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The Na+-dependent phosphate cotransporter-2A (NPT2A, SLC34A1) is a primary regulator of extracellular phosphate homeostasis. Its most prominent structural element is a carboxy-terminal PDZ ligand that binds Na+/H+ Exchanger Regulatory Factor-1 (NHERF1, SLC9A3R1). NHERF1, a multidomain PDZ protein, establishes NPT2A membrane localization and is required for hormone-inhibitable phosphate transport. NPT2A also possesses an uncharacterized internal PDZ ligand. Two recent clinical reports describe congenital hypophosphatemia in children harboring Arg495His or Arg495Cys variants within the internal PDZ motif. The wild-type internal 494TRL496 PDZ ligand binds NHERF1 PDZ2, which we consider a regulatory domain. Ablating the internal PDZ ligand with a 494AAA496 substitution blocked hormone-inhibitable phosphate transport. Complementary approaches, including CRISPR/Cas9 technology, site-directed mutagenesis, confocal microscopy, and modeling, showed that NPT2A Arg495His or Arg495Cys variants do not support PTH or FGF23 action on phosphate transport. Coimmunoprecipitation experiments indicate that both variants bind NHERF1 similarly to WT NPT2A. However, in contrast with WT NPT2A, NPT2A Arg495His, or Arg495Cys variants remain at the apical membrane and are not internalized in response to PTH. We predict that Cys or His substitution of the charged Arg495 changes the electrostatics, preventing phosphorylation of the upstream Thr494, interfering with phosphate uptake in response to hormone action, and inhibiting NPT2A trafficking. We advance a model wherein the carboxy-terminal PDZ ligand defines apical localization NPT2A, while the internal PDZ ligand is essential for hormone-triggered phosphate transport.

The Na+-dependent phosphate cotransporter-2A (NPT2A, SLC34A1) is a primary regulator of extracellular phosphate homeostasis. Its most prominent structural element is a carboxy-terminal PDZ ligand that binds Na+/H+Exchanger Regulatory Factor-1 (NHERF1, SLC9A3R1). NHERF1, a multidomain PDZ protein,establishes NPT2A membrane localization and is required for hormone-sensitive phosphate transport. NPT2A also possesses an uncharacterized internal PDZ ligand. Two recent clinical reports describe congenital hypophosphatemia in children harboring Arg495His or Arg495Cys variants within the internal PDZ motif. The wild-type internal494TRL496PDZ ligand binds NHERF1 PDZ2, which we consider a regulatory domain. Ablating the internal PDZ ligand with a494AAA496substitution blocked hormone-sensitive phosphate transport. Complementary approaches, including CRISPR/Cas9 technology, site-directed mutagenesis, confocal microscopy, and modeling, showed that NPT2A Arg495His or Arg495Cys variants do not support PTH or FGF23 action on phosphate transport. Coimmunoprecipitation experiments indicate that both variants bind NHERF1 similarly to WT NPT2A. However, in contrast to WT NPT2A, NPT2A Arg495His or Arg495Cys variants remain at the apical membrane and are not internalized in response to PTH. We predict that Cys or His substitution of the charged Arg495changes the electrostatics, preventing phosphorylation of the upstream Thr494, interfering with phosphate uptake in response to hormone action, and inhibiting NPT2A trafficking. We advance a model wherein the carboxyterminal PDZ ligand defines apical localization NPT2A, while the internal PDZ ligand is essential for hormone-triggered phosphate transport.

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.

SARS-CoV-2 is responsible for the global COVID-19 pandemic. Angiotensin converting enzyme 2 (ACE2) is the membrane-delimited receptor for SARS-CoV-2. Lung, intestine and kidney, major sites of viral infection, express ACE2 that harbours an intracellular, carboxy-terminal PDZ-recognition motif. These organs prominently express the PDZ protein Na+/H+ exchanger regulatory factor-1 (NHERF1). Here, we report NHERF1 tethers ACE2 and augments SARS-CoV-2 cell entry. ACE2 directly binds both NHERF1 PDZ domains. Disruption of either NHERF1 PDZ core-binding motif or the ACE2 PDZ-recognition sequence eliminates interaction. Proximity ligation assays establish that ACE2 and NHERF1 interact at constitutive expression levels in human lung and intestine cells. Ablating ACE2 interaction with NHERF1 accelerated SARS-CoV-2 cell entry. Conversely, elimination of the ACE2 C-terminal PDZ binding motif decreased ACE2 membrane residence and reduced pseudotyped virus entry. We conclude that the PDZ interaction of ACE2 with NHERF1 facilitates SARS-CoV-2 internalization. β-arrestin is likely indispensable, as with G protein-coupled receptors., Graphical Abstract

The type II sodium-dependent phosphate cotransporter (NPT2A) mediates renal phosphate uptake. The NPT2A is regulated by parathyroid hormone (PTH) and fibroblast growth factor 23, which requires Na+/H+ exchange regulatory factor-1 (NHERF1), a multidomain PDZ-containing phosphoprotein. Phosphocycling controls the association between NHERF1 and the NPT2A. Here, we characterize the critical involvement of G protein–coupled receptor kinase 6A (GRK6A) in mediating PTH-sensitive phosphate transport by targeted phosphorylation coupled with NHERF1 conformational rearrangement, which in turn allows phosphorylation at a secondary site. GRK6A, through its carboxy-terminal PDZ recognition motif, binds NHERF1 PDZ1 with greater affinity than PDZ2. However, the association between NHERF1 PDZ2 and GRK6A is necessary for PTH action. Ser162, a PKCα phosphorylation site in PDZ2, regulates the binding affinity between PDZ2 and GRK6A. Substitution of Ser162 with alanine (S162A) blocks the PTH action but does not disrupt the interaction between NHERF1 and the NPT2A. Replacement of Ser162 with aspartic acid (S162D) abrogates the interaction between NHERF1 and the NPT2A and concurrently PTH action. We used amber codon suppression to generate a phosphorylated Ser162(pSer162)-PDZ2 variant. KD values determined by fluorescence anisotropy indicate that incorporation of pSer162 increased the binding affinity to the carboxy terminus of GRK6A 2-fold compared with WT PDZ2. Molecular dynamics simulations predict formation of an electrostatic network between pSer162 and Asp183 of PDZ2 and Arg at position −1 of the GRK6A PDZ-binding motif. Our results suggest that PDZ2 plays a regulatory role in PTH-sensitive NPT2A-mediated phosphate transport and phosphorylation of Ser162 in PDZ2 modulates the interaction with GRK6A.

We examined whether the scaffolding protein sodium-hydrogen exchanger regulatory factor 1 (NHERF1) interacts with the calcium pump PMCA2 and the tyrosine kinase receptor ErbB2/HER2 in normal mammary epithelial cells and breast cancer cells. NHERF1 interacts with the PDZ-binding motif in PMCA2 in both normal and malignant breast cells. NHERF1 expression is increased in HER2-positive breast cancers and correlates with HER2-positive status in human ductal carcinoma in situ (DCIS) lesions and invasive breast cancers as well as with increased mortality in patients. NHERF1 is part of a multiprotein complex that includes PMCA2, HSP90, and HER2 within specific actin-rich and lipid raft-rich membrane signaling domains. Knocking down NHERF1 reduces PMCA2 and HER2 expression, inhibits HER2 signaling, dissociates HER2 from HSP90, and causes the internalization, ubiquitination, and degradation of HER2. These results demonstrate that NHERF1 acts with PMCA2 to regulate HER2 signaling and membrane retention in breast cancers.

Phosphate homeostasis is critical for many cellular processes and is tightly regulated. The sodium-dependent phosphate cotransporter, NaPi2a, is the major regulator of urinary phosphate reabsorption in the renal proximal tubule. Its activity is dependent upon its brush border localization that is regulated by fibroblast growth factor 23 (FGF23) and PTH. High levels of FGF23, as are seen in the Hyp mouse model of human X-linked hypophosphatemia, lead to renal phosphate wasting. Long-term treatment of Hyp mice with 1,25-dihydroxyvitamin D (1,25D) or 1,25D analogues has been shown to improve renal phosphate wasting in the setting of increased FGF23 mRNA expression. Studies were undertaken to define the cellular and molecular basis for this apparent FGF23 resistance. 1,25D increased FGF23 protein levels in the cortical bone and circulation of Hyp mice but did not impair FGF23 cleavage. 1,25D attenuated urinary phosphate wasting as early as one hour postadministration, without suppressing FGF23 receptor/coreceptor expression. Although 1,25D treatment induced expression of early growth response 1, an early FGF23 responsive gene required for its phosphaturic effects, it paradoxically enhanced renal phosphate reabsorption and NaPi2a protein expression in renal brush border membranes (BBMs) within one hour. The Na-H+ exchange regulatory factor 1 (NHERF1) is a scaffolding protein thought to anchor NaPi2a to the BBM. Although 1,25D did not alter NHERF1 protein levels acutely, it enhanced NHERF1-NaPi2a interactions in Hyp mice. 1,25D also prevented the decrease in NHERF1/NaPi2a interactions in PTH-treated wild-type mice. Thus, these investigations identify a novel role for 1,25D in the hormonal regulation of renal phosphate handling.

RGS14 is a multifunctional scaffolding protein that integrates G protein, MAPK, and Ca++/CaM signaling pathways. RGS14 contains an RGS domain, tandem Ras/Rap-binding domains, and a G protein regulatory motif. Human/primate RGS14 differ from rodent proteins by possessing a carboxy-terminal extension encoding a Type I PDZ ligand. RGS14 has been implicated in disordered phosphate metabolism. The human RGS14 gene is adjacent to SLC34A1 that encodes the NPT2A sodium-phosphate cotransporter. Hormone-regulated NPT2A requires the PDZ protein NHERF1 which contains two PDZ domains (PDZ1 and PDZ2). NHERF1 binds the PDZ ligand carboxy tail of NPT2A to regulate phosphate uptake, and this NPT2A:NHERF1 complex is inhibited by parathyroid hormone (PTH). Studies here define roles for RGS14 in NHERF1-dependent, PTH-sensitive phosphate transport. We found that RGS14 binds to NHERF1 via the PDZ2 domain. PTH inhibits NPT2A-mediated phosphate transport and RGS14 blocked this action. Several rare human mutations have been reported in the RGS14 PDZ ligand located at residues 563 (D563N, D563G) and 565 (A565S, A565V). D563N disrupted RGS14 binding to NHERF1 and did not interfere with PTH action, whereas D563G, A565S, and A565V bound NHERF1 and were functionally equivalent to wild-type RGS14. Computational analysis and molecular dynamics modeling of NHERF1 PDZ2 binding to the RGS14 C-terminal PDZ ligands refined the structural determinants of this interaction. Additional studies demonstrated that RGS14 is expressed in human kidney proximal and distal tubule cells. Together, our findings are consistent with the view that RGS14 contributes to PTH-sensitive phosphate transport in humans. RGS14 coding variants may cause disordered phosphate metabolism.

International audience; Na+-H+ exchanger regulatory factor-1 (NHERF1) is a PDZ protein that scaffolds membrane proteins, including sodium-phosphate co-transport protein 2A (NPT2A) at the plasma membrane. NHERF1 is a phosphoprotein with 40 Ser and Thr residues. Here, using tandem MS analysis, we characterized the sites of parathyroid hormone (PTH)-induced NHERF1 phosphorylation and identified 10 high-confidence phosphorylation sites. Ala replacement at Ser(46), Ser(162), Ser(181), Ser(269), Ser(280), Ser(291), Thr(293), Ser(299), and Ser(302) did not affect phosphate uptake, but S290A substitution abolished PTH-dependent phosphate transport. Unexpectedly, Ser(290) was rapidly dephosphorylated and rephosphorylated after PTH stimulation, and we found that protein phosphatase 1 (PP1), which binds NHERF1 through a conserved VxF/W PP1 motif, dephosphorylates Ser(290). Mutating (VPF259)-V-257 eliminated PP1 binding and blunted dephosphorylation. Tautomycetin blocked PP1 activity and abrogated PTH-sensitive phosphate transport. Using fluorescence lifetime imaging (FLIM), we observed that PTH paradoxically and transiently elevates intracellular phosphate. Added phosphate blocked PP1-mediated Ser(290) dephosphorylation of recombinant NHERF1. Hydrogen-deuterium exchange MS revealed that -sheets in NHERF1's PDZ2 domain display lower deuterium uptake than those in the structurally similar PDZ1, implying that PDZ1 is more cloistered. Dephosphorylated NHERF1 exhibited faster exchange at C-terminal residues suggesting that NHERF1 dephosphorylation precedes Ser(290) rephosphorylation. Our results show that PP1 and NHERF1 form a holoenzyme and that a multiprotein kinase cascade involving G protein-coupled receptor kinase 6A controls the Ser(290) phosphorylation status of NHERF1 and regulates PTH-sensitive, NPT2A-mediated phosphate uptake. These findings reveal how reversible phosphorylation modifies protein conformation and function and the biochemical mechanisms underlying PTH control of phosphate transport.

International audience; The G protein-coupled parathyroid hormone receptor (PTHR) regulates mineral-ion homeostasis and bone remodeling. Upon parathyroid hormone (PTH) stimulation, the PTHR internalizes into early endosomes and subsequently traffics to the retromer complex, a sorting platform on early endosomes that promotes recycling of surface receptors. The C terminus of the PTHR contains a type I PDZ ligand that binds PDZ domain-containing proteins. Mass spectrometry identified sorting nexin 27 (SNX27) in isolated endosomes as a PTHR binding partner. PTH treatment enriched endosomal PTHR. SNX27 contains a PDZ domain and serves as a cargo selector for the retromer complex. VPS26, VPS29, and VPS35 retromer subunits were isolated with PTHR in endosomes from cells stimulated with PTH. Molecular dynamics and protein binding studies establish that PTHR and SNX27 interactions depend on the PDZ recognition motif in PTHR and the PDZ domain of SNX27. Depletion of either SNX27 or VPS35 or actin depolymerization decreased the rate of PTHR recycling following agonist stimulation. Mutating the PDZ ligand of PTHR abolished the interaction with SNX27 but did not affect the overall rate of recycling, suggesting that PTHR may directly engage the retromer complex. Coimmunoprecipitation and overlay experiments show that both intact and mutated PTHR bind retromer through the VPS26 protomer and sequentially assemble a ternary complex with PTHR and SNX27. SNX27-independent recycling may involve N-ethylmaleimide-sensitive factor, which binds both PDZ intact and mutant PTHRs. We conclude that PTHR recycles rapidly through at least two pathways, one involving the ASRT complex of actin, SNX27, and retromer and another possibly involving N-ethylmaleimide-sensitive factor.

G protein–coupled receptor (GPCR) signaling and trafficking are essential for cellular function and regulated by phosphorylation, β-arrestin, and ubiquitination. The GPCR parathyroid hormone receptor (PTHR) exhibits time-dependent reversible ubiquitination. The exact ubiquitination sites in PTHR are unknown, but they extend upstream of its intracellular tail. Here, using tandem MS, we identified Lys(388) in the third loop and Lys(484) in the C-terminal tail as primary ubiquitination sites in PTHR. We found that PTHR ubiquitination requires β-arrestin and does not display a preference for β-arrestin1 or -2. PTH stimulated PTHR phosphorylation at Thr(387)/Thr(392) and within the Ser(489)–Ser(493) region. Such phosphorylation events may recruit β-arrestin, and we observed that chemically or genetically blocking PTHR phosphorylation inhibits its ubiquitination. Specifically, Ala replacement at Thr(387)/Thr(392) suppressed β-arrestin binding and inhibited PTHR ubiquitination, suggesting that PTHR phosphorylation and ubiquitination are interdependent. Of note, Lys-deficient PTHR mutants promoted normal cAMP formation, but exhibited differential mitogen-activated protein kinase (MAPK) signaling. Lys-deficient PTHR triggered early onset and delayed ERK1/2 signaling compared with wildtype PTHR. Moreover, ubiquitination of Lys(388) and Lys(484) in wildtype PTHR strongly decreased p38 signaling, whereas Lys-deficient PTHR retained signaling comparable to unstimulated wildtype PTHR. Lys-deficient, ubiquitination-refractory PTHR reduced cell proliferation and increased apoptosis. However, elimination of all 11 Lys residues in PTHR did not affect its internalization and recycling. These results pinpoint the ubiquitinated Lys residues in PTHR controlling MAPK signaling and cell proliferation and survival. Our findings suggest new opportunities for targeting PTHR ubiquitination to regulate MAPK signaling or manage PTHR-related disorders.

Parathyroid hormone (PTH) and FGF23 are the primary hormones regulating acute phosphate homeostasis. Human renal proximal tubule cells (RPTECs) were used to characterize the mechanism and signaling pathways of PTH and FGF23 on phosphate transport and the role of the PDZ protein NHERF1 in mediating PTH and FGF23 effects. RPTECs express the NPT2A phosphate transporter, αKlotho, FGFR1, FGFR3, FGFR4, and the PTH receptor. FGFR1 isoforms are formed from alternate splicing of exon 3 and of exon 8 or 9 in Ir-like loop 3. Exon 3 was absent, but mRNA containing both exons 8 and 9 is present in cytoplasm. Using an FGFR1c-specific antibody together with mass spectrometry analysis, we show that RPTECs express FGFR-β1C. The data are consistent with regulated FGFR1 splicing involving a novel cytoplasmic mechanism. PTH and FGF23 inhibited phosphate transport in a concentration-dependent manner. At maximally effective concentrations, PTH and FGF23 equivalently decreased phosphate uptake and were not additive, suggesting a shared mechanism of action. Protein kinase A or C blockade prevented PTHbut notFGF23actions. Conversely, inhibitingSGK1, blocking FGFR dimerization, or knocking down Klotho expression disruptedFGF23actions but did not interfere withPTHeffects. C-terminal FGF23(180-251) competitively and selectively blocked FGF23 action without disruptingPTHeffects. However, bothPTH and FGF23-sensitive phosphate transport were abolished by NHERF1 shRNA knockdown. Extended treatment with PTH or FGF23 down-regulated NPT2A without affecting NHERF1. We conclude that FGFR1c and PTHR signaling pathways converge on NHERF1 to inhibit PTH- and FGF23-sensitive phosphate transport and down-regulate NPT2A. Fil: Sneddon, W. Bruce. University of Pittsburgh; Estados Unidos Fil: Ruiz, Giovanni W.. University of Pittsburgh; Estados Unidos Fil: Gallo, Luciana Ines. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. University of Pittsburgh; Estados Unidos Fil: Xiao, Kunhong. University of Pittsburgh; Estados Unidos Fil: Zhang, Qiangmin. University of Pittsburgh; Estados Unidos Fil: Rbaibi, Youssef. University of Pittsburgh; Estados Unidos Fil: Weisz, Ora A.. University of Pittsburgh; Estados Unidos Fil: Apodaca, Gerard L.. University of Pittsburgh; Estados Unidos Fil: Friedman, Peter A.. University of Pittsburgh; Estados Unidos

Parathyroid hormone (PTH) regulates calcium homeostasis and bone remodeling through its cognitive receptor (PTHR). We describe here a PTHR isoform harboring an in-frame 42-bp deletion of exon 14 (Δe14-PTHR) that encodes transmembrane domain 7. Δe14-PTHR was detected in human kidney and buccal epithelial cells. We characterized its topology, cellular localization, and signaling, as well as its interactions with PTHR. The C-terminus of the Δe14-PTHR is extracellular, and cell surface expression is strikingly reduced compared with the PTHR. Δe14-PTHR displayed impaired trafficking and accumulated in endoplasmic reticulum. Signaling and activation of cAMP and ERK by Δe14-PTHR was decreased significantly compared with PTHR. Δe14-PTHR acts as a functional dominant-negative by suppressing the action of PTHR. Cells cotransfected with both receptors exhibit markedly reduced PTHR cell membrane expression, colocalization with Δe14-PTHR in endoplasmic reticulum, and diminished cAMP activation and ERK phosphorylation in response to challenge with PTH. Δe14-PTHR forms heterodimers with PTHR, which may account for cytoplasmic retention of PTHR in the presence of Δe14-PTHR. Analysis of the PTHR heteronuclear RNA suggests that base-pair complementarity in introns surrounding exon 14 causes exon skipping and accounts for generation of the Δe14-PTHR isoform. Thus Δe14-PTHR is a poorly functional receptor that acts as a dominant-negative of PTHR trafficking and signaling and may contribute to PTH resistance. © 2011 American Society for Bone and Mineral Research.

PTH regulates renal calcium homeostasis by actions on the distal nephron. PTH-induced calcium transport in mouse distal convoluted tubule (DCT) cells requires activation of ERK1/2. ERK activation by beta-adrenergic receptors occurs in a biphasic manner and involves receptor internalization. An early rapid phase is beta-arrestin (betaAr) independent, whereas prolonged activation is betaAr dependent. We characterized PTH-stimulated ERK activation and the involvement of receptor internalization and betaAr dependence. In DCT cells, PTH transiently activated ERK maximally at 5 min and then returned to baseline. betaAr dependence of PTH receptor (PTH1R)-mediated ERK stimulation was assessed using mouse embryonic fibroblasts (MEFs) from betaAr1- and -2-null mice. In wild-type MEFs, PTH(1-34)-stimulated ERK activation peaked after 5 min, was 50% maximal after 15 min, and then recovered to 80% of maximal stimulation by 30 min. In MEFs null for betaAr1 and -2, PTH-stimulated ERK activation peaked by 5 min and returned to baseline. The effect was identical in betaAr2-null MEFs. In betaAr1-null MEFs, ERK exhibited delayed activation and remained elevated. PTH-stimulated ERK activation and receptor endocytosis were not inhibited by the clathrin-binding domain of betaAr1 [Ar(319-418)]. Coexpression of the sodium proton exchanger regulatory factor 1 (NHERF1) with Ar(319-418) blocked PTH1R internalization. We conclude that PTH-stimulated ERK activation in DCT cells proceeds with a rapid but transient phase that may involve betaAr1. Furthermore, the betaAr-dependent late phase of ERK activation by PTH requires the participation of betaAr2 and PTH1R internalization.

The PTH receptor (PTH1R) activates multiple signaling pathways, including extracellular signal-regulated kinases 1 and 2 (ERK1/2). The role of epidermal growth factor receptor (EGFR) transactivation in ERK1/2 activation by PTH in distal kidney cells, a primary site of PTH action, was characterized. ERK1/2 phosphorylation was stimulated by PTH and blocked by the EGFR inhibitor, AG1478. Upon PTH stimulation, metalloprotease cleavage of membrane-bound heparin-binding fragment (HB-EGF) induced EGFR transactivation of ERK. Conditioned media from PTH-treated distal kidney cells activated ERK in HEK-293 cells. AG1478 added to HEK-293 cells ablated transactivation by conditioned media. HB-EGF directly activated ERK1/2 in HEK-293 cells. Pretreatment of distal kidney cells with the metalloprotease inhibitor GM-6001 abolished transactivation of ERK1/2 by PTH. The role of the PTH1R COOH terminus in PTX-sensitive ERK1/2 activation was characterized in HEK-293 cells transfected with wild-type PTH1R, with a PTH1R mutated at its COOH terminus, or with PTH1R truncated at position 480. PTH stimulated ERK by wild-type, mutated and truncated PTH1Rs 21-, 27- and 57-fold, respectively. Thus, the PTH1R COOH terminus exerts an inhibitory effect on ERK activation. EBP50, a scaffolding protein that binds to the PDZ recognition domain of the PTH1R, impaired PTH but not isoproterenol or calcitonin-induced ERK activation. Pertussis toxin inhibited PTH-stimulated ERK1/2 by mutated and truncated PTH1Rs and abolished ERK1/2 activation by wild-type PTH1R. We conclude that ERK phosphorylation in distal kidney cells by PTH requires PTH1R activation of Gi, which leads to stimulation of metalloprotease-mediated cleavage of HB-EGF and transactivation of the EGFR and is regulated by EBP50.

The sequences in the rat osteocalcin gene that lie 3′ to the vitamin D response element (VDRE) contain a GGTTTGG motif (-420 to -414) that is essential for transcriptional activation of osteocalcin-CAT (OC-CAT) fusion genes by 1,25(OH)2D3. A second copy of this motif, present on the antisense strand is unable to compete for nuclear protein binding to the VDRE-associated motif, suggesting that the core element extends beyond the GGTTTGG motif. In order to examine the base requirements for both function and nuclear protein interactions with the VDRE-associated GGTTTGG enhancer motif, deletion and substitution of flanking sequences was performed in the context of both the native osteocalcin promoter and a heterologous viral promoter. These data demonstrate that the base requirements for protein-DNA interactions and transactivation are located between -430 and -414. The position of the element with respect to the VDRE is flexible and insertion of additional copies either 5′ or 3′ to the VDRE further enhances transactivation, both in the context of the native osteocalcin promoter and a heterologous viral promoter. These data demonstrate that VDR-dependent transactivation of the rat osteocalcin gene requires not only the VDRE (-456 to -442) but also sequences between -430 and -414. The protein(s) that interacts with these sequences is capable of enhancing transcription in both a position and orientation-independent fashion. J. Cell. Biochem. 73:400–407, 1999. © 1999 Wiley-Liss, Inc.

The sequences in the rat osteocalcin gene that lie 3' to the vitamin D response element (VDRE) have been shown to augment transcriptional activation by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. These DNA sequences, however, are unable to bind the VDR or mediate 1,25-(OH)2D3 responsiveness independently of the VDRE. To further characterize this region, the functional properties of a series of mutant oligonucleotides were examined in transiently transfected ROS 17/2.8 cells. When these mutant oligonucleotides were expressed upstream of the heterologous herpes simplex virus thymidine kinase promoter, the bases between -420 and -414 of the rat osteocalcin gene were identified as critical for maximal transactivation by 1,25-(OH)2D3. Furthermore, mutation of these sequences in the context of the native osteocalcin promoter and enhancer totally abolished the ability of the VDRE to mediate 1,25-(OH)2D3 responsiveness. These bases, which are essential for the 1,25-(OH)2D3 responsiveness of the rat osteocalcin gene, are also present in a similar position, relative to the VDRE, in the human osteocalcin gene. To explore whether these sequences could enhance transactivation by other inducible transcription factors, they were examined for their ability to synergize with the chick vitellogenin estrogen response element and the rat somatostatin cAMP response element. When placed upstream to the herpes simplex virus thymidine kinase promoter and transfected into ROS 17/2.8 cells, these sequences were able to enhance transcriptional responsiveness to 17beta-estradiol and forskolin, respectively, demonstrating that they also contribute to transactivation by other inducible transcription factors.

Bone growth and remodeling depend upon the opposing rates of bone formation and resorption. These functions are regulated by intrinsic seven transmembrane-spanning receptors, the parathyroid hormone receptor (PTH1R) and frizzled (FZD), through their respective ligands, parathyroid hormone (PTH) and Wnt. FZD activation of canonical beta-catenin signaling requires the adapter protein Dishevelled (Dvl). We identified a Dvl-binding motif in the PTH1R. Here, we report that the PTH1R activates the beta-catenin pathway by directly recruiting Dvl, independent of Wnt or LRP5/6. PTH1R coimmunoprecipitated with Dvl. Deleting the carboxyl-terminal PTH1R PDZ-recognition domain did not abrogate PTH1R-Dvl interactions; nor did truncating the receptor at position 480. However, further deletion eliminating the putative Dvl recognition domain abolished PTH1R interactions with Dvl. PTH activated beta-catenin in a time- and concentration-dependent manner and translocated beta-catenin to the nucleus. beta-Catenin activation was inhibited by Dvl2 dominant negatives and by short hairpin RNA sequences targeted against Dvl2. PTH-induced osteoclastogenesis was also inhibited by Dvl2 dominant negative mutants. These findings demonstrate that G protein-coupled receptors other than FZD directly activate beta-catenin signaling, thereby mimicking many of the functions of the canonical Wnt-FZD pathway. The distinct modes whereby FZD and PTH1R activate beta-catenin control convergent or divergent effects on osteoblast differentiation, and osteoclastogenesis may arise from PTH1R-induced second messenger phosphorylation.

Transgenic mice lacking calcium channel β3 subunits (CaVβ3) were used to determine the involvement of a multimeric calcium channel in mediating stimulated renal calcium absorption. We measured the ability of calcium channel β3 subunit-null (CaVβ3−/−) and wild-type (CaVβ3+/+) mice to increase renal calcium absorption in response to the calcium-sparing diuretic chlorothiazide (CTZ). Control rates of fractional sodium excretion were comparable in CaVβ3−/− and CaVβ3+/+ mice and CTZ increased sodium excretion similarly in both groups. CTZ enhanced calcium absorption only in wild-type CaVβ3+/+ mice. This effect was specific for diuretics acting on distal tubules because both CaVβ3−/− and CaVβ3+/+ mice responded comparably to furosemide. The absence of β3 subunits resulted in compensatory increases of TrpV5 calcium channels, the plasma membrane Ca-ATPase, NCX1 Na/Ca exchanger protein, and calbindin-D9k but not calbindin-D28k. We conclude that TrpV5 mediates basal renal calcium absorption and that a multimeric calcium channel that includes CaVβ3 mediates stimulated calcium transport.

Gamma-aminobutyric acid (GABA)-mediated chloride uptake was determined in synaptoneurosomes prepared from the brains of entorhinal cortex-kindled and paired control rats. Subjects were sacrificed either 24 h or 28 days after the experimental animals had exhibited their sixth stage 5 convulsion. in the cortex and cerebellum, no difference was observed between kindled and control animals at either time period. In the brain-stem, however, a significant reduction (approximately 50%) in chloride uptake was observed in kindled subjects both 24 h and 28 days after the last seizure. These findings are consistent with the hypothesis that the ‘kindled state’ results from a long-lasting decrease in GABA-mediated inhibitory activity.

The sodium-hydrogen exchange regulatory factor 1 (NHERF-1/EBP50) interacts with the C terminus of several G protein-coupled receptors (GPCRs). We examined the role of NHERF-1 and the cytoskeleton on the distribution, dynamics, and trafficking of the beta(2)-adrenergic receptor (beta(2)AR; a type A receptor), the parathyroid hormone receptor (PTH1R; type B), and the calcium-sensing receptor (CaSR; type C) using fluorescence recovery after photobleaching, total internal reflection fluorescence, and image correlation spectroscopy. beta(2)AR bundles were observed only in cells that expressed NHERF-1, whereas the PTH1R was localized to bundles that parallel stress fibers independently of NHERF-1. The CaSR was never observed in bundles. NHERF-1 reduced the diffusion of the beta(2)AR and the PTH1R. The addition of ligand increased the diffusion coefficient and the mobile fraction of the PTH1R. Isoproterenol decreased the immobile fraction but did not affect the diffusion coefficient of the beta(2)AR. The diffusion of the CaSR was unaffected by NHERF-1 or the addition of calcium. NHERF-1 reduced the rate of ligand-induced internalization of the PTH1R. This phenomenon was accompanied by a reduction of the rate of arrestin binding to PTH1R in ligand-exposed cells. We conclude that some GPCRs, such as the beta(2)AR, are attached to the cytoskeleton primarily via the binding of NHERF-1. Others, such as the PTH1R, bind the cytoskeleton via several interacting proteins, one of which is NHERF-1. Finally, receptors such as the CaSR do not interact with the cytoskeleton in any significant manner. These interactions, or the lack thereof, govern the dynamics and trafficking of the receptor.

Internalization of the PTH type I receptor (PTH1R) is regulated in a cell- and ligand-specific manner. We previously demonstrated that the sodium/proton exchanger regulatory factor type 1 (NHERF1; EBP50) is pivotal in determining the range of peptides that internalize the PTH1R. Antagonist PTH fragments can internalize the PTH1R in some kidney and bone cell models. PTH(7-34), which binds to, but does not activate, the PTH1R, internalizes the PTH1R in kidney distal tubule (DT) cells, where NHERF1 is not expressed. The effect of antagonist PTHrP peptides has not, to this point, been assessed. PTH1R internalization was measured by real-time confocal fluorescence microscopy of DT cells stably expressing 105 EGFP-tagged PTH1R/cell. PTHrP(7-34) internalized the PTH1R in a manner indistinguishable from PTH(7-34). Introduction of NHERF1 into DT cells, however, blocked PTH(7-34)-, but not PTHrP(7-34)-, induced PTH1R internalization. To delineate the sequences within PTHrP that determine whether PTH1R internalization is affected by NHERF1, chimeric PTH/PTHrP fragments were tested for their ability to induce PTH1R internalization. PTH(7-21)/PTHrP (22-34), PTH(7-32)/PTHrP(33-34), and PTH(7-33)/PTHrP(34) at 1 microM each internalized the PTH1R 50-70% in a NHERF1-independent manner. When the C terminus of PTHrP was replaced with homologous amino acids from PTH, NHERF1 inhibited PTH1R internalization. It was determined that simply mutating F34 to A in PTH induced PTH1R internalization in a NHERF1-independent manner. None of the chimeric peptides activated the PTH1R but all effectively competed for 1 nM PTH(1-34) in cyclic AMP assays. In addition, all chimeric peptides competed for radiolabeled PTH(1-34) in binding assays in DT cells. PTH(1- 34) and PTHrP(7-34), but not PTH(7-34), efficiently recruited beta-arrestin1 to plasma membrane PTH1Rs. We, therefore, conclude that PTH(1-34) and PTHrP(7-34) induce a conformational change in the PTH1R that promotes arrestin binding and dissociates NHERF1 from PTH1R internalization.

G protein-coupled receptors (GPCRs) mediate the action of many hormones, cytokines, and sensory and chemical signals. It is generally thought that receptor desensitization and internalization require occupancy and activation of the GPCR. PTH and PTHrP receptor (PTH1R) belongs to GPCR class B and is the major regulator of extracellular calcium homeostasis. Using kidney distal convoluted tubule cells transfected with a human PTH1R/enhanced green fluorescent protein fusion protein, quantitative, real-time fluorescence microscopy was used to analyze receptor internalization. In these cells, which are the target of the calcium-sparing action of PTH, PTH(1-34) activated adenylyl cyclase (AC) and phospholipase C (PLC) and PTH1R endocytosis. PTH(1-31), however, stimulated AC and PLC but not PTH1R endocytosis. Conversely, PTH(7-34) rapidly stimulated PTH1R internalization without activating AC or PLC. PTH(2-34) and (3-34) caused PTH1R internalization intermediate between PTH(1-34) and (7-34). PTH1R sequestration occurred in a dynamin- and clathrin-dependent manner. Directly activating AC inhibited PTH1R internalization in response to PTH(7-34). PTH1R endocytosis was sensitive to protein kinase C inhibition. PTH(1-34), (7-34), and (1-31) evoked PTH1R phosphorylation. Removal of most of the C terminus of the PTH1R eliminated receptor phosphorylation and the cAMP/protein kinase C sensitivity of internalization. PTH(1-34) and (7-34) internalized the truncated PTH1R with identical kinetics, and the response was unaffected by forskolin. Thus, the PTH1R C terminus contains regulatory sequences that are involved in, but not required for, PTH1R internalization. The results demonstrate that receptor activation and internalization can be selectively dissociated.

Parathyroid hormone (PTH) regulates extracellular calcium homeostasis through the type 1 PTH receptor (PTH1R) expressed in kidney and bone. The PTH1R undergoes beta-arrestin/dynamin-mediated endocytosis in response to the biologically active forms of PTH, PTH-(1-34), and PTH-(1-84). We now show that amino-truncated forms of PTH that do not activate the PTH1R nonetheless induce PTH1R internalization in a cell-specific pattern. Activation-independent PTH1R endocytosis proceeds through a distinct arrestin-independent mechanism that is operative in cells lacking the adaptor protein Na/H exchange regulatory factor 1 (NHERF1) (ezrin-binding protein 50). Using a combination of radioligand binding experiments and quantitative, live cell confocal microscopy of fluorescently tagged PTH1Rs, we show that in kidney distal tubule cells and rat osteosarcoma cells, which lack NHERF1, the synthetic antagonist PTH-(7-34) and naturally circulating PTH-(7-84) induce internalization of PTH1R in a beta-arrestin-independent but dynamin-dependent manner. Expression of NHERF1 in these cells inhibited antagonist-induced endocytosis. Conversely, expression of dominant-negative forms of NHERF1 conferred internalization sensitivity to PTH-(7-34) in cells expressing NHERF1. Mutation of the PTH1R PDZ-binding motif abrogated interaction of the receptor with NHERF1. These mutated receptors were fully functional but were now internalized in response to PTH-(7-34) even in NHERF1-expressing cells. Removing the NHERF1 ERM domain or inhibiting actin polymerization allowed otherwise inactive ligands to internalize the PTH1R. These results demonstrate that NHERF1 acts as a molecular switch that legislates the conditional efficacy of PTH fragments. Distinct endocytic pathways are determined by NHERF1 that are operative for the PTH1R in kidney and bone cells.