A link between Cdc42 and syntaxin is involved in mastoparan-stimulated insulin release

Mastoparan, a hormone receptor-mimetic peptide isolated from wasp venom, stimulates insulin release from pancreatic beta-cells in a Ca2+-independent but GTP-dependent manner. In this report, the role of the Rho family GTP-binding protein Cdc42, in the mastoparan stimulus-secretion pathway, was examined. Overexpression of wild-type Cdc42 in betaHC-9 cells, an insulin-secreting mouse-derived cell line, resulted in a 2-fold increase in mastoparan-stimulated insulin release over vector-transfected betaHC-9 cells. This effect was not seen with secretagogues such as glucose that stimulate secretion via Ca2+- dependent pathways. GDP/GTP exchange assay data and studies with pertussis (PTX) toxin suggest that mastoparan may work directly to activate Cdc42 and not via PTX-sensitive heterotrimeric GTP-binding proteins. Using bacterial glutathione S-transferase-Cdc42 fusion proteins and co-immunoprecipitation and transient transfection studies, Cdc42 was shown to be an upstream regulator of the exocytotic protein, syntaxin. These results suggest that the GTP-dependent signal underlying the mastoparan effect acts at a "distal site" in stimulus-secretion coupling on one of the SNARE proteins essential for exocytosis. In vitro binding assays, using purified Cdc42 and syntaxin proteins, show that Cdc42 mediates the GTP signal through an indirect association with syntaxin. The H3 domain at the C-terminus of syntaxin, which participates in the formation of the ternary SNARE complex with the core proteins, SNAP-25 and synaptobrevin, is also required for the association with Cdc42. Thus, these studies indicate that Cdc42 could be a putative GTP-binding protein thought to be involved in the mastoparan-stimulated GTP-dependent pathway of insulin release.