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

Kwan, DH;Yung, LY;Ye, RD;Wong, YH;

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 G(q) family may possess the ability to activate Ras/ERK by interacting with the adaptor protein tetratricopeptide repeat 1 (TPR1). Within the G(q) family, the highly promiscuous G(14) can relay signals from numerous receptors. Here, we examined if G(14) interacts with TPR1 to stimulate Ras signaling pathways. Expression of the constitutively active G(14) QL mutant in HEK293 cells led to the formation of GTP-bound Ras as well as increased phosphorylations of downstream signaling molecules including ERK and IB kinase. Stimulation of endogenous G(14) -coupled somatostatin type 2 and (2) -adrenergic receptors produced similar responses in human hepatocellular HepG2 carcinoma cells. Co-immunoprecipitation assays using HEK293 cells demonstrated a stronger association of TPR1 for G(14) QL than G(14) , suggesting that TPR1 preferentially binds to the GTP-bound form of G(14) . Activated G(14) 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 G(14) to induce Ras signaling in native HepG2 or transfected HEK293 cells. Although expression of the dominant negative mutant of TPR1 suppressed G(14) QL-induced phosphorylations of ERK and IB kinase, it did not affect G(14) QL-induced stimulation of phospholipase C or c-Jun N-terminal kinase. Our results suggest that TPR1 is required for G(14) to stimulate Ras-dependent signaling pathways, but not for the propagation of signals along Ras-independent pathways.