Activation of ras-dependent signaling pathways by G14-coupled receptors requires the adaptor protein TPR1

Many Gq-coupled receptors mediate mitogenic signals by stimulating extracellular signal-regulated protein kinases (ERKs) that are typically regulated by the small GTPase Ras. Recent studies have revealed that members of the Gaq family may possess the ability to activate Ras/ERK by interacting with the adaptor protein tetratricopeptide repeat 1 (TPR1). Within the Gaq family, the highly promiscuous Ga14 can relay signals from numerous receptors. Here, we examined if Ga14 interacts with TPR1 to stimulate Ras signaling pathways. Expression of the constitutively active Ga14QL mutant in HEK293 cells led to the formation of GTP-bound Ras as well as increased phosphorylations of downstream signaling molecules including ERK and I?B kinase. Stimulation of endogenous G14-coupled somatostatin type 2 and a2-adrenergic receptors produced similar responses in human hepatocellular HepG2 carcinoma cells. Co-immunoprecipitation assays using HEK293 cells demonstrated a stronger association of TPR1 for Ga14QL than Ga14, suggesting that TPR1 preferentially binds to the GTP-bound form of Ga14. Activated Ga14 also interacted with the Ras guanine nucleotide exchange factors SOS1 and SOS2. Expression of a dominant negative mutant of TPR1 or siRNA-mediated knockdown of TPR1 effectively abolished the ability of Ga14 to induce Ras signaling in native HepG2 or transfected HEK293 cells. Although expression of the dominant negative mutant of TPR1 suppressed Ga14QL-induced phosphorylations of ERK and I?B kinase, it did not affect Ga14QL-induced stimulation of phospholipase C beta or c-Jun N-terminal kinase. Our results suggest that TPR1 is required for Ga14 to stimulate Ras-dependent signaling pathways, but not for the propagation of signals along Ras-independent pathways. J. Cell. Biochem. 113: 34863497, 2012. (C) 2012 Wiley Periodicals, Inc.