Abstract
Although ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is a PDZ domain-containing protein known to bind to various channels, receptors, cytoskeletal elements, and cytoplasmic proteins, there is still very little evidence for a role of EBP50 in the regulation of receptor signal transduction. In this report, we show that EBP50 inhibits the phospholipase C (PLC)-beta-mediated inositol phosphate production of a Galpha(q)-coupled receptor as well as PLC-beta activation by the constitutively active Galpha(q)-R183C mutant. Coimmunoprecipitation experiments revealed that EBP50 interacts with Galpha(q) and to a greater extent with Galpha(q)-R183C. Agonist stimulation of the thromboxane A(2) receptor (TP receptor) resulted in an increased interaction between EBP50 and Galpha(q), suggesting that EBP50 preferentially interacts with activated Galpha(q). We also demonstrate that EBP50 inhibits Galpha(q) signaling by preventing the interaction between Galpha(q) and the TP receptor and between activated Galpha(q) and PLC-beta1. Investigation of the EBP50 regions involved in Galpha(q) binding indicated that its two PDZ domains are responsible for this interaction. This study constitutes the first demonstration of an interaction between a G protein alpha subunit and another protein through a PDZ domain, with broad implications in the regulation of diverse physiological systems.
Highlights
EBP501, a 55-kDa phosphoprotein, was first identified as a cofactor essential for protein kinase A-mediated inhibition of Naϩ/Hϩ exchanger isoform 3 (NHE3)
Inhibition of G␣q-mediated Signaling by ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) —Because the TP receptors activate phospholipases C (PLC)- through the G␣q protein, we looked at whether EBP50 could directly inhibit G␣q signaling
We have shown that EBP50 inhibited the inositol phosphate production induced by TP␣ and TP agonist stimulation
Summary
EBP501 ( known as NHERF1), a 55-kDa phosphoprotein, was first identified as a cofactor essential for protein kinase A-mediated inhibition of Naϩ/Hϩ exchanger isoform 3 (NHE3). Our experiments revealed that EBP50 regulates the G␣q signaling pathway by preferentially binding through its PDZ domains to the activated G␣q and by preventing its interaction with PLC-.
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