Caveolin-1 functions as a novel Cdc42 guanine nucleotide dissociation inhibitor in pancreatic beta-cells.
The cycling of the small Rho family GTPase Cdc42 is required for insulin granule exocytosis, although the regulatory proteins involved in Cdc42 cycling in pancreatic beta-cells are unknown. Here we demonstrate that the caveolar protein caveolin-1 (Cav-1) is a Cdc42-binding protein in beta-cells. Cav-1 associated with Cdc42-VAMP2-bound granules present near the plasma membrane under basal conditions. However, stimulation with glucose induced the dissociation of Cav-1 from Cdc42-VAMP2 complexes, coordinate with the timing of Cdc42 activation. Analyses of the Cav-1 scaffolding domain revealed a motif conserved in guanine nucleotide dissociation inhibitors (GDIs), which suggested a novel role for Cav-1 as a Cdc42 GDI in beta-cells. The novel role was further supported by: 1) in vitro binding analyses that demonstrated a direct interaction between Cav-1 and Cdc42; 2) GST-Cdc42 interaction assays showing preferential Cav-1 binding to GDP-Cdc42 over that of GTP-Cdc42; 3) Cav-1 depletion studies resulting in an inappropriate 40% induction of activated Cdc42 in the absence of stimuli and also a 40% increase in basal insulin release from both MIN6 cells and islets. Expression of wild-type Cav-1 in Cav-1-depleted cells restored basal level secretion to normal, whereas expression of a scaffolding domain mutant of Cav-1 failed to normalize secretion. Taken together, these data suggest that Cav-1 functions as a Cdc42 GDI in beta-cells, maintaining Cdc42 in an inactive state and regulating basal secretion in the absence of stimuli. Through its interaction with the Cdc42-VAMP2-bound insulin granule complex, Cav-1 may contribute to the specific targeting of granules to "active sites" of exocytosis organized by caveolae.
Nevins
Angela K
AK
Department of Biochemistry and Molecular Biology and Center for Diabetes Research, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
Thurmond
Debbie C
DC
D000595:Amino Acid Sequence; D000818:Animals; D051242:Caveolin 1 / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D002460:Cell Line; D005089:Exocytosis; D006147:Guanine / Q000737:chemistry; D050417:Insulin-Secreting Cells / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry; D013347:Subcellular Fractions / Q000378:metabolism; D050988:Vesicle-Associated Membrane Protein 2 / Q000378:metabolism; D020764:cdc42 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
D051242:Caveolin 1; D011994:Recombinant Proteins; D050988:Vesicle-Associated Membrane Protein 2; D006147:Guanine; D020764:cdc42 GTP-Binding Protein