Localization of the RhoGEF Effector Activating Surface of Gα 13 to its Carboxy Terminus

Abstract

The monomeric GTPase Rho regulates a variety of cellular processes, such as actin cytoskeletal rearrangement and gene expression. Two members of the heterotrimeric GTP-binding protein (G-protein) family, Gα12 and Gα13, have been shown to mediate signals from G-protein coupled receptors leading to Rho activation. In this novel signaling pathway, one of the guanine nucleotide exchange factors (GEFs) for Rho, p115RhoGEF, serves as a direct link between Gα13 and Rho. The goal of the present study is to isolate the region of Gα13 responsible for stimulating p115’s RhoGEF activity. To do this, three sets of Gα12/13 and Gα13/12 chimeras were constructed, in which the fusion point between Gα12 and Gα13 (or Gα13 and Gα12) was moved progressively towards the C-terminus of the chimera. The resulting constructs represent chimeras with fusion points before and after the switch regions of Gα12 and Gα13, as well as in the α4 helix. The results of in vitro experiments suggest that the region of Gα13 responsible for stimulation of RhoGEF activity is localized to the C-terminus of the switch regions. A precise delineation of the signaling pathway involving Gα12 and Gα13, p115, and Rho will allow for a more complete understanding of the biochemical mechanism and physiological significance of Rho activation through G-proteins. sources of support: National Institutes of Health (grant Gm 61454) and National Institutes of Health Pharmacological Sciences Training Program (grant Gm 070388)