Gαq-coupled Receptor Internalization Specifically Induced by Gαq Signaling

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 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...

LPA2 receptor mediates mitogenic signals in human colon cancer cells

“…Phospho-Akt, total Akt, phospho-Erk1/2, and total Erk1/2 were determined as described in Fig. 2 known to alter not only GPCR-mediated signaling, but also the signaling that regulates downstream targets, such as NHE3 and CFTR (14,21,25,26,40). In the present study, the knockdown of NHERF2 in Caco-2 cells severely compromised cellular signaling mediated by LPA 2 .…”

Moesin contributes an essential structural role inDrosophilaphotoreceptor morphogenesis

“…It is plausible that the ability of Moesin to bind F-actin and its regulators may contribute to the establishment of the RTW. Rhabdomere membrane is in dynamic exchange across the rhabdomere base with endosomes of the photoreceptor cytoplasm (Bahner et al, 2002), and it is notable that ERM proteins bind to regulators of membrane recycling (Rochdi and Parent, 2003). Moesin may participate in the membrane exchange that supports a dynamic sensory membrane.…”

Novel Dominant Rhodopsin Mutation Triggers Two Mechanisms of Retinal Degeneration and Photoreceptor Desensitization

“…5), which leads to a persistent Ca 2ϩ influx and photoreceptor desensitization. Second, activated G q ␣-protein may stimulate the internalization of specific G-protein-coupled receptors in the absence of arrestin binding (Rochdi and Parent, 2003). Thus, constitutively active NINAE pp100 would activate G q ␣, which could drive internalization of both wild-type and NINAE pp100 rhodopsin molecules to desensitize the photoreceptor cell.…”