Scientific article
Open access

Subcellular distribution and function of Rab3A, B, C, and D isoforms in insulin-secreting cells

Published inMolecular endocrinology, vol. 13, no. 2, p. 202-212
Publication date1999

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.

  • Animals
  • Blotting, Western
  • Cells, Cultured
  • Centrifugation, Density Gradient
  • Cytoplasmic Granules/secretion
  • Densitometry
  • Enzyme-Linked Immunosorbent Assay
  • Exocytosis
  • GTP-Binding Proteins/metabolism
  • Humans
  • Image Processing, Computer-Assisted
  • Insulin/secretion
  • Islets of Langerhans/cytology/secretion
  • Microscopy, Fluorescence
  • Mutagenesis, Site-Directed
  • PC12 Cells
  • Rats
  • Recombinant Fusion Proteins/metabolism
  • Transfection
  • Rab3 GTP-Binding Proteins
Citation (ISO format)
IEZZI-BAKHTIARI, Mariella et al. Subcellular distribution and function of Rab3A, B, C, and D isoforms in insulin-secreting cells. In: Molecular endocrinology, 1999, vol. 13, n° 2, p. 202–212. doi: 10.1210/mend.13.2.0228
Main files (1)
Article (Published version)
ISSN of the journal0888-8809

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