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Subcellular Distribution and Function of Rab3A, B, C, and D Isoforms in Insulin-Secreting Cells

Division de Biochimie Clinique (M.I., C.B.W.) Département de Médicine Interne and Département de Morphologie (P.M., A.C.) Université de Genève Geneva, Switzerland 1211
Institut de Biologie Cellulaire et de Morphologie (G.E., R.R.) Université de Lausanne Lausanne, Switzerland 1005
Department of Anatomy and Cell Biology (G.B.) Columbia University College of Physicians and Surgeons New York, New York 10032
Institut de Biologie Physico-Chimique (F.D.) Centre Nationale de la Recherche Scientifique ERS 575 Paris, France 75005

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 ß-cells and neuroendocrine cells, the four Rab3 isoforms play a similar role in exocytosis. A Rab3A/C chimera in which the carboxy-terminal 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.

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