The Rab-binding protein Noc2 is associated with insulin-containing secretory granules and is essential for pancreatic beta-cell exocytosis.
The small GTPases Rab3 and Rab27 are associated with secretory granules of pancreatic beta-cells and regulate insulin exocytosis. In this study, we investigated the role of Noc2, a potential partner of these two GTPases, in insulin secretion. In the beta-cell line INS-1E wild-type Noc2, Noc265E, and Noc258A, a mutant capable of interacting with Rab27 but not Rab3, colocalized with insulin-containing vesicles. In contrast, two mutants (Noc2138S,141S and Noc2154A,155A,156A) that bind neither Rab3 nor Rab27 did not associate with secretory granules and were uniformly distributed throughout the cell cytoplasm. Overexpression of wild-type Noc2, Noc265E, or Noc258A inhibited hormone secretion elicited by insulin secretagogues. In contrast, overexpression of the mutants not targeted to secretory granules was without effect. Silencing of the Noc2 gene by RNA interference led to a strong impairment in the capacity of INS-1E cells to respond to insulin secretagogues, indicating that appropriate levels of Noc2 are essential for pancreatic beta-cell exocytosis. The defect was already detectable in the early secretory phase (0-10 min) but was particularly evident during the sustained release phase (10-45 min). Protein-protein binding studies revealed that Noc2 is a potential partner of Munc13, a component of the machinery that controls vesicle priming and insulin exocytosis. These data suggest that Noc2 is involved in the recruitment of secretory granules at the plasma membrane possibly via the interaction with Munc13.
Institut de Biologie Cellulaire et de Morphologie, University of Lausanne, Switzerland.
Séverine
S
Cheviet
Thierry
T
Coppola
Lee P
LP
Haynes
Robert D
RD
Burgoyne
Romano
R
Regazzi
D000818:Animals; D002458:Cell Fractionation; D002460:Cell Line; D005089:Exocytosis / Q000502:physiology*; D049452:Green Fluorescent Proteins; D019382:Human Growth Hormone / Q000235:genetics; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D047908:Intracellular Signaling Peptides and Proteins; D007515:Islets of Langerhans / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics; D016296:Mutagenesis; D009419:Nerve Tissue Proteins / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins; D034741:RNA, Small Interfering; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D022142:Secretory Vesicles / Q000378:metabolism*; D014162:Transfection; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000075745:rab27 GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism
D007328:Insulin; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D009419:Nerve Tissue Proteins; D011506:Proteins; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; C109307:Rph3al protein, rat; C485548:UNC13B protein, human; C103912:Unc13a protein, rat; D000075745:rab27 GTP-Binding Proteins; D019382:Human Growth Hormone; D049452:Green Fluorescent Proteins; C066694:RAB27A protein, human; C512961:Rab27a protein, rat; D020691:rab GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins