NHERF1 Regulates Parathyroid Hormone Receptor Desensitization: Interference with -Arrestin Binding

Type 1 parathyroid hormone receptor (PTH1R) activation, desensitization, internalization, and recycling proceed in a cyclical manner. The Na /H exchange regulatory factor 1 (NHERF1) is a cytoplasmic adapter protein that regulates trafficking and signaling of several G protein-coupled receptors (GPCRs) including the PTH1R. The mineral ion wasting and bone phenotype of NHERF1-null mice suggests that PTH1R may interact with NHERF1. The objective of this study was to examine the effect of NHERF1 on PTH1R desensitization. Using rat osteosarcoma T6-N4 cells expressing the endogenous PTH1R, in which NHERF1 expression could be induced by tetracycline, PTH1R desensitization was assessed by measuring adenylyl cyclase activity after successive PTH challenges. PTH1Rmediated adenylyl cyclase responses were desensitized by repetitive PTH challenges in a concentration-dependent manner, and desensitization was inhibited by NHERF1. NHERF1 blocked PTH-induced dissociation of the PTH1R from G s. Blocking PTH1R endocytosis did not mitigate PTH1R desensitization. Reducing constitutive NHERF1 levels in human osteosarcoma SAOS2 cells, which express both endogenous PTH1R and NHERF1, with short hairpin RNA directed against NHERF1 restored PTH1R desensitization. Mutagenesis of the PDZbinding domains or deletion of the NHERF1 MERM domain demonstrated that both are required for inhibition of receptor desensitization. A phosphorylation-deficient PTH1R exhibited reduced desensitization and interaction with -arrestin2 compared with wild-type PTH1R. NHERF1 inhibited -arrestin2 binding to wtPTH1R but had no effect on -arrestin2 association with pdPTH1R. Such an effect may protect against PTH resistance or PTH1R down-regulation in cells harboring NHERF1. The parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTH1R) belongs to Class B of the seventransmembrane family of G protein-coupled receptors (GPCRs) (Gensure et al., 2005). PTH1R is present primarily in the bone and kidney. Interaction with its cognate ligands, PTH, PTHrP, or biologically active peptide fragments leads to activation of Gs and Gq with consequent stimulation of adenylate cyclase and phospholipase C (Abou-Samra et al., 1992; Friedman et al., 1996). A cascade of cell-specific events ensues that regulates virtually all aspects of extracellular calcium and phosphate homeostasis. As with most GPCRs, the response of PTH1R to agonists is regulated by multiple mechanisms, including a well characterized and highly conserved process involving receptor phosphorylation by G protein-coupled receptor kinase 2 (GRK2) (Dicker et al., 1999; Flannery and Spurney, 2001) and arrestin recruitment (Ferrari et al., 1999; Tawfeek and Abou-Samra, 1999; Vilardaga et al., 2002). Arrestins are cytoplasmic adaptor proteins that bind to phosphorylated GPCRs and uncouple them from their cognate G proteins, thereby producing a nonsignaling, desensitized receptor (Krupnick and Benovic, 1998). -Arrestin1 and -arrestin2 are widely expressed and regulate the functions of many GPCRs, including the PTH1R (Malecz et al., 1998; Ferrari et al., 1999). In mice lacking -arrestin2, PTH treatment promotes sustained cAMP signaling in primary osteoblasts in vitro and altered skeletal response to PTH in vivo (Ferrari et al., 2005). These processes contribute directly to PTH1R desensitization by facilitating the uncoupling of the receptor from its cognate G proteins. This work was supported by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grants DK69998, DK063211, DK11794]. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.108.054486. □S The online version of this article (available at http://molpharm. aspetjournals.org) contains supplemental material. ABBREVIATIONS: PTH, parathyroid hormone; PTHrP, parathyroid hormone-related peptide; PTH1R, type 1 PTH receptor; GPCR, G proteincoupled receptor; GRK2, G protein-coupled receptor kinase 2; NHERF1, Na /H exchange regulatory factor 1; PDZ, postsynaptic density 95/discs large/zona occludens; MERM, merlin-ezrin-radixin-moesin; GFP, green fluorescent protein; BSA, bovine serum albumin; ROS, rat osteosarcoma; shRNA, short hairpin RNA; siRNA, small interfering RNA; IBMX, 3-isobutyl-1-methylxanthine; Tet, tetracycline; wtPTH1R, wild-type PTH1R; PKC, protein kinase C; PD, phosphorylation deficient; HA, hemagglutinin. 0026-895X/09/7505-1189–1197$20.00 MOLECULAR PHARMACOLOGY Vol. 75, No. 5 Copyright © 2009 The American Society for Pharmacology and Experimental Therapeutics 54486/3458544 Mol Pharmacol 75:1189–1197, 2009 Printed in U.S.A. 1189 http://molpharm.aspetjournals.org/content/suppl/2009/02/02/mol.108.054486.DC1 Supplemental material to this article can be found at: at A PE T Jornals on N ovem er 7, 2017 m oharm .aspeurnals.org D ow nladed from The interaction of -arrestin1 or -arrestin2 with phosphorylated PTH1R is the likely mechanism of desensitization of the PTH1R-activated responses (Tawfeek et al., 2002). After -arrestin interactions, the PTH1R is endocytosed and either targeted for degradation, leading to receptor downregulation (Tian et al., 1994; Ureña et al., 1994; Massry and Smogorzewski, 1998), or recycled to the membrane, leading to receptor resensitization (Chauvin et al., 2002). Na /H exchange regulatory factor 1 (NHERF1), also known as ezrin-radixin-moesin-binding phosphoprotein-50, is a cytoplasmic scaffolding protein. NHERF1 recruits various cellular receptors, ion transporters, and other proteins to the plasma membrane of epithelia and other cells (Voltz et al., 2001; Bretscher et al., 2002; Shenolikar et al., 2004; Tan et al., 2004). NHERF1 contains tandem postsynaptic density 95/discs large/ zona occludens (PDZ) domains and a merlin-ezrin-radixinmoesin (MERM) domain. PTH1R was reported to interact with PDZ1 and PDZ2 with similar affinities (Sun and Mierke, 2005; Wang et al., 2007). The MERM domain binds to respective actin-associated MERM proteins. NHERF1 tethers the PTH1R to the actin cytoskeleton through the MERM domain. The mineral ion-wasting and bone phenotype of NHERF1null mice or patients with NHERF1 coding region mutations suggests that the PTH1R is the principal GPCR interacting with NHERF1 (Shenolikar et al., 2002). Humans with NHERF1 mutations present with renal stones or bone demineralization (Karim et al., 2008) underscoring the primary role of NHERF1 in associating with and modulating PTH1R activity. Expression of NHERF1 restores both PTH(1–34)-mediated inhibition of the Npt2 sodium-phosphate cotransporter (Mahon et al., 2003) and the increase of intracellular calcium (Mahon and Segre, 2004) in OK/H cells, which express low levels of NHERF1 and are resistant to the action of PTH. Our previous data established that NHERF1 inhibited PTH1R internalization without affecting receptor recycling in kidney cells and osteoblasts, and in cells heterologously expressing the PTH1R and NHERF1 (Wang et al., 2007). The effect of NHERF1 on PTH1R desensitization has not been defined. We speculated that PTH1R desensitization may involve facilitated interaction of -arrestin1 or arrestin2, the PTH1R in cells lacking or with diminished expression of NHERF1 compared with cells expressing high levels of NHERF1. Using several different cell models, we show that NHERF1 potently inhibits PTH1R desensitization. This effect requires both intact PDZ and MERM domains in NHERF1. NHERF1 reduces PTH1R desensitization by preventing or displacing -arrestin2 binding to the receptor and inhibits PTH1R dissociation from G s. Materials and Methods Materials. NHERF1 rabbit polyclonal antibody was purchased from Affinity Bioreagents (Golden, CO). HA.11 ascites monoclonal antibody and HA.11 monoclonal affinity matrix were obtained from Covance Research Products (Princeton, NJ). Horseradish peroxidase-conjugated goat anti-rabbit secondary antibody was from Pierce (Rockord, IL). Horseradish peroxidase-conjugated sheep anti-mouse antibody was from GE Healthcare (Chalfont St. Giles, Buckinghamshire, UK). Tetracycline hydrochloride was purchased from American Bioanalytical (Natick, MA). Lipofectamine 2000, Zeocin, blasticidin, polyclonal GFP antibody, and protein A-Sepharose 4B conjugate were obtained from Invitrogen (Carlsbad, CA). Protease inhibitor mixture Set I was from Calbiochem (San Diego, CA). Human PTH(1–34) was purchased from Bachem California (Torrance, CA). GFP agarose, polyclonal actin antibody, dynamin siRNA, and monoclonal dynamin antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). cAMP-Glo assay kit was obtained from Promega (Madison, WI). FuGENE 6 was purchased from Roche Applied Science (Indianapolis, IN). Polyclonal G s antibody was obtained from Millipore (Billerica, MA). All other reagents were from Sigma-Aldrich (St. Louis, MO). Cell Culture. Rat osteosarcoma ROS 17/2.8 cells and human osteosarcoma SAOS2 cells were cultured in Dulbecco’s modified Eagle’s medium/Ham’s F-12 medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 g/ml streptomycin. COS-7 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 g/ml streptomycin. All the cells were maintained at 37°C in a humidified atmosphere of 5% CO2, 95% air. Construction of pcDNA4/TO-NHERF1. His-tagged rabbit NHERF1 in pcDNA3 (provided by Dr. E. J. Weinman, University of Maryland, Baltimore, MD) was cut with KpnI and XhoI, and a 1.1-kilobase pair fragment without epitope was subcloned into the pcDNA4/TO vector (Invitrogen), which has two tetracycline operator sequences between the TATA box of the cytomegalovirus promoter and the transcriptional start site. The fidelity of the plasmids was confirmed by sequencing (ABI PRISM 377; Applied Biosystems, Fos-