules. Rab3D was also mainly associated with secretory granules, but a fraction of this isoform was localized on lighter organelles. Analyses by confocal microscopy of immunostained HIT-T15 cells transfected with epitope-tagged constructs confirmed the distribution of the Rab3 isoforms. Transfection of HIT-T15 cells with GTPase-deficient mutants of the Rab3 isoforms decreased nutrient-induced insulin release to different degrees (D>B>A>>C), while overexpression of Rab3 wild types had minor or no effects. Expression of the same Rab3 mutants in PC12 cells provoked an inhibition of K+-stimulated secretion of dense core vesicles, indicating that, in beta-cells and neuroendocrine cells, the four Rab3 isoforms play a similar role in exocytosis. A Rab3A/C chimera in which the carboxyterminal domain of A was replaced with the corresponding region of C inhibited insulin secretion as Rab3A. In contrast, a Rab3C/A chimera containing the amino-terminal domain of C was less potent and reduced exocytosis as Rab3C. This suggests that the degree of inhibition obtained after transfection of the Rab3 isoforms is determined by differences in the variable amino-terminal region.
Iezzi
M
M
Département de Médecine Interne, Université de Genève, Geneva, Switzerland.
Escher
G
G
Meda
P
P
Charollais
A
A
Baldini
G
G
Darchen
F
F
Wollheim
C B
CB
Regazzi
R
R
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D003594:Cytoplasmic Granules / Q000378:metabolism; D003720:Densitometry; D004797:Enzyme-Linked Immunosorbent Assay; D005089:Exocytosis; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D016716:PC12 Cells; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D020692:rab3 GTP-Binding Proteins