A Link between Cdc42 and Syntaxin Is Involved in Mastoparan-Stimulated Insulin Release

Abstract

Mastoparan, a hormone receptor-mimetic peptide isolated from wasp venom, stimulates insulin release from pancreatic beta-cells in a Ca(2+)-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 beta HC-9 cells, an insulin-secreting mouse-derived cell line, resulted in a 2-fold increase in mastoparan-stimulated insulin release over vector-transfected beta HC-9 cells. This effect was not seen with secretagogues such as glucose that stimulate secretion via Ca(2+)-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.

A dual role for IQGAP1 in regulating exocytosis

“…First, our data affirmed a previous finding that stable expression of CDC42WT inhibits glucose-stimulated insulin secretion (Fig. 9A, last two columns) (Daniel et al, 2002) and further showed that expression of IQGAP1-F (F1) had no measurable effect on the inhibition (Fig. 9A, first columns).…”

“…This agrees with the finding that stable expression of CDC42, while enhancing mastoparanactivated CDC42 insulin secretion, inhibited glucose-stimulated secretion (Fig. 9) (Daniel et al, 2002). Mastoparan is a toxin from wasp venom that stimulates insulin release independent of Ca 2+ .…”

“…Early passages were used for insulin-secretion assays. Equal numbers of cells were grown and washed twice with Krebs-Ringer bicarbonate (KRB) buffer (129 mM NaCl, 5 mM NaHCO 3 , 4.8 mM KCL, 1.2 mM KH2PO 2 , 2.0 mM CaCl 2 , 1.2 mM MgSO 4 , 0.2% BSA, 10 mM HEPES, pH 7.4, and 0.1 mM glucose), incubated at 37°C in the same buffer for 30 minutes and treated with buffer alone (basal) or with 30 mM glucose (stimulated) as previously described (Daniel et al, 2002). Both sets were incubated at 37°C for another 30 minutes.…”

“…First, we found that IQGAP1, the exocyst and septins are abundant proteins in these cells, offering insulin secretion as a physiologically relevant functional assay for exocytosis. Second, mastoparan, a tetradecapeptide from wasp venom, was reported to enhance insulin secretion in ␤HC-9 cell lines that overexpress wild-type CDC42 by stimulating its exchange activity (Daniel et al, 2002). Mastoparan is known to stimulate exocytosis independent of Ca 2+ by activating G-proteins in a number of mammalian cell types, including ␤-cells (reviewed in Kowluru, 2003).…”

Dominant-Negative α-Subunit of Farnesyl- and Geranyltransferase Inhibits Glucose-Stimulated, but Not KCl-Stimulated, Insulin Secretion in INS 832/13 Cells

“…Original observations from multiple laboratories, including our own, demonstrated critical involvement of small G-proteins, such as Rac1, Cdc42, Rap1, and ARF6 (ADP-ribosylation factor 6) in GSIS from normal rat islets, human islets, and clonal ␤-cell preparations (3,(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). Such conclusions were primarily based on data from experiments using 1) inhibitors of requisite posttranslational modifications of these G-proteins; 2) clostridial toxins, which monoglucosylate and inactivate specific G-proteins; and 3) gene depletion (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). Despite this compelling evidence, the precise biochemical mechanisms underlying glucose-mediated activation of these signaling proteins leading to GSIS remain only partially understood.…”