RACK1 Regulates the Cell Surface Expression of the G Protein-Coupled Receptor for Thromboxane A2

We used the yeast two-hybrid system to screen for proteins that interact with the C-terminus of the β isoform of the thromboxane A₂ receptor (TPβ). This screen identified receptor for activated C-kinase 1 (RACK1) as a new TPβ-interacting protein. Here, we show that RACK1 directly binds to the C-terminus and the first intracellular loop of TPβ. The TPβ–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 TPβ 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 TPβ and suggested that the receptors remained predominantly localized in the endoplasmic reticulum (ER) in RACK1-depleted cells. Confocal microscopy also revealed that a transient TPβ–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 β₂-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.