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 Gα(14) can relay signals from numerous receptors. Here, we examined if Gα(14) interacts with TPR1 to stimulate Ras signaling pathways. Expression of the constitutively active Gα(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 IκB kinase. Stimulation of endogenous G(14) -coupled somatostatin type 2 and α(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 Gα(14) QL than Gα(14) , suggesting that TPR1 preferentially binds to the GTP-bound form of Gα(14) . Activated Gα(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 Gα(14) to induce Ras signaling in native HepG2 or transfected HEK293 cells. Although expression of the dominant negative mutant of TPR1 suppressed Gα(14) QL-induced phosphorylations of ERK and IκB kinase, it did not affect Gα(14) QL-induced stimulation of phospholipase Cβ or c-Jun N-terminal kinase. Our results suggest that TPR1 is required for Gα(14) to stimulate Ras-dependent signaling pathways, but not for the propagation of signals along Ras-independent pathways.
Division of Life Science and the Biotechnology Research Institute, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China.
Dawna H T
DH
Kwan
Lisa Y
LY
Yung
Richard D
RD
Ye
Yung H
YH
Wong
D033942:Adaptor Proteins, Vesicular Transport / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D056945:Hep G2 Cells; D020962:Heterotrimeric GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051550:I-kappa B Kinase / Q000235:genetics / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D054799:Phospholipase C beta / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000235:genetics; D020837:SOS1 Protein / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics*; D020664:Son of Sevenless Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
D033942:Adaptor Proteins, Vesicular Transport; D034741:RNA, Small Interfering; D020837:SOS1 Protein; C485509:SOS2 protein, human; D020664:Son of Sevenless Proteins; C475144:TTC1 protein, human; D051550:I-kappa B Kinase; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D054799:Phospholipase C beta; C119109:GNA14 protein, human; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins; D018631:ras Proteins