RACK1 regulates the cell surface expression of the G protein-coupled receptor for thromboxane A(2).
We used the yeast two-hybrid system to screen for proteins that interact with the C-terminus of the beta isoform of the thromboxane A(2) receptor (TPbeta). This screen identified receptor for activated C-kinase 1 (RACK1) as a new TPbeta-interacting protein. Here, we show that RACK1 directly binds to the C-terminus and the first intracellular loop of TPbeta. The TPbeta-RACK1 association was further confirmed by co-immunoprecipitation studies in HEK293 cells and was not modulated by stimulation of the receptor. We observed that cell surface expression of TPbeta was increased when RACK1 was overexpressed, while it was inhibited when endogenous RACK1 expression was knocked down by small interfering RNA. Confocal microscopy confirmed the impaired cell surface expression of TPbeta and suggested that the receptors remained predominantly localized in the endoplasmic reticulum (ER) in RACK1-depleted cells. Confocal microscopy also revealed that a transient TPbeta-RACK1 association takes place in the ER. The effect of RACK1 on receptor trafficking to the cell surface appears to be selective to some G protein-coupled receptors (GPCRs) because inhibition of RACK1 expression also affected cell surface targeting of the angiotensin II type 1 receptor and CXCR4 but not of beta(2)-adrenergic and prostanoid DP receptors. Our data demonstrate for the first time a direct interaction between RACK1 and a GPCR and identify a novel role for RACK1 in the regulation of the transport of a membrane receptor from the ER to the cell surface.
Parent
Audrey
A
Service de Rhumatologie, Département de Médecine, Faculté de Médecine, Université de Sherbrooke, Fleurimont, Quebec, J1H 5N4, Canada. jean-luc.parent@usherbrooke.ca
Laroche
Geneviève
G
Hamelin
Emilie
E
Parent
Jean-Luc
JL
D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D034281:Dynamins / Q000037:antagonists & inhibitors / Q000235:genetics; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D025941:Protein Interaction Mapping; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D034622:RNA Interference; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D044244:Receptors, Thromboxane A2, Prostaglandin H2 / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D020798:Two-Hybrid System Techniques
D007211:Indoles; D008301:Maleimides; D009363:Neoplasm Proteins; D047428:Protein Kinase Inhibitors; C419368:RACK1 protein, human; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D044244:Receptors, Thromboxane A2, Prostaglandin H2; D011994:Recombinant Proteins; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; D034281:Dynamins; C070515:bisindolylmaleimide I