Disruption of Rab3-calmodulin interaction, but not other effector interactions, prevents Rab3 inhibition of exocytosis.
Rab GTPases regulate membrane traffic between the cellular compartments of eukaryotic cells. Rab3 is associated with secretory vesicles of neuronal and endocrine cells and controls the Ca(2+)-triggered release of neurotransmitters and hormones. To clarify the mode of action of Rab3 we generated mutants of the GTPase that do not interact efficiently with its putative effectors Rabphilin and RIM. Surprisingly, these mutants transfected in PC12 cells were still capable of inhibiting Ca(2+)-evoked secretion. Rab3 was shown previously to bind to calmodulin in a Ca(2+)-dependent manner. By replacing two arginines conserved between Rab3 isoforms, we generated a mutant with a reduced affinity for calmodulin. This mutant retained the capacity to interact with the Rab3 regulatory proteins, Rabphilin, RIM, Mss4 and RabGDI, and was correctly targeted to dense-core secretory granules. However, replacement of the two arginines abolished the ability of the GTP-bound form of Rab3 to inhibit exocytosis of catecholamine- and insulin-secreting cells. We propose that a Rab3-calmodulin complex generated by elevated Ca(2+) concentrations mediated at least some of the effects of the GTPase and limited the number of exocytotic events that occurred in response to secretory stimuli.
Coppola
T
T
Institut de Biologie Cellulaire et de Morphologie, University of Lausanne, Rue de Bugnon 9, 1005 Lausanne, Switzerland.
Perret-Menoud
V
V
Lüthi
S
S
Farnsworth
C C
CC
Glomset
J A
JA
Regazzi
R
R
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism*; D003594:Cytoplasmic Granules / Q000378:metabolism; D005089:Exocytosis / Q000235:genetics*; D019204:GTP-Binding Proteins / Q000378:metabolism; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors; D008856:Microscopy, Fluorescence; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000378:metabolism; D016716:PC12 Cells; D011485:Protein Binding / Q000235:genetics; D011506:Proteins / Q000378:metabolism; D051381:Rats; D014162:Transfection; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D020692:rab3 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000097624:Rabphilin-3A
D048868:Adaptor Proteins, Signal Transducing; D002118:Calcium; D002147:Calmodulin; D019204:GTP-Binding Proteins; D020730:Guanine Nucleotide Dissociation Inhibitors; D020662:Guanine Nucleotide Exchange Factors; D009419:Nerve Tissue Proteins; D011506:Proteins; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins; D000097624:Rabphilin-3A; C086796:GDP dissociation inhibitor 1; C079550:Rabif protein, rat; C107293:Rim protein, mammalian; C491489:Rims1 protein, rat