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Submitted on January 22, 2003
Accepted on June 10, 2003
TC3 and INS-1 cells: Evidence for its Regulation by Rho Subfamily of G-Proteins
1 Department of Pharmaceutical Sciences and Departments of Pharmacology, Physiology, Radiology and Biomedical Engineering, Wayne State University and Cell Biochemistry Research Laboratory, John D. Dingell VA Medical Center, Detroit, MI 48201, John D. Dingell VA Medical Center and Argonne National Laboratory, and Cardiovascular Research Institute, National University Medical Institutes, National University of Singapore, 10 Medical Drive, Singapore 117597.
* To whom correspondence should be addressed. E-mail: akowluru{at}med.wayne.edu.
Mastoparan, a tetradecapeptide from wasp venom, stimulates insulin secretion from the islet 
cells, presumably via activation of trimeric G-proteins. Herein, we used Clostridial toxins, which selectively modify and inactivate 
subfamily of G-proteins, to examine whether mastoparan-induced insulin secretion also involves activation of these signaling proteins. Mastoparan, but not mastoparan 17 [an inactive analog of mastoparan], significantly stimulated insulin secretion from TC3 and INS-1 cells. Preincubation of TC3 cells with either Clostridium difficille toxin B, which inactivates , Cdc42 and Rac, or Clostridium sordellii toxin, which inactivates Ras, Rap, and Rac, markedly attenuated the mastoparan-induced insulin secretion, implicating Rac in this phenomenon. Mastoparan-stimulated insulin secretion was resistant to GGTI-2147, a specific inhibitor of geranylgeranylation of G-proteins [e.g. Rac], suggesting that mastoparan induces direct activation of Rac via GTP/GDP exchange. This was confirmed by a pull-down assay, which quantifies the binding of activated [i.e. GTP-bound] Rac to p21-activated kinase. However, glucose-induced insulin secretion from these cells was abolished by toxin B or GGTI-2147, suggesting that geranylgeranylation step is critical for glucose-stimulated secretion. Mastoparan significantly increased translocation of cytosolic Rac and Cdc42 to the membrane fraction. Confocal light microscopy revealed a substantial degree of colocalization of Rac [and Cdc42 to a lesser degree] with insulin in cells exposed to mastoparan. Further, stable expression of a dominant negative [N17Rac] form of Rac into INS-1 cells resulted in a significant reduction in mastoparan-stimulated insulin secretion from these cells. Taken together, our findings implicate G-proteins, specifically Rac, in mastoparan- induced insulin release.
Cell •
confocal light microscopy
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