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Regulation of GTP-binding Protein αq(Gαq) Signaling by the Ezrin-Radixin-Moesin-binding Phosphoprotein-50 (EBP50)*

  • Moulay Driss Rochdi
    Footnotes
    Affiliations
    Service de Rhumatologie, Faculté de Médecine and Centre de Recherche Clinique-CHUS, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada and
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  • Valérie Watier
    Affiliations
    Service de Rhumatologie, Faculté de Médecine and Centre de Recherche Clinique-CHUS, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada and
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  • Carole La Madeleine
    Affiliations
    Service de Rhumatologie, Faculté de Médecine and Centre de Recherche Clinique-CHUS, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada and
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  • Hiroko Nakata
    Affiliations
    Department of Pharmacology, University of Illinois, Chicago, Illinois 60612
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  • Tohru Kozasa
    Affiliations
    Department of Pharmacology, University of Illinois, Chicago, Illinois 60612
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  • Jean-Luc Parent
    Correspondence
    Recipient of a New Investigator Award from the Canadian Institutes of Health Research. To whom correspondence should be addressed: Service de Rhumatologie, Faculté de Médecine, Université de Sherbrooke, 3001, 12e Avenue Nord, Fleurimont, Québec J1H 5N4, Canada. Tel.: 819-564-5264; Fax: 819-564-5265;
    Affiliations
    Service de Rhumatologie, Faculté de Médecine and Centre de Recherche Clinique-CHUS, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada and
    Search for articles by this author
  • Author Footnotes
    * This work was supported in part by grants (to J.-L. P.) from the Canadian Institutes of Health Research and the Kidney Foundation of Canada.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    § Supported by a doctoral fellowship from the “Fonds de la Recherche en Santé du Québec.”
Open AccessPublished:August 21, 2002DOI:https://doi.org/10.1074/jbc.M207910200
      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)-β-mediated inositol phosphate production of a Gαq-coupled receptor as well as PLC-β activation by the constitutively active Gαq-R183C mutant. Coimmunoprecipitation experiments revealed that EBP50 interacts with Gαq and to a greater extent with Gαq-R183C. Agonist stimulation of the thromboxane A2 receptor (TP receptor) resulted in an increased interaction between EBP50 and Gαq, suggesting that EBP50 preferentially interacts with activated Gαq. We also demonstrate that EBP50 inhibits Gαq signaling by preventing the interaction between Gαq and the TP receptor and between activated Gαq and PLC-β1. Investigation of the EBP50 regions involved in Gαq 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 α subunit and another protein through a PDZ domain, with broad implications in the regulation of diverse physiological systems.
      EBP50
      The abbreviations used are: EBP50, ezrin-radixin-moesin-binding phosphoprotein 50; ERM, ezrin-radixin-moesin; GAP, GTPase-activating protein; GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; NHE3, Na+/H+ exchanger isoform 3; NHERF, NHE regulatory factor; PLC, phospholipase C; TP receptor, thromboxane A2 receptor; TXA2, thromboxane A2; CFTR, cystic fibrosis transmembrane conductance regulator; Ni-NTA, nickel-nitrilotriacetic acid; HA, hemagglutinin; RGS, regulators of G protein signaling.
      1The abbreviations used are: EBP50, ezrin-radixin-moesin-binding phosphoprotein 50; ERM, ezrin-radixin-moesin; GAP, GTPase-activating protein; GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; NHE3, Na+/H+ exchanger isoform 3; NHERF, NHE regulatory factor; PLC, phospholipase C; TP receptor, thromboxane A2 receptor; TXA2, thromboxane A2; CFTR, cystic fibrosis transmembrane conductance regulator; Ni-NTA, nickel-nitrilotriacetic acid; HA, hemagglutinin; RGS, regulators of G protein signaling.
      (also known as NHERF1), a 55-kDa phosphoprotein, was first identified as a cofactor essential for protein kinase A-mediated inhibition of Na+/H+ exchanger isoform 3 (NHE3) (
      • Weinman E.J.
      • Steplock D.
      • Wang Y.
      • Shenolikar S.
      ). EBP50 contains two PDZ domains (PDZ1 and PDZ2) implicated in multiple protein-protein interactions, and an ERM domain, which binds to the actin-associated ERM proteins (ezrin, radixin, moesin, and merlin) (
      • Murthy A.
      • Gonzalez-Agosti C.
      • Cordero E.
      • Pinney D.
      • Candia C.
      • Solomon F.
      • Gusella J.
      • Ramesh V.
      ,
      • Reczek D.
      • Berryman M.
      • Bretscher A.
      ). EBP50 was also found to interact with a small number of transmembrane proteins such as the cystic fibrosis transmembrane conductance regulator (CFTR) (
      • Moyer B.D.
      • Duhaime M.
      • Shaw C.
      • Denton J.
      • Reynolds D.
      • Karlson K.H.
      • Pfeiffer J.
      • Wang S.
      • Mickle J.E.
      • Milewski M.
      • Cutting G.R.
      • Guggino W.B., Li., M.
      • Stanton B.A.
      ), the P2Y1 purinergic receptor (
      • Hall R.A.
      • Ostedgaard L.S.
      • Premont R.T.
      • Blitzer J.T.
      • Rahman N.
      • Welsh M.J.
      • Lefkowitz R.J.
      ), the platelet-derived growth factor receptor (
      • Maudsley S.
      • Zamah A.M.
      • Rahman N.
      • Blitzer J.T.
      • Luttrell L.M.
      • Lefkowitz R.J.
      • Hall R.A.
      ), the β2-adrenergic receptor (
      • Hall R.A.
      • Ostedgaard L.S.
      • Premont R.T.
      • Blitzer J.T.
      • Rahman N.
      • Welsh M.J.
      • Lefkowitz R.J.
      ), the B1 subunit of the H+-ATPase (
      • Breton S.
      • Wiederhold T.
      • Marshansky V.
      • Nsumu N.N.
      • Ramesh V.
      • Brown D.
      ), and the type IIa sodium phosphate cotransporter (
      • Gisler S.M.
      • Stagljar I.
      • Traebert M.
      • Bacic D.
      • Biber J.
      • Murer H.
      ). EBP50 also associates with the phospholipases C (PLC)-β1/β2, and with the TRP4 and TRP5 calcium channels to form a PLC-β1/2-TRP4/5-EBP50 protein complex (
      • Tang Y.
      • Tang J.
      • Chen Z.
      • Trost C.
      • Flockerzi V., Li, M.
      • Ramesh V.
      • Zhu M.X.
      ). The physiological role of this interaction on the regulation of PLC-β1/2 remains undefined. The EBP50 protein can also bind through its PDZ domains to various intracellular proteins, including GRK6A (
      • Hall R.A.
      • Spurney R.F.
      • Premont R.T.
      • Rahman N.
      • Blitzer J.T.
      • Pitcher J.A.
      • Lefkowitz R.J.
      ), EPI64 (
      • Reczek D.
      • Bretscher A.
      ), and Yes-associated protein 65 (12). A close relative of EBP50 has been identified and is known as E3KARP (
      • Yun C.H., Oh, S.
      • Zizak M.
      • Steplock D.
      • Tsao S.
      • Tse C.M.
      • Weinman E.J.
      • Donowitz M.
      ), SIP-1 (
      • Poulat F.
      • Barbara P.S.
      • Desclozeaux M.
      • Soullierk S.
      • Moniotk B.
      • Bonneaudk N.
      • Boizetk B.
      • Berta P.
      ), and NHERF2 (
      • Hwang J.I.
      • Heo K.
      • Shin K.J.
      • Kim E.
      • Yun C.
      • Ryu S.H.
      • Shin H.S.
      • Suh P.G.
      ). EBP50 and NHERF2 share 52% amino acid identity and a conserved domain architecture (
      • Yun C.H., Oh, S.
      • Zizak M.
      • Steplock D.
      • Tsao S.
      • Tse C.M.
      • Weinman E.J.
      • Donowitz M.
      ). It has been shown recently that the PDZ domains of EBP50 can homo-oligomerize and also hetero-oligomerize with the PDZ domains of NHERF2 (
      • Lau A.G.
      • Hall R.A.
      ). Despite the growing evidence suggesting the role of EBP50 as a scaffolding protein involved in the formation of signaling complexes, there is still little evidence for a role of EBP50 in the regulation of transmembrane receptor signaling pathways.
      Our laboratory has been interested in the regulation of the thromboxane A2 (TXA2) receptors (TP receptors). TXA2 has a variety of pharmacological effects that modulate the physiological responses of several cells and tissues (
      • Kinsella B.T.
      ). Binding of TXA2 to its receptor induces platelet aggregation, constriction of vascular and bronchiolar smooth muscle cells, as well as mitogenesis and hypertrophy of vascular smooth muscle cells (
      • Narumiya S.
      • Sugimoto Y.
      • Ushikubi F.
      ). The TP receptor is part of the G protein-coupled receptor (GPCR) superfamily. Two TP receptor isoforms were identified that are generated by the alternative splicing of a single gene, TPα (343 amino acids) and TPβ (407 amino acids), which share the first 328 amino acids (
      • Hirata M.
      • Hayashi Y.
      • Ushikubi F.
      • Yokota Y.
      • Kageyama R.
      • Nakanishi S.
      • Narumiya S.
      ,
      • Raychowdhury M.K.
      • Yukawa M.
      • Collins L.J.
      • McGrail S.H.
      • Kent K.C.
      • Ware J.A.
      ). Different experiments demonstrated that agonist-induced production of the second messenger inositol phosphates by the TP receptors results from the activation of the Gq/11 family of the Gα subunits (
      • Kinsella B.T.
      ). Gαq-mediated production of inositol phosphates involves the stimulation of PLC-β isoforms in the following order of potency: PLC-β1 ≥ PLC-β3 > PLC-β2 (
      • Smrcka A.V.
      • Sternweis P.C.
      ). Furthermore, PLC-β isoenzymes accelerate the intrinsic GTP hydrolysis by Gα subunits acting as a GTPase-activating protein (GAP) (
      • Chidiac P.
      • Ross E.M.
      ). More recently, it was shown that PLC-βs form dimers in vivo and suggested that dimerization could mediate their interaction with GTP-bound Gαq (
      • Singer A.U.
      • Waldo G.L.
      • Harden T.K.
      • Sondek J.
      ).
      Although the signaling pathways of the TP receptors are being described in increasing detail, their regulation remains to be characterized. Knowing that the TP receptor signaling cascade involves the activation of Gαq and the stimulation of the downstream effector PLC-β, we investigated the effect of EBP50 on the signaling of the agonist-stimulated TP receptor through the Gαq pathway. Surprisingly, our experiments revealed that EBP50 regulates the Gαq signaling pathway by preferentially binding through its PDZ domains to the activated Gαq and by preventing its interaction with PLC-β. Furthermore, we show that EBP50 also interferes in the TP receptor-Gαq coupling. This novel interaction between PDZ domains and Gαq has broad implications in intracellular signaling regulation. Growing evidence demonstrates the role of EBP50 in a wide variety of physiological events. The regulation of these events could possibly be modulated by the Gαq-EBP50 interaction.

      DISCUSSION

      This study demonstrates a direct regulation of the Gαq signaling pathway by EBP50. We have shown that EBP50 inhibited the inositol phosphate production induced by TPα and TPβ agonist stimulation. We showed that EBP50 decreased PLC-β-dependent production of inositol phosphates induced by Gαq-R183C, a constitutively active mutant of Gαq. We demonstrated that EBP50 interacts in a greater extent with Gαq-R183C than with Gαq. Moreover, stimulation of the TPβ receptor promoted the Gαq-EBP50 interaction, strongly indicating that EBP50 interacts better with the activated Gαq. It was observed that, beyond 5 min of agonist stimulation of TPβ, the amount of EBP50 that coimmunoprecipitated with Gαq decreased to levels similar to what was observed in the absence of agonist. It is known that the activation of Gαq by GPCRs is a rapid process involving the release of GDP by Gαq and its exchange for a GTP. Thereafter, rapid hydrolysis of the GTP by the intrinsic GTPase activity of Gαq results in the inactivation of the Gαq subunit and its return to the inactive GDP-bound state. In addition, rapid desensitization of GPCRs occurs after agonist stimulation resulting in their inability to activate G protein subunits. Taken together, this could explain the loss of EBP50-Gαq interaction after prolonged stimulation of TPβ. These results strongly suggest that the regulation of the TP receptors signaling by EBP50 is caused by the binding of the activated Gαq to EBP50. However, because activation of the Gα subunit results in its dissociation from the βγ subunits, the strong binding of the active form of Gαq to EBP50 could be due to its dissociation from the βγ subunits and not to its GTP-bound active state per se. Arguing against the last statement is the fact that the overexpressed inactive Gαqdid not interact as well as Gαq-R183C with EBP50, unless the endogenous βγ subunits can account for the difference. Furthermore, the activated Gαq-EBP50 interaction after receptor stimulation is transient, suggesting that the Gαq comes off the EBP50 protein upon GTP hydrolysis, which could indicate that EBP50 indeed binds preferentially to the active form of Gαq. More experiments need to be performed to verify if the βγ subunits compete with EBP50 for the binding of Gαq.
      Our results showed that EBP50 interferes with the coupling of the TP receptors to Gαq which could be explained by the binding of EBP50 to both the active and the inactive forms of Gαqleading to the possible sequestration of Gαq. It can be argued that EBP50 could also bind directly to the receptor, as reported for other GPCRs (
      • Hall R.A.
      • Ostedgaard L.S.
      • Premont R.T.
      • Blitzer J.T.
      • Rahman N.
      • Welsh M.J.
      • Lefkowitz R.J.
      ), and further interfere in the Gαqcoupling this way. The EBP50-TPβ receptor interaction is currently studied in our laboratory.
      GTP-bound Gαq preferentially activates PLC-β1 as described in the Introduction. Singer et al. (
      • Singer A.U.
      • Waldo G.L.
      • Harden T.K.
      • Sondek J.
      ) have shown that the GTP bound-Gαq directly binds and activates PLC-β. Because on the one hand EBP50 inhibited the inositol phosphate production and on the other hand had a greater affinity for the active form of Gαq, the effect of EBP50 on the Gαq-PLC-β1 interaction was investigated. We observed an almost complete inhibition in the interaction of Gαq-R183C with PLC-β1 in the presence of EBP50. Taken together, the results described above indicate that EBP50 regulates the TP receptor signaling through Gαq at different levels. First, we have shown that EBP50 interferes with the coupling of the TP receptors to Gαq by binding and preventing the GDP-bound Gαq (inactive form) from binding to the TP receptor. Second, we have shown that EBP50 also interferes with the coupling of the activated Gαq to the downstream effector PLC-β1.
      GAPs regulate the heterotrimeric G protein signaling by increasing the rate of GTP hydrolysis by Gα subunits up to 2000-fold. Both RGS proteins (regulators of G protein signaling) and G protein effectors (such as PLC-β and p115RhoGEF, which lack an RGS domain) belong to the family of GAP proteins. RGS proteins specifically bind the GTP-bound Gα subunits and share an RGS domain involved in the regulation of the intrinsic GTPase activity of Gα. Because our results have shown that EBP50 binds preferentially the GTP-bound Gαq, and that this interaction is transient after activation of a GPCR, there was the possibility that EBP50 could modulate the intrinsic GTP hydrolysis activity of Gαq. Analysis of the EBP50 amino acid sequence and its alignment with the RGS proteins failed to show any significant homology, indicating that EBP50 does not contain an RGS domain. Our results showed that EBP50 does not modulate the intrinsic GTPase activity of Gαq. Regulation of Gαq signaling by EBP50 must then rely principally on the mechanisms discussed above. We cannot, however, totally rule out the GAP activity of EBP50. Indeed, it was shown that the GRK2 GAP activity is apparent only in the presence of an activated GPCR (
      • Carman C.V.
      • Parent J.L.
      • Day P.W.
      • Pronin A.N.
      • Sternweis P.M.
      • Wedegaertner P.B.
      • Gilman A.G.
      • Benovic J.L.
      • Kozasa T.
      ), which suggests the possibility that receptors could have a critical role in potentiating GAP activity in cells. Moreover, it has been demonstrated that the ability of RGS2 to inhibit Gαq-mediated signals in cells is highly dependent on the nature of the receptors that are being stimulated (
      • Xu X.
      • Zeng W.
      • Popov S.
      • Berman D.M.
      • Davignon I., Yu, K.
      • Yowe D.
      • Offermanns S.
      • Muallem S.
      • Wilkie T.M.
      ). The authors of this study suggested that regulatory selectivity may be conferred by specific receptor-RGS complexes. Thus, further investigation of the role of receptors in modulating EBP50-Gαq interactions seem warranted.
      EBP50 binds to NHE3 and is involved in its regulation by cAMP, as well as in its down-regulation (
      • Weinman E.J.
      • Minkoff C.
      • Shenolikar S.
      ). EBP50 may also be required to localize the H+-ATPase in both apical and basolateral membranes in renal intercalated cells (
      • Weinman E.J.
      • Minkoff C.
      • Shenolikar S.
      ). Furthermore, EBP50 serves as a membrane retention signal for CFTR (
      • Moyer B.D.
      • Denton J.
      • Karlson K.H.
      • Reynolds D.
      • Wang S.
      • Mickle J.E.
      • Milewski M.
      • Cutting G.R.
      • Guggino W.B., Li, M.
      • Stanton B.A.
      ) and facilitates the dimerization of CFTR that promotes the full expression of chloride channel activity (
      • Raghuram V.
      • Mak D.D.
      • Foskett J.K.
      ). Moreover, recent experiments (
      • Maudsley S.
      • Zamah A.M.
      • Rahman N.
      • Blitzer J.T.
      • Luttrell L.M.
      • Lefkowitz R.J.
      • Hall R.A.
      ) have shown that EBP50 is involved in the dimerization of the platelet-derived growth factor receptor. Among its numerous functions, EBP50 also regulates the sorting of the β2-adrenergic receptor to either recycling endosomes or lysosomal degradatory pathways (
      • Cao T.T.
      • Deacon H.W.
      • Reczek D.
      • Bretscher A.
      • Zastrow M.V.
      ).
      Since its isolation, EBP50 has been associated with a broad array of biological systems, all of which depend on the interaction of EBP50 through its PDZ1 and PDZ2 domains with the different target proteins. Our study identifies Gαq as a novel interacting partner for the PDZ domains of EBP50. This finding is important in terms of G protein signaling regulation. The study of possible interactions between other PDZ domain-containing proteins with Gα subunits constitutes a new and exciting field of research. It is also easy to envision the great impact of the Gαq-EBP50 interaction in signaling “cross-talk” because an EBP50 molecule bound to Gαq could possibly be unable to engage in the interaction with another binding partner. Indeed, one can imagine that multiple biological systems associated with EBP50 could be modulated by the activated Gαq-EBP50 interaction.