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

Abstract

We used the yeast two-hybrid system to screen for proteins that interact with the C-terminus of the b isoform of the thromboxane A 2 receptor (TPb). This screen identified receptor for activated C-kinase 1 (RACK1) as a new TPb-interacting protein. Here, we show that RACK1 directly binds to the C-terminus and the first intracellular loop of TPb. The TPb-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 TPb 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 TPb and suggested that the receptors remained predominantly localized in the endoplasmic reticulum (ER) in RACK1-depleted cells. Confocal microscopy also revealed that a transient TPb-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 b 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.

Rack1 promotes epithelial cell–cell adhesion by regulating E-cadherin endocytosis

“…For example, Rack1 regulates the distribution of the Na þ /H þ exchanger, NHE6, between endosomes and the plasma membrane, and maintains the luminal pH of the endocytic recycling compartments (Ohgaki et al, 2008). Rack1 also regulates the transport of the G-protein-coupled receptors for thromboxane-A2, CXCR4 and angiotensin-II type-1 from the endoplasmic reticulum to the cell surface (Parent et al, 2008). Our results showing that Rack1 regulates the endocytosis and recycling of Ecadherin to the cell surface and cell-cell contacts (Figures 4 and 5), together with these other studies, identify an important function of Rack1 in regulating the trafficking of cell-surface proteins.…”

Thromboxane A2 modulates cisplatin-induced apoptosis through a Siva1-dependent mechanism

“…The yeast two-hybrid screen was performed as described previously. 3 Briefly, a plasmid containing the C-terminus of TPb (pAS2-1-TPbCT) was transformed into the yeast strain pJ69-4a according to the lithium yeast transformation protocol. This stably transformed clone was then transformed with a Human HeLa MATCHMAKER cDNA Library or with the empty pGad424 plasmid (Clontech).…”

“…Generation of the TP constructs in the pGEX-4T-1 vector (GE Healthcare) was described previously. 3 Myc-Mdm2, Myc-TRAF2 and Myc-XIAP were all cloned by RT-PCR from HeLa cells mRNA and inserted into the pcDNA3 expression vector (Invitrogen).…”

“…2 We performed a yeast two-hybrid screen looking for proteins interacting with the C-terminal tail of TPb. 3 We report here that the Siva1 protein directly interacts with the C-tail of TPb.…”

“…We have previously shown that the C-terminus of TPb (C-tail) contains regulatory domains responsible for the trafficking properties of the receptor. 3 To identify putative proteins that interact with the receptor, we performed a yeast two-hybrid screen using the TPb C-tail as bait. A total of 1.5 Â 10 6 independent clones were screened, yielding over 300 positives, seven of which contained the full-length Siva1 coding sequence (data not shown).…”