PTH, FGF-23, Klotho and Vitamin D as regulators of calcium and phosphorus: Genetics, epigenetics and beyond.

The actions of several bone-mineral ion regulators, namely PTH, FGF23, Klotho and 1,25(OH)2 vitamin D (1,25(OH)2D), control calcium and phosphate metabolism, and each of these molecules has additional biological effects related to cell signaling, metabolism and ultimately survival. Therefore, these factors are tightly regulated at various levels - genetic, epigenetic, protein secretion and cleavage. We review the main determinants of mineral homeostasis including well-established genetic and post-translational regulators and bring attention to the epigenetic mechanisms that affect the function of PTH, FGF23/Klotho and 1,25(OH)2D. Clinically relevant epigenetic mechanisms include methylation of cytosine at CpG-rich islands, histone deacetylation and micro-RNA interference. For example, sporadic pseudohypoparathyroidism type 1B (PHP1B), a disease characterized by resistance to PTH actions due to blunted intracellular cAMP signaling at the PTH/PTHrP receptor, is associated with abnormal methylation at the GNAS locus, thereby leading to reduced expression of the stimulatory G protein α-subunit (Gsα). Post-translational regulation is critical for the function of FGF-23 and such modifications include glycosylation and phosphorylation, which regulate the cleavage of FGF-23 and hence the proportion of available FGF-23 that is biologically active. While there is extensive data on how 1,25(OH)2D and the vitamin D receptor (VDR) regulate other genes, much more needs to be learned about their regulation. Reduced VDR expression or VDR mutations are the cause of rickets and are thought to contribute to different disorders. Epigenetic changes, such as increased methylation of the VDR resulting in decreased expression are associated with several cancers and infections. Genetic and epigenetic determinants play crucial roles in the function of mineral factors and their disorders lead to different diseases related to bone and beyond.

Actions of Parathyroid Hormone Ligand Analogues in Humanized PTH1R Knockin Mice.

Rodent models are commonly used to evaluate parathyroid hormone (PTH) and PTH-related protein (PTHrP) ligands and analogues for their pharmacologic activities and potential therapeutic utility toward diseases of bone and mineral ion metabolism. Divergence, however, in the amino acid sequences of rodent and human PTH receptors (rat and mouse PTH1Rs are 91% identical to the human PTH1R) can lead to differences in receptor-binding and signaling potencies for such ligands when assessed on rodent vs human PTH1Rs, as shown by cell-based assays in vitro. This introduces an element of uncertainty in the accuracy of rodent models for performing such preclinical evaluations. To overcome this potential uncertainty, we used a homologous recombination-based knockin (KI) approach to generate a mouse (in-host strain C57Bl/6N) in which complementary DNA encoding the human PTH1R replaces a segment (exon 4) of the murine PTH1R gene so that the human and not the mouse PTH1R protein is expressed. Expression is directed by the endogenous mouse promoter and hence occurs in all biologically relevant cells and tissues and at appropriate levels. The resulting homozygous hPTH1R-KI (humanized) mice were healthy over at least 10 generations and showed functional responses to injected PTH analog peptides that are consistent with a fully functional human PTH1R in target bone and kidney cells. The initial evaluation of these mice and their potential utility for predicting behavior of PTH analogues in humans is reported here.

Ligand-Dependent Effects of Methionine-8 Oxidation in Parathyroid Hormone Peptide Analogues.

LA-PTH is a long-acting parathyroid hormone (PTH) peptide analogue in preclinical development for hypoparathyroidism (HP). Like native PTH, LA-PTH contains a methionine at position 8 (Met8) that is predicted to be critical for function. We assessed the impact of Met oxidation on the functional properties of LA-PTH and control PTH ligands. Oxidation of PTH(1-34) resulted in marked (~20-fold) reductions in binding affinity on the PTH receptor-1 (PTHR1) in cell membranes, similarly diminished potency for 3',5'-cyclic AMP signaling in osteoblastic cell lines (SaOS-2 and UMR106), and impaired efficacy for raising blood calcium in mice. Surprisingly, oxidation of LA-PTH resulted in little or no change in these functional responses. The signaling potency of oxidized-LA-PTH was, however, reduced approximately 40-fold compared to LA-PTH in cells expressing a PTHR1 construct that lacks the N-terminal extracellular domain (ECD). Molecular modeling revealed that while Met8 of both LA-PTH and PTH(1-34) is situated within the orthosteric ligand-binding pocket of the receptor's transmembrane domain bundle (TMD), the Met8 sidechain position is shifted for the 2 ligands so that on Met8 oxidation of PTH(1-34), steric clashes occur that are not seen with oxidized LA-PTH. The findings suggest that LA-PTH and PTH(1-34) engage the receptor differently in the Met8-interaction environment of the TMD bundle, and that this interaction environment can be allosterically influenced by the ECD component of the ligand-receptor complex. The findings should be useful for the future development of novel PTH-based peptide therapeutics for diseases of bone and mineral ion metabolism.

[Study on mechanism of Drynariae Rhizoma in treating osteoporosis with integrative pharmacology perspective].

Drynariae Rhizoma has great significance in the clinical practice of osteoporosis treatment. Based on the perspective of integrative pharmacology, the study explored the mechanism of action of Drynariae Rhizoma in the treatment of osteoporosis. Six active components in Drynariae Rhizoma were obtained, mainly including glycosides and sterols. Taking the median of 2 times of "node connectivity" as the card value, the core node of the Chinese medicine target disease gene interaction network was selected. Based on this, three topological structural eigenvalues, such as "node connectivity" "node tightness" and "node connectivity" were calculated, thereby screening out four core targets of Drynariae Rhizoma treatment for osteoporosis, including thyroid parathyroid hormone 1 receptor (PTH1R), parathyroid hormone 2 receptor (PTH2R), calcitonin receptor gene (CALCR), and SPTBN1 gene (SPTBN1). Based on the gene ontology database GO and KEGG pathway database, the molecular function, intracellular localization, and biological reactions and pathways of proteins encoded by drug target genes were determined. Combined with enrichment calculation, it is predicted that osteoporosis may play a role in biosynthetic processes, such as circulatory system, nervous system, energy metabolism, prolactin signal pathway, GnRH signaling pathway, neurotrophic factor signaling pathway and other pathway. The conclusion of this study is certain with the existing research results, and the new target and new pathway could also be used as a theoretical basis for the further verification of osteoporosis.

In vitro and preclinical assessment of an intranasal spray formulation of parathyroid hormone PTH 1-34 for the treatment of osteoporosis.

Osteoporosis treatment with PTH 1-34 injections significantly reduces the incidence of bone fracture. Potential further reductions in fracture rate should be observed through nasal spray delivery to address the poor compliance associated with patient dislike of repeated PTH 1-34 subcutaneous injections. In vitro human osteoblast-like Saos-2 cell intracellular cAMP levels were used to define PTH 1-34 nasal spray formulation bioactivity. The chemically synthesised PTH 1-34 had an EC50 of 0.76nM. Absorption enhancers polyethylene glycol (15)-hydroxystearate (Solutol® HS15), poloxamer 407, chitosan or sodium hyaluronate did not diminish the bioactivity of PTH 1-34 within an in vitro cell culture model (p >0.05). We also demonstrated the effectiveness of the transmucosal absorption enhancer Solutol® HS15 in a nasal spray formulation using a preclinical pharmacokinetic model. In Sprague-Dawley rats without the absorption enhancer the uptake of PTH 1-34 into the blood via intranasal delivery produced a Cmax of 2.1±0.5ng/ml compared to 13.7±1.6ng/ml with Solutol® HS15 enhancer (p=0.016) and a Cmax14.8±8ng/ml in subcutaneous injections. Together these data illustrate that the nasal spray formulation bioactivity in vitro is not affected by the nasal spray absorption enhancers investigated, and the Solutol® HS15 nasal spray formulation had an equivalent pharmacokinetic profile to subcutaneous injection in the rat model. The Solutol® HS15 formulation therefore demonstrated potential as a PTH 1-34 nasal spray formulation for the treatment of osteoporosis.

Serum calcium and bone: effect of PTH, phosphate, vitamin D and uremia

Hyperparathyroidism develops in chronic kidney disease (CKD). A decreased calcemic response to parathyroid hormone (PTH) contributes to the development of hyperparathyroidism and is presumed due to reduced calcium efflux from bone. Contributing factors to the decreased calcemic response to PTH in CKD include: 1) hyperphosphatemia; 2) decreased serum calcitriol; 3) downregulation of the PTH1 receptor; 4) large, truncated amino-terminal PTH fragments acting at the carboxy-PTH receptor; and 5) uremic toxins. Also, prolonged high dose calcitriol administration may decrease the exchangeable pool of bone calcium independent of PTH. The goal of the review is to provide a better understanding of how the above cited factors affect calcium efflux from bone in CKD. In conclusion, much remains to be learned about the role of bone in the regulation of serum calcium (AU)

El hiperparatiroidismo se desarrolla en la enfermedad renal crónica (ERC). La disminución de la respuesta calcémica a la hormona paratiroidea (PTH) contribuye al desarrollo de hiperparatiroidismo y es probable que se deba a una reducción de la emisión de calcio de los huesos. Entre los factores que contribuyen a la disminución de la respuesta calcémica a la PTH en la ERC se encuentran: 1) la hiperfosfatemia; 2) la disminución del calcitriol sérico; 3) la desensibilización del receptor PTHR1; 4) la presencia de fragmentos de gran tamaño de los extremos aminoterminales de la hormona paratiroidea que actúan en el receptor carboxi-PTH y 5) las toxinas urémicas. Asimismo, la administración prolongada de una dosis elevada de calcitriol podría disminuir la reserva intercambiable de calcio independiente de la hormona paratiroidea. El objetivo de esta revisión es facilitar la comprensión de cómo afectan los factores mencionados anteriormente a la emisión de calcio procedente del hueso en la ERC. Como conclusión, aún queda mucho por aprender acerca del papel de los huesos en la regulación del calcio sérico (AU)

Small molecule inhibition of the Na(+)/H(+) exchange regulatory factor 1 and parathyroid hormone 1 receptor interaction.

We have identified a series of small molecules that bind to the canonical peptide binding groove of the PDZ1 domain of NHERF1 and effectively compete with the association of the C-terminus of the parathyroid hormone 1 receptor (PTH1R). Employing nuclear magnetic resonance and molecular modeling, we characterize the mode of binding that involves the GYGF loop important for the association of the C-terminus of PTH1R. We demonstrate that the common core of the small molecules binds to the PDZ1 domain of NHERF1 and displaces a (15)N-labeled peptide corresponding to the C-terminus of PTH1R. The small size (molecular weight of 192) of this core scaffold makes it an excellent candidate for further elaboration in the development of an inhibitor for this important protein-protein interaction.

Species-specific responses of N homeostasis and electrolyte handling to low N intake: a comparative physiological approach in a monogastric and a ruminant species.

In our former studies low crude protein (LCP) intake influenced N homeostasis and electrolyte handling in goats. We hypothesised that due to rumino-hepatic nitrogen (N) recycling adaptation of N homeostasis and adjustment of electrolyte handling to LCP intake differs between goats and monogastric animals. Therefore, an experiment similar to that with goats was conducted with rats. Two feeding groups received a diet either containing 20 or 8 % crude protein (as fed basis) for 5 weeks and intake and excretion of N, calcium (Ca) and phosphorus (P) were determined. To detect systemic and endocrine adaptation to LCP intake plasma concentrations of urea, Ca, phosphate (Pi), insulin-like growth factor 1 (IGF-1), 1,25-dihydroxyvitamin D3 (calcitriol), parathyroid hormone (PTH) and cross-linked telopeptide of type I collagen (CTX) were measured. Adjustment of renal electrolyte transport was assessed by detecting protein expression of key proteins of renal Pi transport. All data were compared with the data of the goat experiment. LCP intake decreased plasma urea concentration stronger in goats than in rats. In both species urinary N excretion declined, but faecal N excretion decreased in goats only. Furthermore, in goats urinary Ca excretion decreased, but in rats urinary Ca concentration increased. Decreased plasma IGF-1 and calcitriol concentrations were found in goats only. Thus, renal Ca excretion appears to be a common target in adaptation of electrolyte homeostasis in both species, but is regulated differently.

Parathyroid hormone [1-34] improves articular cartilage surface architecture and integration and subchondral bone reconstitution in osteochondral defects in vivo.

OBJECTIVE: The 1-34 amino acid segment of the parathyroid hormone (PTH [1-34]) mediates anabolic effects in chondrocytes and osteocytes. The aim of this study was to investigate whether systemic application of PTH [1-34] improves the repair of non-osteoarthritic, focal osteochondral defects in vivo. DESIGN: Standardized cylindrical osteochondral defects were bilaterally created in the femoral trochlea of rabbits (n = 8). Daily subcutaneous injections of 10 µg PTH [1-34]/kg were given to the treatment group (n = 4) for 6 weeks, controls (n = 4) received saline. Articular cartilage repair was evaluated by macroscopic, biochemical, histological and immunohistochemical analyses. Reconstitution of the subchondral bone was assessed by micro-computed tomography. Effects of PTH [1-34] on synovial membrane, apoptosis, and expression of the PTH receptor (PTH1R) were determined. RESULTS: Systemic PTH [1-34] increased PTH1R expression on both, chondrocytes and osteocytes within the repair tissue. PTH [1-34] ameliorated the macro- and microscopic aspect of the cartilaginous repair tissue. It also enhanced the thickness of the subchondral bone plate and the microarchitecture of the subarticular spongiosa within the defects. No significant correlations were established between these coexistent processes. Apoptotic levels, synovial membrane, biochemical composition of the repair tissue, and type-I/II collagen immunoreactivity remained unaffected. CONCLUSIONS: PTH [1-34] emerges as a promising agent in the treatment of focal osteochondral defects as its systemic administration simultaneously stimulates articular cartilage and subchondral bone repair. Importantly, both time-dependent mechanisms of repair did not correlate significantly at this early time point and need to be followed over prolonged observation periods.

The bone-protective effect of genistein in the animal model of bilateral ovariectomy: roles of phytoestrogens and PTH/PTHR1 against post-menopausal osteoporosis.

Genistein, a major phytoestrogen of soy, is considered a potential drug for the prevention and treatment of post-menopausal osteoporosis. Mounting evidence suggested a positive correlation between genistein consumption and bone health both in vivo and in vitro. Earlier studies have revealed that genistein acted as a natural estrogen analogue which activated estrogen receptor and exerted anti-osteoporotic effect. However, it remains unclear whether PTH, the most crucial hormone that regulates mineral homeostasis, participates in the process of genistein-mediated bone protection. In the present study, we compared the therapeutic effects between genistein and nilestriol and investigated whether PTH and its specific receptor PTHR1 altered in response to genistein-containing diet in the animal model of ovariectomy. Our results showed that genistein administration significantly improved femoral mechanical properties and alleviates femoral turnover. Genistein at all doses (4.5 mg/kg, 9.0 mg/kg and 18.0 mg/kg per day, respectively) exerted improved bending strength and b-ALP limiting effects than nilestriol in the present study. However, genistein administration did not exert superior effects on bone protection than nilestriol. We also observed circulating PTH restoration in ovariectomized rats receiving genistein at the dose of 18 mg/kg per day. Meanwhile, PTHR1 abnormalities were attenuated in the presence of genistein as confirmed by RT-PCR, Western blot and immunohistochemistry. These findings strongly support the idea that besides serving as an estrogen, genistein could interact with PTH/PTHR1, causing a superior mineral restoring effect than nilestriol on certain circumstance. In conclusion, our study reported for the first time that the anti-osteoporotic effect of genistein is partly PTH/PTHR1-dependent. Genistein might be a potential option in the prevention and treatment of post-menopausal osteoporosis with good tolerance, more clinical benefits and few undesirable side effects.

Hormonal regulation of phosphate homeostasis in goats during transition to rumination.

Regulatory processes in phosphorus (P) homeostasis in small ruminants are quite different compared to monogastric animals. Adaptive responses of modulating hormones [parathyroid hormone (PTH) and calcitriol] to feeding variable amounts of P are lacking. Therefore, the aim of this study was to examine the influence of high dietary P intake (control diet: 4 g kg(-1) dry matter; high-P diet: 8 g kg(-1) dry matter) on the expression levels of PTH receptor (PTHR), vitamin D receptor (VDR) and Na+-dependent Pi transporters (NaPi II) in kidney and jejunum of goats starting rumination. After 3 months of feeding, plasma phosphate (Pi) and PTH concentrations were increased in the high-P diet group, whereas calcium and calcitriol were not changed. The intestinal Na+-dependent Pi transport capacity was not influenced by a high-P diet and the expression of jejunal VDR, PTHR and NaPi IIb was not modified. Interestingly, renal Na+-dependent Pi transport capacity was significantly reduced and concomitantly the expression of PTHR and NaPi IIa was decreased. In conclusion, the adaptive response of renal Pi reabsorption in goats, which were in transition from non-ruminant to ruminant stage was comparable to that of monogastric animals. In contrast, the modulation of the intestinal Pi absorption was like in adult ruminants.

Application of the BD ACTOne technology for the high-throughput screening of Gs-coupled receptor antagonists.

A novel technology for monitoring the changes of 3,'5'-adenosine cyclic monophosphate (cAMP) in live cells suitable for drug screening relies on the use of cyclic nucleotide-gated channels as biosensors coexpressed with the appropriate target receptor. The technique (termed BD ACTOne) offers measurement of cAMP-dependent calcium influx or membrane depolarization with conventional fluorescent methods both in kinetic and in endpoint modes, optimal for high-throughput and subsequent compound screening. The utility of the technique is reported here based on assay development and high-throughput screening for small-molecule antagonists of the peptide parathyroid hormone 2 receptor (PTH2R). The dual-signaling properties of the receptor were retained in the recombinant system, and the observed pharmacological profile corresponded to data from radiolabeled cAMP determination. The membrane-potential-based high-throughput assay produced reproducible actives and led to the identification of several chemical scaffolds with potential utility as PTH2R ligands.

Discovery of a small molecule antagonist of the parathyroid hormone receptor by using an N-terminal parathyroid hormone peptide probe.

Once-daily s.c. administration of either human parathyroid hormone (PTH)-(1-84) or recombinant human PTH-(1-34) provides for dramatic increases in bone mass in women with postmenopausal osteoporosis. We initiated a program to discover orally bioavailable small molecule equivalents of these peptides. A traditional high-throughput screening approach using cAMP activation of the PTH/PTH-related peptide receptor (PPR) as a readout failed to provide any lead compounds. Accordingly, we designed a new screen for this receptor that used a modified N-terminal fragment of PTH as a probe for small molecule binding to the transmembrane region of the PPR, driven by the assumption that the pharmacological properties (agonist/antagonist) of compounds that bound to this putative signaling domain of the PPR could be altered by chemical modification. We developed DPC-AJ1951, a 14 amino acid peptide that acts as a potent agonist of the PPR, and characterized its activity in ex vivo and in vivo assays of bone resorption. In addition, we studied its ability to initiate gene transcription by using microarray technology. Together, these experiments indicated that the highly modified 14 amino acid peptide induces qualitatively similar biological responses to those produced by PTH-(1-34), albeit with lower potency relative to the parent peptide. Encouraged by these data, we performed a screen of a small compound collection by using DPC-AJ1951 as the ligand. These studies led to the identification of the benzoxazepinone SW106, a previously unrecognized small molecule antagonist for the PPR. The binding of SW106 to the PPR was rationalized by using a homology receptor model.

Distinct beta-arrestin- and G protein-dependent pathways for parathyroid hormone receptor-stimulated ERK1/2 activation.

Parathyroid hormone (PTH) regulates calcium homeostasis via the type I PTH/PTH-related peptide (PTH/PTHrP) receptor (PTH1R). The purpose of the present study was to identify the contributions of distinct signaling mechanisms to PTH-stimulated activation of the mitogen-activated protein kinases (MAPK) ERK1/2. In Human embryonic kidney 293 (HEK293) cells transiently transfected with hPTH1R, PTH stimulated a robust increase in ERK activity. The time course of ERK1/2 activation was biphasic with an early peak at 10 min and a later sustained ERK1/2 activation persisting for greater than 60 min. Pretreatment of HEK293 cells with the PKA inhibitor H89 or the PKC inhibitor GF109203X, individually or in combination reduced the early component of PTH-stimulated ERK activity. However, these inhibitors of second messenger dependent kinases had little effect on the later phase of PTH-stimulated ERK1/2 phosphorylation. This later phase of ERK1/2 activation at 30-60 min was blocked by depletion of cellular beta-arrestin 2 and beta-arrestin 1 by small interfering RNA. Furthermore, stimulation of hPTH1R with PTH analogues, [Trp1]PTHrp-(1-36) and [d-Trp12,Tyr34]PTH-(7-34), selectively activated G(s)/PKA-mediated ERK1/2 activation or G protein-independent/beta-arrestin-dependent ERK1/2 activation, respectively. It is concluded that PTH stimulates ERK1/2 through several distinct signal transduction pathways: an early G protein-dependent pathway meditated by PKA and PKC and a late pathway independent of G proteins mediated through beta-arrestins. These findings imply the existence of distinct active conformations of the hPTH1R responsible for the two pathways, which can be stimulated by unique ligands. Such ligands may have distinct and valuable therapeutic properties.

Calcium-sensing receptor regulation of PTH-inhibitable proximal tubule phosphate transport.

Inorganic phosphate (Pi) is absorbed by proximal tubules through a cellular pathway that is inhibited by parathyroid hormone (PTH). The calcium-sensing receptor (CaSR) is expressed on apical membranes of proximal tubules. In the present studies, we determined the effect of luminal and/or basolateral PTH on phosphate absorption and tested the hypothesis that CaSR activation blocks PTH-inhibitable phosphate absorption. Single proximal S3 tubules were dissected from the kidneys of mice and studied by the Burg technique. Tubules were bathed with DMEM culture media supplemented with 6% BSA and perfused with an ultrafiltrate prepared from the bathing solution. 33P and FITC-inulin were added to the luminal perfusate to measure phosphate absorption (JPi) and fluid absorption (Jv), respectively. JPi averaged 2.9 pmol.min-1.mm-1 under control conditions and decreased by 20% upon addition of serosal PTH. PTH had no effect on Jv. Inclusion of PTH in the luminal perfusate reduced JPi to 2.1 pmol. min-1. mm-1. Combined addition of PTH to perfusate and bathing solutions reduced JPi to 1.5 pmol. min-1. mm-1 without affecting Jv. Indirect immunofluorescence studies revealed abundant PTH receptor (PTH1R) expression on brush-border membranes, with lower amounts on basolateral membranes. CaSRs were localized primarily, but not exclusively, to brush-border membranes. CaSR activation with luminal Gd3+ abolished the inhibitory action of PTH on JPi. Addition of Gd3+ to the serosal bathing solution had no effect on PTH-sensitive JPi. Gd3+ i.e., PTH-independent JPi. Gd3+ did not affect basal, had no effect on Jv when added to lumen or bath. Dopamine-inhibitable JPi was not affected by Gd3+. Experiments with proximal-like opossum kidney cells showed that elevated extracellular Ca2+ or NPS R467, a type II calcimimetic, inhibited PTH action on Pi uptake. In conclusion, PTH1Rs are expressed on apical and basolateral membranes of mouse proximal tubules. Stimulating apical or basolateral PTH1R inhibits phosphate absorption. CaSR activation specifically regulates PTH-suppressible phosphate absorption.

Activation-independent parathyroid hormone receptor internalization is regulated by NHERF1 (EBP50).

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.