Activation of ras-dependent signaling pathways by G(14) -coupled receptors requires the adaptor protein TPR1

Many G_q‐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 Gα_q family may possess the ability to activate Ras/ERK by interacting with the adaptor protein tetratricopeptide repeat 1 (TPR1). Within the Gα_q family, the highly promiscuous Gα₁₄ can relay signals from numerous receptors. Here, we examined if Gα₁₄ interacts with TPR1 to stimulate Ras signaling pathways. Expression of the constitutively active Gα₁₄QL mutant in HEK293 cells led to the formation of GTP‐bound Ras as well as increased phosphorylations of downstream signaling molecules including ERK and IκB kinase. Stimulation of endogenous G₁₄‐coupled somatostatin type 2 and α₂‐adrenergic receptors produced similar responses in human hepatocellular HepG2 carcinoma cells. Co‐immunoprecipitation assays using HEK293 cells demonstrated a stronger association of TPR1 for Gα₁₄QL than Gα₁₄, suggesting that TPR1 preferentially binds to the GTP‐bound form of Gα₁₄. Activated Gα₁₄ 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 Gα₁₄ to induce Ras signaling in native HepG2 or transfected HEK293 cells. Although expression of the dominant negative mutant of TPR1 suppressed Gα₁₄QL‐induced phosphorylations of ERK and IκB kinase, it did not affect Gα₁₄QL‐induced stimulation of phospholipase Cβ or c‐Jun N‐terminal kinase. Our results suggest that TPR1 is required for Gα₁₄ to stimulate Ras‐dependent signaling pathways, but not for the propagation of signals along Ras‐independent pathways. J. Cell. Biochem. 113: 3486–3497, 2012. © 2012 Wiley Periodicals, Inc.