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RGS14 regulates PTH- and FGF23-sensitive NPT2A-mediated renal phosphate uptake via binding to the NHERF1 scaffolding protein

2022; Elsevier BV; Volume: 298; Issue: 5 Linguagem: Inglês

10.1016/j.jbc.2022.101836

ISSN

1083-351X

Autores

Peter A. Friedman, W. Bruce Sneddon, Tatyana Mamonova, Carolina Montañez‐Miranda, Suneela Ramineni, Nicholas H. Harbin, Katherine E. Squires, Julia Gefter, Clara E. Magyar, David R. Emlet, John R. Hepler,

Tópico(s)

Fibroblast Growth Factor Research

Resumo

Phosphate homeostasis, mediated by dietary intake, renal absorption, and bone deposition, is incompletely understood because of the uncharacterized roles of numerous implicated protein factors. Here, we identified a novel role for one such element, regulator of G protein signaling 14 (RGS14), suggested by genome-wide association studies to associate with dysregulated Pi levels. We show that human RGS14 possesses a carboxy-terminal PDZ ligand required for sodium phosphate cotransporter 2a (NPT2A) and sodium hydrogen exchanger regulatory factor-1 (NHERF1)–mediated renal Pi transport. In addition, we found using isotope uptake measurements combined with bioluminescence resonance energy transfer assays, siRNA knockdown, pull-down and overlay assays, and molecular modeling that secreted proteins parathyroid hormone (PTH) and fibroblast growth factor 23 inhibited Pi uptake by inducing dissociation of the NPT2A–NHERF1 complex. PTH failed to affect Pi transport in cells expressing RGS14, suggesting that it suppresses hormone-sensitive but not basal Pi uptake. Interestingly, RGS14 did not affect PTH-directed G protein activation or cAMP formation, implying a postreceptor site of action. Further pull-down experiments and direct binding assays indicated that NPT2A and RGS14 bind distinct PDZ domains on NHERF1. We showed that RGS14 expression in human renal proximal tubule epithelial cells blocked the effects of PTH and fibroblast growth factor 23 and stabilized the NPT2A–NHERF1 complex. In contrast, RGS14 genetic variants bearing mutations in the PDZ ligand disrupted RGS14 binding to NHERF1 and subsequent PTH-sensitive Pi transport. In conclusion, these findings identify RGS14 as a novel regulator of hormone-sensitive Pi transport. The results suggest that changes in RGS14 function or abundance may contribute to the hormone resistance and hyperphosphatemia observed in kidney diseases. Phosphate homeostasis, mediated by dietary intake, renal absorption, and bone deposition, is incompletely understood because of the uncharacterized roles of numerous implicated protein factors. Here, we identified a novel role for one such element, regulator of G protein signaling 14 (RGS14), suggested by genome-wide association studies to associate with dysregulated Pi levels. We show that human RGS14 possesses a carboxy-terminal PDZ ligand required for sodium phosphate cotransporter 2a (NPT2A) and sodium hydrogen exchanger regulatory factor-1 (NHERF1)–mediated renal Pi transport. In addition, we found using isotope uptake measurements combined with bioluminescence resonance energy transfer assays, siRNA knockdown, pull-down and overlay assays, and molecular modeling that secreted proteins parathyroid hormone (PTH) and fibroblast growth factor 23 inhibited Pi uptake by inducing dissociation of the NPT2A–NHERF1 complex. PTH failed to affect Pi transport in cells expressing RGS14, suggesting that it suppresses hormone-sensitive but not basal Pi uptake. Interestingly, RGS14 did not affect PTH-directed G protein activation or cAMP formation, implying a postreceptor site of action. Further pull-down experiments and direct binding assays indicated that NPT2A and RGS14 bind distinct PDZ domains on NHERF1. We showed that RGS14 expression in human renal proximal tubule epithelial cells blocked the effects of PTH and fibroblast growth factor 23 and stabilized the NPT2A–NHERF1 complex. In contrast, RGS14 genetic variants bearing mutations in the PDZ ligand disrupted RGS14 binding to NHERF1 and subsequent PTH-sensitive Pi transport. In conclusion, these findings identify RGS14 as a novel regulator of hormone-sensitive Pi transport. The results suggest that changes in RGS14 function or abundance may contribute to the hormone resistance and hyperphosphatemia observed in kidney diseases. Parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23) control serum Pi levels by inhibiting the sodium phosphate cotransporter 2a (NPT2A) in the kidney, thereby promoting regulated Pi excretion. Hormonal control of this process requires the PDZ scaffold protein sodium hydrogen exchanger regulatory factor-1 (NHERF1) (1Hernando N. Gagnon K.B. Lederer E.D. Phosphate transport in epithelial and nonepithelial tissue.Physiol. Rev. 2020; 101: 1-35Crossref PubMed Scopus (23) Google Scholar). Mice lacking NHERF1 and humans harboring NHERF1 mutations are hypophosphatemic, consequent to urinary Pi loss, and osteopenia because of insufficient serum Pi reserves. The cognate type 1 PTH G protein–coupled receptor (PTHR) mediates PTH actions, whereas FGF23 operates through the FGF receptor 1 (FGFR1) receptor tyrosine kinase. Though the two hormones act through structurally unrelated receptor forms, PTH and FGF23 initiate signaling pathways that converge on the NPT2A–NHERF1 complex in kidney cells to inhibit Pi uptake. In the absence of NHERF1, Pi excretion continues unabated with attendant loss of PTH and FGF23 regulation. Despite advances in understanding the biochemical events underlying this process, critical gaps exist in our knowledge of hormone regulatory actions because of additional unidentified components. Here, we introduce evidence that regulator of G protein signaling 14 (RGS14) is one such factor. Numerous genome-wide association studies (GWASs) implicate RGS14 in kidney diseases (2Kestenbaum B. Glazer N.L. Kottgen A. Felix J.F. Hwang S.J. Liu Y. Lohman K. Kritchevsky S.B. Hausman D.B. Petersen A.K. Gieger C. Ried J.S. Meitinger T. Strom T.M. Wichmann H.E. et al.Common genetic variants associate with serum phosphorus concentration.J. Am. Soc. 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Cummings B.B. Tukiainen T. Birnbaum D.P. Kosmicki J.A. Duncan L.E. Estrada K. et al.Analysis of protein-coding genetic variation in 60,706 humans.Nature. 2016; 536: 285-291Crossref PubMed Scopus (6261) Google Scholar) in the human RGS14 PDZ-binding motif, as we reported (9Squires K.E. Montanez-Miranda C. Pandya R.R. Torres M.P. Hepler J.R. Genetic analysis of rare human variants of regulators of g protein signaling proteins and their role in human physiology and disease.Pharmacol. Rev. 2018; 70: 446-474Crossref PubMed Scopus (36) Google Scholar), though the impact of these variants on RGS14 function is unknown. Potential interactions and functional consequences of RGS14 engagement of PDZ proteins have not been described. RGSs are GTPase-activating proteins that function primarily as central components of G protein–coupled receptor and G protein signaling pathways (10Hollinger S. Hepler J.R. Cellular regulation of RGS proteins: Modulators and integrators of G protein signaling.Pharmacol. Rev. 2002; 54: 527-559Crossref PubMed Scopus (590) Google Scholar, 11Ross E.M. Wilkie T.M. GTPase-activating proteins for heterotrimeric G proteins: regulators of G protein signaling (RGS) and RGS-like proteins.Annu. Rev. Biochem. 2000; 69: 795-827Crossref PubMed Scopus (911) Google Scholar). RGS14 is an unusual multifunctional scaffolding protein that integrates G protein, mitogen-activated protein kinase, and Ca2+/calmodulin signaling pathways (12Evans P.R. Dudek S.M. Hepler J.R. Regulator of G Protein signaling 14: A molecular Brake on synaptic plasticity linked to learning and memory.Prog. Mol. Biol. Transl. Sci. 2015; 133: 169-206Crossref PubMed Scopus (13) Google Scholar, 13Evans P.R. Gerber K.J. Dammer E.B. Duong D.M. Goswami D. Lustberg D.J. Zou J. Yang J.J. Dudek S.M. Griffin P.R. Seyfried N.T. Hepler J.R. Interactome analysis reveals regulator of G Protein signaling 14 (RGS14) is a novel calcium/calmodulin (Ca2+/CaM) and CaM Kinase II (CaMKII) binding partner.J. Proteome Res. 2018; 17: 1700-1711Crossref PubMed Scopus (11) Google Scholar). It is the only RGS protein that harbors a canonical PDZ-recognition motif. Rgs14 1Human proteins are indicated by UniProt 3-letter uppercase abbreviation; genes are in italics. Only the first letter is uppercase for the corresponding rodent protein or gene.1Human proteins are indicated by UniProt 3-letter uppercase abbreviation; genes are in italics. Only the first letter is uppercase for the corresponding rodent protein or gene. is most highly expressed in rodents in the brain, lung, heart, and spleen (14Hollinger S. Taylor J.B. Goldman E.H. Hepler J.R. RGS14 is a bifunctional regulator of Gαi/o activity that exists in multiple populations in brain.J. Neurochem. 2001; 79: 941-949Crossref PubMed Scopus (55) Google Scholar). Rgs14 actions are best understood in the rodent brain, where it tonically suppresses synaptic plasticity and hippocampal-based learning (12Evans P.R. Dudek S.M. Hepler J.R. Regulator of G Protein signaling 14: A molecular Brake on synaptic plasticity linked to learning and memory.Prog. Mol. Biol. Transl. Sci. 2015; 133: 169-206Crossref PubMed Scopus (13) Google Scholar, 13Evans P.R. Gerber K.J. Dammer E.B. Duong D.M. Goswami D. Lustberg D.J. Zou J. Yang J.J. Dudek S.M. Griffin P.R. Seyfried N.T. Hepler J.R. Interactome analysis reveals regulator of G Protein signaling 14 (RGS14) is a novel calcium/calmodulin (Ca2+/CaM) and CaM Kinase II (CaMKII) binding partner.J. Proteome Res. 2018; 17: 1700-1711Crossref PubMed Scopus (11) Google Scholar, 15Lee S.E. Simons S.B. Heldt S.A. Zhao M. Schroeder J.P. Vellano C.P. Cowan D.P. Ramineni S. Yates C.K. Feng Y. Smith Y. Sweatt J.D. Weinshenker D. Ressler K.J. Dudek S.M. et al.RGS14 is a natural suppressor of both synaptic plasticity in CA2 neurons and hippocampal-based learning and memory.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 16994-16998Crossref PubMed Scopus (114) Google Scholar), in the heart, where it diminishes myocardial remodeling (16Li Y. Tang X.H. Li X.H. Dai H.J. Miao R.J. Cai J.J. Huang Z.J. Chen A.F. Xing X.W. Lu Y. Yuan H. Regulator of G protein signalling 14 attenuates cardiac remodelling through the MEK-ERK1/2 signalling pathway.Basic Res. Cardiol. 2016; 111: 47Crossref PubMed Scopus (19) Google Scholar), and in the brown adipose tissue metabolism linked to longevity (17Vatner D.E. Zhang J. Oydanich M. Guers J. Katsyuba E. Yan L. Sinclair D. Auwerx J. Vatner S.F. Enhanced longevity and metabolism by brown adipose tissue with disruption of the regulator of G protein signaling 14.Aging Cell. 2018; 17e12751Crossref PubMed Scopus (17) Google Scholar). Much less is known about human RGS14. Human and rodent Rgs14 share a common domain structure, which includes an amino-terminal RGS domain that binds Gαi/o-GTP and acts as a GTPase-activating protein to limit G protein signaling (14Hollinger S. Taylor J.B. Goldman E.H. Hepler J.R. RGS14 is a bifunctional regulator of Gαi/o activity that exists in multiple populations in brain.J. Neurochem. 2001; 79: 941-949Crossref PubMed Scopus (55) Google Scholar, 18Cho H. Kozasa T. Takekoshi K. De Gunzburg J. Kehrl J.H. RGS14, a GTPase-activating protein for Giα, attenuates Giα- and G13iα-mediated signaling pathways.Mol. Pharmacol. 2000; 58: 569-576Crossref PubMed Scopus (77) Google Scholar); two tandem Ras/Rap-binding domains that bind active H-Ras and Rap2 (19Traver S. Bidot C. Spassky N. Baltauss T. De Tand M.F. Thomas J.L. Zalc B. Janoueix-Lerosey I. Gunzburg J.D. RGS14 is a novel Rap effector that preferentially regulates the GTPase activity of galphao.Biochem. J. 2000; 350 Pt 1: 19-29Crossref PubMed Google Scholar, 20Willard F.S. Willard M.D. Kimple A.J. Soundararajan M. Oestreich E.A. Li X. Sowa N.A. Kimple R.J. Doyle D.A. Der C.J. Zylka M.J. Snider W.D. Siderovski D.P. Regulator of G-protein signaling 14 (RGS14) is a selective H-Ras effector.PLoS One. 2009; 4e4884Crossref Scopus (33) Google Scholar, 21Shu F.J. Ramineni S. Hepler J.R. RGS14 is a multifunctional scaffold that integrates G protein and Ras/Raf MAPkinase signalling pathways.Cell. Signal. 2010; 22: 366-376Crossref PubMed Scopus (57) Google Scholar); and a G protein regulator (also referred to as GoLoco) motif that binds inactive Gαi1/3 to anchor Rgs14 at membranes (22Shu F.J. Ramineni S. Amyot W. Hepler J.R. Selective interactions between Giα1 and Giα3 and the GoLoco/GPR domain of RGS14 influence its dynamic subcellular localization.Cell Signal. 2007; 19: 163-176Crossref PubMed Scopus (36) Google Scholar). Human, primate, and ovine RGS14 differ from the rodent protein because they contain a carboxy-terminal class I PDZ-recognition sequence. Potential interaction and functional consequences of RGS14 engagement with PDZ proteins have not been described. PDZ proteins, named for the common structural domain shared by the postsynaptic density protein 95 (PSD95), Drosophila disc large tumor suppressor (DlgA), and zonula occludens 1 protein (ZO1), constitute a family of 200 to 300 members (23Hung A.Y. Sheng M. PDZ domains: Structural modules for protein complex assembly.J. Biol. Chem. 2002; 277: 5699-5702Abstract Full Text Full Text PDF PubMed Scopus (583) Google Scholar, 24Romero G. Von Zastrow M. Friedman P.A. Role of PDZ proteins in regulating trafficking, signaling, and function of GPCRs. Means, motif, and opportunity.Adv. Pharmacol. 2011; 62: 279-314Crossref PubMed Scopus (116) Google Scholar). These adapter molecules transiently assemble a variety of membrane-associated proteins, including transporters, receptors, ion channels, and signaling molecules in short-lived functional units. PDZ modules consist of 80 to 90 amino acids forming a 3-dimensional globular domain that is composed of six β-sheets (βA–βF) and two α-helices (αA and αB) within the larger protein (23Hung A.Y. Sheng M. PDZ domains: Structural modules for protein complex assembly.J. Biol. Chem. 2002; 277: 5699-5702Abstract Full Text Full Text PDF PubMed Scopus (583) Google Scholar). Scaffolding proteins harboring PDZ domains may contain single or multiple PDZ modules and can also include other protein–protein interaction motifs (24Romero G. Von Zastrow M. Friedman P.A. Role of PDZ proteins in regulating trafficking, signaling, and function of GPCRs. Means, motif, and opportunity.Adv. Pharmacol. 2011; 62: 279-314Crossref PubMed Scopus (116) Google Scholar). Class I PDZ ligands take the form Asp/Glu-Ser/Thr-X-Φ (D/E-S/T-X-Φ), where X is promiscuous and Φ is a hydrophobic residue. The human RGS14 PDZ-recognition sequence is -Asp-Ser-Ala-Leu (-DSAL). NPT2A is expressed at luminal membranes of proximal kidney tubules, where it mediates the bulk of Pi absorption from the urine (25Beck L. Karaplis A.C. Amizuka N. Hewson A.S. Ozawa H. Tenenhouse H.S. Targeted inactivation of Npt2 in mice leads to severe renal phosphate wasting, hypercalciuria, and skeletal abnormalities.Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5372-5377Crossref PubMed Scopus (497) Google Scholar). NPT2A activity is regulated by PTH and FGF23 and requires NHERF1 (26Cunningham R. Xiaofei E. Steplock D. Shenolikar S. Weinman E.J. Defective PTH regulation of sodium-dependent phosphate transport in NHERF-1-/- renal proximal tubule cells and wild-type cells adapted to low phosphate media.Am. J. Physiol. Ren. Physiol. 2005; 289: F933-F938Crossref PubMed Scopus (40) Google Scholar, 27Andrukhova O. Zeitz U. Goetz R. Mohammadi M. Lanske B. Erben R.G. FGF23 acts directly on renal proximal tubules to induce phosphaturia through activation of the ERK1/2-SGK1 signaling pathway.Bone. 2012; 51: 621-628Crossref PubMed Scopus (140) Google Scholar). NPT2A also contains a carboxy-terminal PDZ motif that facilitates its binding to NHERF1 (28Gisler S.M. Stagljar I. Traebert M. Bacic D. Biber J. Murer H. Interaction of the type IIa Na/Pi cotransporter with PDZ proteins.J. Biol. Chem. 2001; 276: 9206-9213Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). NHERF1 possesses two tandem PDZ domains (PDZ1 and PDZ2) with identical core-binding sequences and a carboxy-terminal Ezrin-binding domain, through which interacting proteins are assembled at the plasma membrane and to cortical actin (29Brone B. Eggermont J. PDZ proteins retain and regulate membrane transporters in polarized epithelial cell membranes.Am. J. Physiol. Cell Physiol. 2005; 288: C20-C29Crossref PubMed Scopus (85) Google Scholar). Recent analysis disclosed that sites upstream of the PDZ-recognition sequence and the 3-dimensional organization of the PDZ module impart greater specificity to the recognition and binding determinants between the PDZ protein and the targeted partner (30Mamonova T. Zhang Q. Khajeh J.A. Bu Z. Bisello A. Friedman P.A. Canonical and noncanonical sites determine NPT2A binding selectivity to NHERF1 PDZ1.PLoS One. 2015; 10e0129554Crossref PubMed Scopus (13) Google Scholar). Based on these considerations, we predicted that RGS14, acting through its PDZ ligand, binds NHERF1 and alters hormone-sensitive Pi transport. We theorized that human RGS14 genetic variants within the PDZ-binding motif might not share this behavior and, at the same time, afford further insight into the structural determinants for binding of the NHERF1 PDZ domains. The goal of the work here was to determine the effect of WT RGS14 and the identified PDZ ligand mutations on basal and hormone-regulated Pi transport and the role of RGS14 interactions with NHERF1 in this process. The described studies use a combination of native RGS14 expression in donor human kidney cells or immortalized kidney cell lines and engineered mutations and heterologous expression to demonstrate that RGS14 suppresses PTH and FGF23 actions of NPT2A by interacting with NHERF1. We first explored the presence and localization of RGS14 along with the other protein components relevant to Pi uptake in the kidney. For this purpose, we employed unused donor human kidney tissue and prepared cultures of cells isolated from these samples. The goal was to define and characterize these proteins at spontaneous constitutive levels. RGS14 and NHERF1 are conspicuously expressed in human kidney (Fig. 1A). Protein lysates prepared from proximal tubule cells isolated from three independent patient samples (#55, #57, and #58) (Fig. 1B) displayed abundant RGS14 as well as NHERF1, NPT2A, and Ezrin, a proximal tubule cell marker that binds NHERF1 (31Reczek D. Bretscher A. The carboxyl-terminal region of EBP50 binds to a site in the amino-terminal domain of Ezrin that is masked in the dormant molecule.J. Biol. Chem. 1998; 273: 18452-18458Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). RGS14 and NHERF1 coimmunoprecipitated in these samples expressing protein at endogenous levels (Fig. 1B). We next localized RGS14 in immortalized human proximal convoluted tubule (HPCT) cells and its binding to NHERF1. Proximity ligation assays (PLA) demonstrated the presence and significant colocalization of RGS14 with NHERF1, again at constitutive expression levels in HPCT cells (Fig. 1C). We next examined the effects of RGS14 on PTH-sensitive Pi uptake cells derived from the donor samples. RGS14 (and RGS12) function as negative regulators of G protein signaling by inactivating Gα subunits. We reasoned that if RGS14 tonically inhibits PTH action, then ablating RGS14 should relieve this blockade. RGS14 siRNA knockdown (siRGS14) completely blocked RGS14 expression in primary kidney cells but did not affect actin expression (Fig. 2A). PTH did not inhibit Pi transport in these primary kidney cells (Fig. 2B), but siRGS14 conspicuously unmasked PTH-sensitive Pi transport (Fig. 2B). In HPCT cells, siRGS14 virtually eliminated RGS14 expression without detectably affecting protein levels of NHERF1, PTHR, or NPT2A (Fig. 2C). RGS14 siRNA knockdown revealed FGF23-sensitive and PTH-sensitive Pi transport (Fig. 2D). There was no hormone modulation of Pi transport in control cells transfected with scrambled siRNA. Treatment of HPCT cells with PF-06869206, a selective NPT2A inhibitor (32Filipski K.J. Sammons M.F. Bhattacharya S.K. Panteleev J. Brown J.A. Loria P.M. Boehm M. Smith A.C. Shavnya A. Conn E.L. Song K. Weng Y. Facemire C. Juppner H. Clerin V. Discovery of orally bioavailable selective inhibitors of the sodium-phosphate cotransporter NaPi2a (SLC34A1).ACS Med. Chem. Lett. 2018; 9: 440-445Crossref PubMed Scopus (17) Google Scholar, 33Clerin V. Saito H. Filipski K.J. Nguyen A.H. Garren J. Kisucka J. Reyes M. Juppner H. Selective pharmacological inhibition of the sodium-dependent phosphate co-transporter NPT2a promotes phosphate excretion.J. Clin. Invest. 2020; 130: 6510-6522Crossref PubMed Scopus (10) Google Scholar), blocked phosphate uptake by greater than 95% (Fig. 2D), indicating that NPT2A mediates virtually all phosphate uptake by HPCT cells. These findings suggested that RGS14 might directly interfere with PTHR signaling, thereby disrupting its physiological action on NPT2A. PTHR couples to Gs to stimulate cAMP production and activate PKA, which, in turn, phosphorylates NHERF1 to uncouple it from NPT2A (34Zizak M. Lamprecht G. Steplock D. Tariq N. Shenolikar S. Donowitz M. Yun C.H. Weinman E.J. cAMP-induced phosphorylation and inhibition of Na+/H+ exchanger 3 (NHE3) are dependent on the presence but not the phosphorylation of NHE regulatory factor.J. Biol. Chem. 1999; 274: 24753-24758Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 35Deliot N. Hernando N. Horst-Liu Z. Gisler S.M. Capuano P. Wagner C.A. Bacic D. O'Brien S. Biber J. Murer H. Parathyroid hormone treatment induces dissociation of type IIa Na+-Pi cotransporter-Na+/H+ exchanger regulatory factor-1 complexes.Am. J. Physiol. Cell Physiol. 2005; 289: C159-C167Crossref PubMed Scopus (92) Google Scholar, 36Zhang Q. Xiao K. Paredes J.M. Mamonova T. Sneddon W.B. Liu H. Wang D. Li S. McGarvey J.C. Uehling D. Al-awar R. Joseph B. Jean-Alphonse F. Orte A. Friedman P.A. Parathyroid hormone initiates dynamic NHERF1 phosphorylation cycling and conformational changes that regulate NPT2A-dependent phosphate transport.J. Biol. Chem. 2019; 294: 4546-4571Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). PTHR can also couple to Gq/11 (37Schwindinger W.F. Fredericks J. Watkins L. Robinson H. Bathon J.M. Pines M. Suva L.J. Levine M.A. Coupling of the PTH/PTHrP receptor to multiple G-proteins. Direct demonstration of receptor activation of Gs' Gq/11' and Gi(1) by [a-32P]GTP-g-azidoanilide photoaffinity labeling.Endocrine. 1998; 8: 201-209Crossref PubMed Scopus (76) Google Scholar, 38Wang B. Ardura J.A. Romero G. Yang Y. Hall R.A. Friedman P.A. Na/H exchanger regulatory factors control PTH receptor signaling by differential activation of Gα protein subunits.J. Biol. Chem. 2010; 285: 26976-26986Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). RGS14 binds directly to active Gαi/o and inactive Gαi1/3 but does not directly engage either Gαs or Gαq (14Hollinger S. Taylor J.B. Goldman E.H. Hepler J.R. RGS14 is a bifunctional regulator of Gαi/o activity that exists in multiple populations in brain.J. Neurochem. 2001; 79: 941-949Crossref PubMed Scopus (55) Google Scholar, 39Vellano C.P. Maher E.M. Hepler J.R. Blumer J.B. G protein-coupled receptors and resistance to inhibitors of cholinesterase-8A (Ric-8A) both regulate the regulator of g protein signaling 14 RGS14.Galphai1 complex in live cells.J. Biol. Chem. 2011; 286: 38659-38669Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). We sought to determine if RGS14 affects PTHR G protein coupling and second messenger signaling directly or indirectly by binding NHERF1. Here, we applied live-cell bioluminescence resonance energy transfer (BRET) bimolecular fluorescence complementation assays using Ven-Gβ1γ2 and a mas-GRKct-Luc biosensor to detect Gβγ release (40Hollins B. Kuravi S. Digby G.J. Lambert N.A. The c-terminus of GRK3 indicates rapid dissociation of G protein heterotrimers.Cell Signal. 2009; 21: 1015-1021Crossref PubMed Scopus (90) Google Scholar, 41Hynes R.O. Metastatic cells will take any help they can get.Cancer Cell. 2011; 20: 689-690Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Human embryonic kidney 293 (HEK293) cells were transfected with hemagglutinin (HA)-PTHR, Gsα, Gβγ-Ven, and GRK-Luc and either FLAG-RGS14 alone or NHERF1 plus FLAG-RGS14 (Fig. 3A). Cells were stimulated with PTH, activating PTHR, causing Gβγ-Ven dissociation from active Gαs, binding to GRK-Luc, and increasing the BRET signal as an index of G protein activation. The results show that RGS14 did not affect PTHR-G activation assessed as BRET from Gβγ-Ven and GRK-Luc (Fig. 3B). Consistent with this finding, RGS14 also did not alter PTH-triggered cAMP production. PTH stimulation of cAMP in cells transfected with the cAMP BRET sensor CAMYEL, PTHR, and either FLAG-RGS14 alone or NHERF1 plus RGS14 was not significantly altered in the presence of either RGS14 or NHERF1 alone, or when the two proteins were added in combination (Fig. 3C). The absence of an effect of RGS14 on PTHR-Gs coupling or cAMP production suggested that RGS14 may exert its regulatory action at a postreceptor site. This interpretation predicts that RGS14 should interfere with PTH-PTHR and FGF23/FGFR1, regulation of Pi transport, and phosphorylate NHERF1 dissociating it from NPT2A. To test this idea, we used human renal proximal tubule epithelial cells (RPTECs) that lack RGS14. Heterologous expression of RGS14 in RPTEC markedly obstructed PTH and FGF23 inhibition of Pi uptake (Fig. 4A), strongly suggesting that RGS14 acts at a common postreceptor locus of action shared by both PTH and FGF23 that directly engages the NPT2A–NHERF1 complex to regulate its function. Consistent with this view, FGF23 (and PTH) inhibited phosphate uptake in HPCT cells (Fig. 2D) supporting the idea that RGS14 actions are downstream of G proteins. When expressed in RPTEC, RGS14 was recovered by coimmunoprecipitation (IP) as a stable complex with NHERF1 and NPT2A (Fig. 4B). Together with findings from Figures 1 and 2, these observations establish that (1) RGS14 is expressed in human kidney and colocalizes with NHERF1; (2) RGS14 forms a stable complex with NHERF1; (3) RGS14 suppresses PTH- and FGF23-sensitive Pi transport; and (4) RGS14 knockdown unmasks hormone-regulated Pi transport. Thus, RGS14 inhibits PTH and FGF23 actions on NPT2A-mediated Pi uptake by engaging NHERF1. We next explored the molecular details by which human RGS14 and NHERF1 bind one another. Human RGS14 possesses a canonical class I carboxy-terminal PDZ ligand consisting of the sequence -DSAL (Fig. 5A) that is absent in rodent Rgs14. To confirm the requirement for the PDZ-recognition motif for binding with NHERF1, we generated constructs for full-length FLAG-tagged human hRGS14 and rat Rgs14 and performed co-IP experiments using HEK293 cells cotransfected with FLAG-RGS14/Rgs14 and HA-NHERF1. The results shown in Figure 5B substantiate that human RGS14 containing a PDZ ligand binds NHERF1, whereas rat Rgs14 lacking the PDZ ligand does not. NHERF1 contains two tandem PDZ domains (Fig. 5A). The two NHERF1 PDZ domains share matching GYGF core-binding motifs but display substrate specificity that is not necessarily identical (42Mamonova T. Zhang Q. Chandra M. Collins B.M. Sarfo E. Bu Z. Xiao K. Bisello A. Friedman P.A. Origins of PDZ binding specificity. A computational and experimental case study using NHERF1 and the parathyroid hormone receptor.Biochemistry. 2017; 56: 2584-2593Crossref PubMed Scopus (6) Google Scholar). We, therefore, inquired if RGS14 exhibited domain selectivity for binding with PDZ1 or PDZ2. For these experiments, we used NHERF1 constructs wherein the GYGF PDZ core-binding motif was mutated to GAGA in PDZ1 (P1), PDZ2 (P2), or both domains (P1 and P2). As shown in Figure 5C, RGS14 binds to WT NHERF1 and NHERF1-P1 but not to NHERF1-P2 or the double mutant (P1P2), indicating that RGS14 selectively binds PDZ2 in cells. As a control, we confirmed that mutant NHERF1 P2 is functional (Fig. 5D) by showing that WT-NHERF1 and NHERF1 mutants P1 and P2 each bind Ezrin. Together, these findings indicate that the -DSAL PDZ ligand unique to human RGS14 selectively and directly binds to the PDZ2 domain of NHERF1. Our findings suggest that human RGS14 binds directly to the PDZ protein NHERF1 (Fig. 5). To

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