Gαq-coupled receptor internalization specifically induced by Gαq signaling. Regulation by EBP50

Abstract

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 A2 β receptor (TPβ receptor). Interestingly, we found that EBP50 almost completely blocked TPβ 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 Gαq, we next studied whether Gαq signaling could induce TPβ receptor internalization, addressing the long standing question about the relationship between GPCR signaling and their internalization. Expression of a constitutively active Gαq mutant (Gαq-R183C) resulted in a robust internalization of the TPβ 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 Cβ and protein kinase C did not appear to significantly contribute to internalization of the TPβ receptor, suggesting that Gαq induces receptor internalization through a phospholipase Cβ- and protein kinase C-independent pathway. Surprisingly, there appears to be specificity in Gα protein-mediated GPCR internalization. Gαq-R183C also induced the internalization of CXCR4 (Gαq-coupled), whereas it failed to do so for the β2-adrenergic receptor (Gαs-coupled). Moreover, Gαs-R201C, a constitutively active form of Gαs, had no effect on internalization of the TPβ, CXCR4, and β2-adrenergic receptors. Thus, we showed that Gα protein signaling can lead to internalization of GPCRs, with specificity in both the Gα proteins and GPCRs that are involved. Furthermore, a new function has been described for EBP50 in its capacity to inhibit receptor endocytosis.