Activated mutant of G alpha(12) enhances the hyperosmotic stress response of NIH3T3 cells

Heterotrimeric G protein G12 stimulates diverse physiological responses inc luding the activities of Na+/H+ exchangers and Jun kinases. We have observe d that the expression of the constitutively activated, GTPase-deficient mut ant of G alpha (12) (G alpha (12)QL) accelerates the hyperosmotic response of NIH3T3 cells as monitored by the hyperosmotic stress-stimulated activity of JNK1. The accelerated response appears to be partly due to the increase d basal activity of JNK since cell lines-such as NIH3T3 cells expressing JN K1-in which JNK activity is elevated, show a similar response. NIH3T3 cells expressing G alpha (12)QL also display heightened sensitivity to hyperosmo tic stress. This is in contrast to JNK1-NIH3T3 cells that failed to enhance sensitivity although they do exhibit an accelerated hyperosmotic response. Reasoning that the increased sensitivity seen in G alpha (12)QL cells is d ue to a signaling component other than JNK, the effect of dimethyamiloride, an inhibitor of Na+/H+ exchanger in this response, was assessed. Treatment of vector control NIH3T3 cells with 50 muM dimethylamiloride potently inhi bited their hyperosmotic response whereas the response was only partially i nhibited in G alpha (12)QL-NIH3T3 cells. These results, for the first time, identify that NHEs are upstream of the JNK module in the hyperosmotic stre ss-signaling pathway and that Ga12 can enhance this response by modulating either or both of these components namely, JNKs and NHEs in NIH3T3 cells. ( C) 2001 Wiley-Liss, Inc.