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

Mol Endocrinol. 1999 Feb;13(2):202-12. doi: 10.1210/mend.13.2.0228.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blotting, Western
  • Cells, Cultured
  • Centrifugation, Density Gradient
  • Cytoplasmic Granules / metabolism
  • Densitometry
  • Enzyme-Linked Immunosorbent Assay
  • Exocytosis
  • GTP-Binding Proteins / metabolism*
  • Humans
  • Image Processing, Computer-Assisted
  • Insulin / metabolism*
  • Insulin Secretion
  • Islets of Langerhans / cytology
  • Islets of Langerhans / metabolism*
  • Microscopy, Fluorescence
  • Mutagenesis, Site-Directed
  • PC12 Cells
  • Rats
  • Recombinant Fusion Proteins / metabolism
  • Transfection
  • rab3 GTP-Binding Proteins

Substances

  • Insulin
  • Recombinant Fusion Proteins
  • GTP-Binding Proteins
  • rab3 GTP-Binding Proteins