Cdc42 and Rac stimulate exocytosis of secretory granules by activating the IP(3)/calcium pathway in RBL-2H3 mast cells.
We have expressed dominant-active and dominant-negative forms of the Rho GTPases, Cdc42 and Rac, using vaccinia virus to evaluate the effects of these mutants on the signaling pathway leading to the degranulation of secretory granules in RBL-2H3 cells. Dominant-active Cdc42 and Rac enhance antigen-stimulated secretion by about twofold, whereas the dominant-negative mutants significantly inhibit secretion. Interestingly, treatment with the calcium ionophore, A23187, and the PKC activator, PMA, rescues the inhibited levels of secretion in cells expressing the dominant-negative mutants, implying that Cdc42 and Rac act upstream of the calcium influx pathway. Furthermore, cells expressing the dominant-active mutants exhibit elevated levels of antigen-stimulated IP(3) production, an amplified antigen-stimulated calcium response consisting of both calcium release from internal stores and influx from the extracellular medium, and an increase in aggregate formation of the IP(3) receptor. In contrast, cells expressing the dominant-negative mutants display the opposite phenotypes. Finally, we are able to detect an in vitro interaction between Cdc42 and PLCgamma1, the enzyme immediately upstream of IP(3) formation. Taken together, these findings implicate Cdc42 and Rac in regulating the exocytosis of secretory granules by stimulation of IP(3) formation and calcium mobilization upon antigen stimulation.
Hong-Geller
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Department of Molecular Medicine, Cornell University, Ithaca, New York 14853, USA.
Cerione
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D000818:Animals; D015220:Calcium Channels / Q000378:metabolism; D020013:Calcium Signaling; D015550:Cell Degranulation; D002460:Cell Line; D002522:Chlorocebus aethiops; D003594:Cytoplasmic Granules / Q000378:metabolism*; D004140:Dinitrophenols / Q000378:metabolism / Q000494:pharmacology; D004533:Egtazic Acid / Q000378:metabolism / Q000494:pharmacology; D004789:Enzyme Activation; D005089:Exocytosis / Q000502:physiology*; D015870:Gene Expression; D005822:Genetic Vectors; D006160:Guanosine Triphosphate / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007527:Isoenzymes / Q000378:metabolism; D008407:Mast Cells / Q000187:drug effects / Q000378:metabolism*; D016296:Mutagenesis; D051966:Phospholipase C gamma; D011493:Protein Kinase C / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000378:metabolism / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism; D014616:Vaccinia virus; D020764:cdc42 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D015220:Calcium Channels; D004140:Dinitrophenols; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007527:Isoenzymes; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; C034638:dinitrophenyl-bovine serum albumin; D012710:Serum Albumin, Bovine; D004533:Egtazic Acid; D015544:Inositol 1,4,5-Trisphosphate; D006160:Guanosine Triphosphate; D011493:Protein Kinase C; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins