The GTPase Rab3a negatively controls calcium-dependent exocytosis in neuroendocrine cells.

Abstract

There is accumulating evidence that small GTPases of the rab family regulate intracellular vesicle traffic along biosynthetic and endocytotic pathways in eukaryotic cells. It has been suggested that Rab3a, which is associated with synaptic vesicles in neurons and with secretory granules in adrenal chromaffin cells, might regulate exocytosis. We report here that overexpression in PC12 cells of Rab3a mutant proteins defective in either GTP hydrolysis or in guanine nucleotide binding inhibited exocytosis, as measured by a double indirect immunofluorescence assay. Moreover, injection of the purified mutant proteins into bovine adrenal chromaffin cells also inhibited exocytosis, as monitored by membrane capacitance measurements. Finally, the electrophysiological approach showed that bovine chromaffin cells which were intracellularly injected with antisense oligonucleotides targeted to the rab3a messenger exhibited an increasing potential to respond to repetitive stimulations. In contrast, control cells showed a phenomenon of desensitization. These results provide clear evidence that Rab3a is involved in regulated exocytosis and suggest that Rab3a is a regulatory factor that prevents exocytosis from occurring unless secretion is triggered. Furthermore, it is proposed that Rab3a is involved in adaptive processes such as response habituation.

Retrograde Transport of KDEL-bearing B-fragment of Shiga Toxin

“…Plasmids-To construct a plasmid expressing B-Glyc-KDEL and BGlyc-KDELGL, a two-step polymerase chain reaction-based strategy was adopted essentially as described previously (22). Polymerase chain reaction primers ShigaC-4 (for B-Glyc-KDEL; 5Ј-ACTAGCTCTGAAA-AGGATGAACTTTGAGAATTCTGACTCAGAATAGCTC-3Ј) or Shiga-C-5 (for B-Glyc-KDELGL; 5Ј-ACTAGCTCTGAAAAGGATGAACTTGG-TCTTTGAGAATTCTGACTCAGAATAGCTC-3Ј) and ShigaC-3 (5Ј-CT-TTTCAGAGCTAGTAGAATTAGGATGATAGCGGCCGCTACGAAAA-ATAACTTCGC-3Ј) were used with plasmid pSU108 (a generous gift from K. N. Timmis, Gesellschaft fü r biotechnologische Forschung, Braunschweig, Germany; (23)) specific primers ShigaAtpE (5Ј-CACTA-CTACGTTTTAAC-3Ј) and Shiga-fd (5Ј-CGGCGCAACTATCGG-3Ј) to produce fragments that were cloned into the SphI and SalI restriction sites of pSU108.…”

Co‐operative regulation of endocytosis by three RAB5 isoforms

“…For example, the Rab3 subgroup accounts for at least four isoforms (Rab3a, Rab3b, Rab3c and Rab3d) sharing high sequence identity (77-85%). However, while Rab3a appears to be only expressed in neurons and neuroendocrine cells and, together with Rab3c, regulates neurotransmitter release [7][8][9][10][11], Rab3b seems to be specifically expressed in polarized epithelial cells [12] and appears to have a divergent function *Corresponding author. Fax: (49) (6221) 387306.…”

Caenorhabditis elegans rab-3Mutant Synapses Exhibit Impaired Function and Are Partially Depleted of Vesicles

“…However, the reduction of rab3B levels using antisense oligonucleotides similarly inhibits evoked secretion from pituitary cells (Lledo et al, 1993). Conversely, rab3A antisense oligonucleotides increase evoked release on repetitive stimulation from adrenal chromaffin cells (Holz et al, 1994;Johannes et al, 1994). Finally, recent physiological experiments with cultured mouse hippocampal cells from rab3A mutants suggest that rab-3 negatively regulates a late step in release (Geppert et al, 1997).…”