The molecular sociology of NHERF1 PDZ proteins controlling renal hormone-regulated phosphate transport

Abstract Parathyroid hormone (PTH) and fibroblast growth factor-23 (FGF23) control extracellular phosphate levels by regulating renal NPT2A-mediated phosphate transport by a process requiring the PDZ scaffold protein NHERF1. NHERF1 possesses two PDZ domains, PDZ1 and PDZ2, with identical core-binding GYGF motifs explicitly recognizing distinct binding partners that play different and specific roles in hormone-regulated phosphate transport. The interaction of PDZ1 and the carboxy-terminal PDZ-binding motif of NPT2A (C-TRL) is required for basal phosphate transport. PDZ2 is a regulatory domain that scaffolds multiple biological targets, including kinases and phosphatases involved in FGF23 and PTH signaling. FGF23 and PTH trigger disassembly of the NHERF1–NPT2A complex through reversible hormone-stimulated phosphorylation with ensuing NPT2A sequestration, down-regulation, and cessation of phosphate absorption. In the absence of NHERF1–NPT2A interaction, inhibition of FGF23 or PTH signaling results in disordered phosphate homeostasis and phosphate wasting. Additional studies are crucial to elucidate how NHERF1 spatiotemporally coordinates cellular partners to regulate extracellular phosphate levels.


Introduction
Phosphate is continuously absorbed from the intestines and primarily stored in bone and teeth.Phosphate wasting or hypophosphatemia associated with malnourishment, chronic kidney disease, and frank resistance to hormone action contributes to exceptionally high mortality rates, especially among the elderly and impoverished [1][2][3].Phosphate serum levels and homeostasis are achieved by parathyroid hormone (PTH) and fibroblast growth factor-23 (FGF23) acting through a bone-kidney axis [4].PTH and FGF23 regulate NPT2A-mediated Na + -phosphate cotransport by a mechanism requiring NHERF1, a PDZ scaffold phosphoprotein [5][6][7][8][9].In the presence of NHERF1, PTH works through its cognate G protein-coupled receptor (PTHR) [10] and FGF23 via a receptor tyrosine kinase (FGFR1) and α-Klotho [11] to block NPT2A-mediated phosphate uptake (Figure 1).PTHR and FGFR1 are members of structurally disparate receptor classes with distinct signaling pathways (Figure 2).Inexplicably, sometimes PTH and FGF23 act in concert but at other times independently [12][13][14][15].The mechanism underlying these events is not understood.Nonetheless, operating through distinct kinases (Figure 1), both pathways converge to activate G protein-coupled receptor kinase 6A (GRK6A) [16][17][18].GRK6A binds NHERF1 and phosphorylates it at Ser 290 , triggering dissociation of NPT2A from NHERF1 and terminating hormone-sensitive phosphate transport [17] (Figure 3).It is a biological riddle of how a GPCR and an FGFR phosphorylate the same obligate residue to regulate phosphate uptake without interfering with each other.
Recently, it was identified that the transmembrane protein 174 (Tmem174) is significantly coexpressed with Slc34a1.Tmem174 is a kidney-specific protein located at the apical membrane of renal proximal tubular cells [37,38].Furthermore, it was suggested that TMEM174 interacts with NPT2A, but not NHERF1, and regulates NPT2A by PTH and FGF23 in human kidney [37,38].We speculate that TMEM174-NPT2A-NHERF1 may form a ternary complex at apical membranes of renal proximal tubules.A multistep mechanism would dissociate the complex in this scenario, permitting FGF23-and PTH-mediated NPT2A internalization.
The specificity of the interaction between NHERF1 PDZ1 and NPT2A relates to Glu 43 and Arg −1 in PDZ1 and the NPT2A C-TR −1 L, respectively [29,32,33].Asp 183 , located at the homologous position in PDZ2, has a shorter side chain compared with Glu 43 to form a direct interaction with Arg −1 [32].PDZ2 with the Asp 183 Glu rescue mutation interacts with Arg −1 , comparable with PDZ1, but does not support hormone-sensitive phosphate transport, therefore underlines the importance of the binding between PDZ1 and NPT2A for hormone regulation [32,33].It should be noted that NPT2C, a SLC34A3 paralog expressed in the kidney, lacks a PDZ ligand, does not bind NHERF1, and supports approximately 30% of Na + -dependent phosphate transport (Npt2a handles 70%).Overall PTH and FGF23 action on Npt2c is still unclear [39,40].

NPT2A
The type-2 sodium-phosphate cotransporter (NPT2A, SLC34A1) is the principal protein mediating hormone-sensitive kidney phosphate absorption [64,65].It is primarily expressed in kidney proximal tubules [66,67] and osteoblasts [68,69].Mice lacking NPT2A display FGF23-independent renal phosphate wasting and hypercalciuria [67].The predicted topology of NPT2A consists of 8 membrane-spanning segments and two helical hairpins [70,71].AlphaFold [72] provided additional structural details of NPT2A (Figure 4).Retrieval from the cell membrane and sequestration controls NPT2A and related SLC34 proteins, unlike most Na + -coupled transporters whose function is regulated directly by posttranslational modifications [73].FGF23 and PTH trigger endocytosis [30,42,65] but only in the presence of NHERF1 because the NPT2A C-TRL binds NHERF1 PDZ1, and this interaction establishes NPT2A apical localization [33].In addition to its canonical carboxy-terminal PDZ ligand, NPT2A possesses a second previously uncharacterized internal PDZ ligand (-Thr −2(494) Arg −1(495) Leu 0(496) ) [33]  (Figure 4).Such an internal motif may establish a stable secondary structure sterically nestled in the binding groove of the PDZ2 domain [74,75].Loss-of-function mutations in this cryptic internal motif (Arg 495 His or Arg 495 Cys) that were not recognized as part of a PDZ ligand cause congenital phosphate wasting and hypophosphatemia [76,77].Unlike WT NPT2A, neither Arg 495 Cys nor Arg 495 His dissociates from NHERF1 upon challenge with PTH [33] and fails to terminate phosphate transport.Concurrently, as demonstrated by confocal fluorescence microscopy, NPT2A Arg 495 Cys and Arg 495 His variants colocalized with NHERF1 at apical cell membranes like WT NPT2A [33].Consistent with the functional results, NPT2A Arg 495 Cys and Arg 495 His variants do not internalize in response to PTH but remain at the apical membrane.Enigmatically, neither Arg 495 Cys nor Arg 495 His mutations are at the -1 locus (Arg 495 ) of the PDZ ligand, considered a permissive position [78].Notably, replacing Arg 495 at the PDZ -1 position with disease-associated mutations Cys or His impairs Thr 494 phosphorylation compared with WT NPT2A [33].Using Alphafold2 and MD simulations, we found that Thr 494 and Leu 496 are solvent-exposed and may interact with NHERF1 [33].Thr 494 phosphorylation was detected in cells transfected with WT NHERF1 but not NHERF1 PDZ1, verifying the requirement for NHERF1 PDZ2 [33].The latter may explain why extensive mutagenesis studies by Murer and colleagues failed to identify phosphorylation residues that accounted for the effect of PTH on the apical membrane abundance of Npt2a [79,80].We advance a model wherein the carboxy-terminal NPT2A PDZ ligand binds NHERF1 PDZ1 and defines apical localization of cotransporter, while the internal NPT2A PDZ ligand controls hormone-triggered phosphate transport through the interaction with PDZ2 [33].
The molecular determinants beyond the canonical binding site disclose a distinct electrostatic network playing a specific role in recognizing the PTHR C-terminus by the PDZ domains of SNX27 [84] and Scribble [85].Currently, there is no information about the exact site of NHERF1 modification or the effect of phosphorylation by PKA.Several studies focused on NHERF1 post-translational modification by PKC [53,[86][87][88], Akt [89], and Cdc2 [90].PKC-induced Ser 77 /Thr 95 phosphorylation in PDZ1 uncouples NPT2A and terminates PTH-sensitive phosphate transport [91,92].Less is known about the physiological importance of Ser 339 and Ser 340 , NHERF1 phosphorylation sites located in the flexible linker between PDZ2 and the EBD.It was shown that the phosphorylation-mimicking NHERF1 S 339 D/S 340 D mutant has a better binding affinity and stoichiometry for the carboxy-terminal PDZ-binding ligand of CFTR [88].These results let us hypothesize that in the cellular environment, phosphorylation of these residues by PKC may promote conformational changes in NHERF1 or disrupt the autoinhibition interaction between NHERF1 PDZ2 and its own the carboxy-terminal PDZ ligand or both, making the PDZ2 domain more available for cellular targets including kinases, receptors, and signaling proteins [88].How NHERF1 conformational rearrangement and plasticity regulate NHERF1-dependent hormone-regulated phosphate transport remains to be investigated.

Kinases (with PDZ ligands) involved in FGF23-regulated phosphate transport
NHERF1 residues phosphorylated by FGF23 have not been described, though FGF23 clearly leads to NHERF1 phosphorylation [13,63].We and others showed that ERK1/2 and SGK1 are downstream modulators of FGF23 signaling [11,36].In agreement with previous studies, the ERK inhibitor, PD98059, blocked PTH and FGF23 actions on phosphate transport.The MAP kinase inhibitor, SB203580, only interfered with FGF23.The JNK inhibitor, SP600125, did not affect phosphate uptake [36].Thus, ERK1/2 participates in mutual PTH/FGF23 regulation.PIK75, a p38γ-specific inhibitor [94], blocked FGF23 action without affecting PTH, as shown by pilot studies (Figure 5).Thus, p38γ may be a new mediator of FGF23-regulated phosphate transport.Similar to GRK6A [18], SGK1 and p38γ MAP kinase have Class I type PDZ-binding motifs at their C-termini, may interact with NHERF1 PDZ domains, and phosphorylate NHERF1 controlling association and disassembly of the NHERF1-NPT2A complex, NPT2A endocytosis and cessation of phosphate transport.

p38γ (MAPK12)
p38γ, unlike p38α, p38β, or p38δ isoforms, uniquely possesses a canonical C-terminal PDZ ligand (-Glu −3 Thr −2 Pro −1 Leu 0(367) ).p38γ forms multiprotein complexes with PDZ domain-containing proteins and phosphatases [95,96].This raises the hypothesis that NHERF1 is a p38γ substrate and that p38γ C-terminal PDZ-ligand binding to NHERF1 promotes NHERF1 phosphorylation required for FGF23-sensitive phosphate transport.Although the p38γ C-terminal sequence (-Glu −3 Thr −2 Pro −1 Leu 0 ) is permissive for binding NHERF1 PDZ1 or PDZ2, we consider PDZ2 as a regulatory domain involved in the interactions with different targets, including p38γ [18], whereas PDZ1 interacts with NPT2A and defines basic phosphate transport [33].NHERF1 has four in silico predicted MAP kinase consensus sites [97,98]. 44PGSP 47 and 184 PDSP 187 sites are in PDZ1 and PDZ2, respectively, and 280 SP 281 and 302 SP 303 are located in the NHERF1 flexible linker between PDZ2 and EBD.Substitution of Ser 46 or Ser 186 by Ala eliminates FGF23-sensitive phosphate uptake without affecting PTH action, as shown by preliminary results (Figure 6), consistent with the idea that the p38γ MAP kinase is involved in FGF23 action on NPT2A-mediated phosphate transport.Phosphoresistant NHERF1 Ser 280 Ala or Ser 302 Ala replacements do not interfere with PTH action on phosphate transport and behave as WT NHERF1 [17], thereby suggesting that Ser 280 and Ser 302 are not involved in PTH-induced phosphorylation.We cannot exclude the possibility that the FGF23-p38γ or FGF23-ERK1/2 pathway impinges on the phosphorylation of Ser 280 and Ser 302 .Whether Ser 46 , Ser 186 , Ser 280 , and Ser 302 are p38γ or ERK1/2 phosphorylation sites contributing to FGF23-stimulated phosphorylation remains to be confirmed.

PIN1
ERKs and p38 MAP kinase are proline-directed kinases (phospho-Ser/Thr-Pro).Proline uniquely adopts cis and trans conformations catalyzed by peptidyl-prolyl isomerases (PPIases) [99].NHERF1 has four 'SerPro' sequences.Peptidyl-prolyl isomerases PIN1 is associated with phosphorylated NHERF1.Dephosphorylation of NHERF1 was blocked in the presence of juglone, a PIN1-selective inhibitor.This study demonstrates that PIN1 regulates phosphorylation-dephosphorylation of NHERF1 [90].Because phosphorylation-dephosphorylation cycling is necessary for hormone-mediated phosphate transport [17], we speculate that PIN1 may be an unrecognized regulator of hormone-mediated inhibition of phosphate uptake.To test this idea, we measured NPT2A-dependent hormone-sensitive phosphate transport in OK cells treated with FGF23 [33] in the presence of juglone, rapamycin, and FK506.The pilot observations demonstrate that FGF23-and PTH-sensitive phosphate uptake was terminated in the presence of PIN1 inhibitors.These data corroborate our previous observation that ERK1/2 is involved in FGF23 and PTH action on phosphate transport [36].
activate ERK1/2, p38γ, or phosphorylate an unidentified NHERF1-interacting protein.It is essential to uncover how SGK1 affects the NHERF1-NPT2A complex and NPT2A localization and determine its upstream and downstream partners in FGF23 action on phosphate transport.

RGS14
Regulators of G protein signaling (RGSs) are GTPase-activating proteins that accelerate GTP hydrolysis and terminate GPCR signaling [108][109][110].RGS14 is an unusual multifunctional scaffolding protein that integrates G protein, mitogen-activated protein kinase, and Ca 2+ /calmodulin signaling pathways [111,112].The best evidence for the activities of Rgs14 comes from studies conducted on rodent brains, where it tonically suppresses hippocampal-based learning and synaptic plasticity [111,112,114], in the heart, where it reduces myocardial remodeling [115], and in brown adipose tissue metabolism associated with longevity [116].Information regarding human RGS14 is far less.Two tandem Ras/Rap-binding domains that bind active H-Ras and Rap2 [118], an amino-terminal RGS domain that binds Gαi/o-GTP and functions as a GTPase-activating protein to limit G protein signaling [113,117], and a G protein regulator (also known as GoLoco) motif that binds inactive Gαi1/3 to anchor Rgs14 at membranes [119] are among the common domain structures shared by human and rodent Rgs14.Human, primate, and ovine RGS14 differ from the rodent protein because they contain a C-terminal Class I type PDZ-recognition sequence (-Asp −3 Ser −2 Ala −1 Leu 0(566) ) [110].The difference arises from a UAG stop codon in exon 15 of all species other than primates on sheep terminating mRNA translation.The corresponding primate codon is CAG, which encodes the Gln (Q) at RGS14 546 [120].The potential interaction and functional consequences of RGS14 engagement with PDZ proteins have not been described.The two NHERF1 PDZ domains share identical GYGF core-binding motifs, but RGS14 selectively binds PDZ2 in cells [110] (Figure 7).Proximity-ligation analysis (PLA) at constitutive expression levels in human proximal convoluted tubule (HPCT) cells demonstrated the presence and significant colocalization of RGS14 with NHERF1.More importantly, RGS14 does not interfere with the interaction between NPT2A and NHERF1 PDZ1 (Figure 7) required for hormone-triggered inhibition of phosphate uptake.PTH failed to affect phosphate transport in cells expressing RGS14, suggesting it tonically suppresses PTH-sensitive but not basal phosphate uptake [110].We expect comparable results for FGF23 action.These findings indicate that RGS14 is a novel regulator of hormone-sensitive phosphate transport.Additional studies are required to understand how RGS14 abundance may contribute to hormone resistance and hyperphosphatemia.RGS12 is a PDZ-containing protein with a C-terminal Class I type PDZ-ligand motif (Thr −2 Phe −1 Val 0 ).We predict that RGS12 with an intact carboxy terminus (residues 667-1447) will block FGF23-and PTH-sensitive phosphate transport.Further studies may shed light on the role of the RGS12 C-terminal PDZ ligand and PDZ domain on NPT2A-dependent hormone-regulated phosphate homeostasis.

Concluding remarks
PTHR and FGFR1, two structurally distinct transmembrane receptors, regulate NPT2A-dependent phosphate homeostasis.Both enable signaling pathways converging on the NPT2A-NHERF1 complex.Mutations in NPT2A or NHERF1 cause elevated renal phosphate excretion and hypophosphatemia in patients, thus highlighting an essential role of the NPT2A-NHERF1 axis in bone and kidney physiology.NHERF1 PDZ1 domain determines the NPT2A apical membrane localization and basal phosphate transport.PDZ2 tethers PDZ and non-PDZ cellular targets, including kinases involved in FGF23 and PTH signaling, and serves as a regulatory domain.Signaling cascades initiated by PTH and FGF23 and controlled NPT2A and NHERF1 phosphorylation are required for hormone-sensitive phosphate transport in health and disease.Identifying FGF23 and PTH downstream regulators and mechanisms underlying hormone-induced phosphorylation will be critical for understanding disordered renal phosphate transport and mineral-ion metabolism associated with chronic kidney disease-mineral and bone disorder (CKD-MBD) and related phosphate-wasting disorders.

Figure 4 .
Figure 4.A computational model of NPT2A AlphaFold2 predicts the structure of NPT2A.Carboxy-terminal (C-TRL) and internal TRL PDZ motifs are depicted in magenta and red, respectively.The insert shows the orientation of the NPT2A internal TRL PDZ motif.Such an internal motif may establish a stable secondary structure sterically nestled in the binding groove of the PDZ domain.

Figure 6 .
Figure 6.Effect of the serine/alanine replacement in NHERF1 on FGF23-and PTH-sensitive phosphate transport NHERF1 Ser 46 and Ser 186 are predicted MAP kinase phosphorylation sites.NHERF1 Ser 46 Ala and Ser 186 Ala substitution selectively abolish FGF23-inhibited phosphate transport at comparable transfection efficiency.OKH cells were transiently transfected with WT-NHERF1 or NHERF1 Ser 46 Ala or Ser 186 Ala variants.Cells were treated with vehicle or 100 nM PTH(1-34) or FGF23.NHERF1