In the present report, we investigated the effect of ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) expression on the agonist-induced internalization of the thromboxane A 2  receptor (TP receptor). Interestingly, we found that EBP50 almost completely blocked TP receptor internalization, which could not be reversed by overexpression of G protein-coupled receptor (GPCR) kinases and arrestins. Because we recently demonstrated that EBP50 can bind to and inhibit G␣ q , we next studied whether G␣ q signaling could induce TP receptor internalization, addressing the long standing question about the relationship between GPCR signaling and their internalization. Expression of a constitutively active G␣ q mutant (G␣ q -R183C) resulted in a robust internalization of the TP receptor, which was unaffected by expression of dominant negative mutants of arrestin-2 and -3, but inhibited by expression of EBP50 or dynamin-K44A, a dominant negative mutant of dynamin. Phospholipase C and protein kinase C did not appear to significantly contribute to internalization of the TP receptor, suggesting that G␣ q induces receptor internalization through a phospholipase C-and protein kinase C-independent pathway. Surprisingly, there appears to be specificity in G␣ protein-mediated GPCR internalization. G␣ q -R183C also induced the internalization of CXCR4 (G␣ q -coupled), whereas it failed to do so for the  2 -adrenergic receptor (G␣ s -coupled). Moreover, G␣ s -R201C, a constitutively active form of G␣ s , had no effect on internalization of the TP, CXCR4, and  2 -adrenergic receptors. Thus, we showed that G␣ protein signaling can lead to internalization of GPCRs, with specificity in both the G␣ proteins and GPCRs that are involved. Furthermore, a new function has been described for EBP50 in its capacity to inhibit receptor endocytosis.
G protein-coupled receptor (GPCR)1 signaling cascades are regulated by different molecular mechanisms. Many proteins have been shown to regulate the different GPCR-related signaling pathways through the direct regulation of the G protein activity (1). Following agonist binding, many GPCRs undergo agonist-induced phosphorylation, internalization, and downregulation, resulting in a decrease of their responsiveness (desensitization) (2). Our studies are mainly interested in the mechanisms that regulate the signaling and the internalization of the thromboxane A 2 receptor (TP receptor). Two TP receptor isoforms were identified which are generated by the alternative splicing of a single gene, TP␣ (343 amino acids) and TP (407 amino acids), which share the first 328 amino acids (3, 4). Previous experiments performed by Parent et al. (5) demonstrated that only TP, but not TP␣, undergoes agonistinduced and tonic (constitutive) internalizations, which are dictated by distinct motifs in the C terminus of the TP receptor. Different experiments showed that agonist-induced production of the second messenger inositol phosphate by the TP receptors results from the activation of the G q/11 family of t...