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.