Differential signaling by the thromboxane receptor isoforms via the novel GTP-binding protein, G(h)

Thromboxane A(2) acts via G protein-coupled receptors; two splice variants of the thromboxane A(2) receptor (TP alpha and TP beta) have been cloned. I t is unknown whether they differ in their capacity to activate intracellula r signaling pathways. Recently, a high molecular weight G protein, G(h), th at can also function as a tissue transglutaminase, has been described. We i nvestigated whether G(h) functions as a signaling protein in association wi th thromboxane receptors, First, we sought G(h) expression in cells known t o express TPs, Reverse transcription-polymerase chain reaction and immunobl otting demonstrated G(h) expression in platelets, megakaryocytic cell lines , and endothelial and vascular smooth muscle cells. Second, immunoprecipita tion of both TP alpha and TP beta in transfected COS-7 cells resulted in th e co-immunoprecipitation of G(h), indicating that TPs may associate G(h) in vice. Finally, agonist activation of TP alpha, but not of TP beta, resulte d in stimulation of phospholipase C-mediated inositol phosphate production in cells cotransfected with G(h), By contrast, agonist activation of both T P iso forms resulted in G(h)-mediated inositol phosphate signaling. G(h) is expressed in platelets and vascular cells and may associate with both TP i soforms. However, stimulation of TP isoforms results in differential activa tion of downstream signaling pathways via this novel G protein.