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The parathyroid hormone receptor type 1 (PTH1R) is a G protein-coupled receptor that plays key roles in regulating calcium homeostasis and skeletal development via binding the ligands, PTH and PTH-related protein (PTHrP), respectively. Eiken syndrome is a rare disease of delayed bone mineralization caused by homozygous PTH1R mutations. Of the three mutations identified so far, R485X, truncates the PTH1R C-terminal tail, while E35K and Y134S alter residues in the receptor's amino-terminal extracellular domain. Here, using a variety of cell-based assays, we show that R485X increases the receptor's basal rate of cAMP signaling and decreases its capacity to recruit β-arrestin2 upon ligand stimulation. The E35K and Y134S mutations each weaken the binding of PTHrP leading to impaired β-arrestin2 recruitment and desensitization of cAMP signaling response to PTHrP but not PTH. Our findings support a critical role for interaction with β-arrestin in the mechanism by which the PTH1R regulates bone formation., (© 2023. The Author(s).)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Context: Five different activating PTH/PTH-related peptide (PTHrP) receptor (PTHR1) mutations have been reported as causes of Jansen metaphyseal chondrodysplasia (JMC), a rare disorder characterized by severe growth plate abnormalities and PTH-independent hypercalcemia., Objectives: Assess the natural history of clinical and laboratory findings in 24 patients with JMC and characterize the disease-causing mutant receptors in vitro., Patients and Methods: The H223R mutation occurred in 18 patients. T410P, I458R and I458K each occurred in single cases; T410R was present in a father and his two sons. Laboratory records were analyzed individually and in aggregate., Results: Postnatal calcium levels were normal in most patients, but elevated between 0.15 and 10 years (11.8 ± 1.37 mg/dL) and tended to normalize in adults (10.0 ± 1.03 mg/dL). Mean phosphate levels were at the lower end of the age-specific normal ranges. Urinary calcium/creatinine (mg/mg) were consistently elevated (children, 0.80 ± 0.40; adults, 0.28 ± 0.19). Adult heights were well below the 3rd percentile for all patients, except for those with the T410R mutation. Most patients with JMC had undergone orthopedic surgical procedures, most had nephrocalcinosis, and two had advanced chronic kidney disease. The five PTHR1 mutants showed varying degrees of constitutive and PTH-stimulated cAMP signaling activity when expressed in HEK293 reporter cells. The inverse agonist [L11,dW12,W23,Y36]PTHrP(7-36) reduced basal cAMP signaling for each PTHR1 mutant., Conclusions: Except for T410R, the other PTHR1 mutations were associated with indistinguishable mineral ion abnormalities and cause similarly severe growth impairment. Hypercalciuria persisted into adulthood. An inverse agonist ligand effectively reduced in vitro PTH-independent cAMP formation at all five PTHR1 mutants, suggesting a potential path toward therapy.

The PTH receptor type 1 (PTHR1) mediates the actions of two endogenous polypeptide ligands, PTH and PTHrP, and thereby plays key roles in bone biology. Based on its capacity to stimulate bone formation, the peptide fragment PTH (1-34) is currently in use as therapy for osteoporosis. Abaloparatide (ABL) is a novel synthetic analog of human PTHrP (1-34) that holds promise as a new osteoporosis therapy, as studies in animals suggest that it can stimulate bone formation with less of the accompanying bone resorption and hypercalcemic effects that can occur with PTH (1-34). Recent studies in vitro suggest that certain PTH or PTHrP ligand analogs can distinguish between two high-affinity PTHR1 conformations, R(0) and RG, and that efficient binding to R(0) results in prolonged signaling responses in cells and prolonged calcemic responses in animals, whereas selective binding to RG results in more transient responses. As intermittent PTH ligand action is known to favor the bone-formation response, whereas continuous ligand action favors the net bone-resorption/calcemic response, we hypothesized that ABL binds more selectively to the RG vs the R(0) PTHR1 conformation than does PTH (1-34), and thus induces more transient signaling responses in cells. We show that ABL indeed binds with greater selectivity to the RG conformation than does PTH (1-34), and as a result of this RG bias, ABL mediates more transient cAMP responses in PTHR1-expressing cells. The findings provide a plausible mechanism (ie, transient signaling via RG-selective binding) that can help account for the favorable anabolic effects that ABL has on bone.

Hypocalcemia and hyperphosphatemia are encountered in idiopathic hypoparathyroidism (IHP) and pseudohypoparathyroidism type Ib (PHP1B). In contrast to PHP1B, which is caused by resistance toward parathyroid hormone (PTH), the genetic defects leading to IHP impair production of this important regulator of mineral ion homeostasis. So far, only five PTH mutations were shown to cause IHP, each of which is located in the hormone's pre-pro leader segment and thus impair hormone secretion. In three siblings affected by IHP, we now identified a homozygous arginine-to-cysteine mutation at position 25 (R25C) of the mature PTH(1-84) polypeptide; heterozygous family members are healthy. Depending on the assay used for evaluating these patients, plasma PTH levels were either low or profoundly elevated, thus leading to ambiguities regarding the underlying diagnosis, namely IHP or PHP1B. Consistent with increased PTH levels, recombinant [Cys25]PTH(1-84) and wild-type PTH(1-84) were secreted equally well by transfected COS-7 cells. However, synthetic [Cys25]PTH(1-34) was found to have a lower binding affinity for the PTH receptor type-1 (PTH1R) than PTH(1-34) and consequently a lower efficiency for stimulating cAMP formation in cells expressing this receptor. Consistent with these in vitro findings, long-term infusion of [Cys25]PTH(1-34) resulted only in minimal calcemic and phosphaturic responses, despite readily detectable levels of [Cys25]PTH(1-34) in plasma. The mineral ion abnormalities observed in the three IHP patients are thus most likely caused by the inherited homozygous missense PTH mutation, which reduces bioactivity of the secreted hormone. Based on these findings, screening for PTH(1-84) mutations should be considered when clinical and laboratory findings are consistent with PHP1B, but GNAS methylation changes have been excluded. Differentiating between IHP and PHP1B has considerable implications for genetic counseling, therapy, and long-term outcome because treatment of IHP patients with inappropriately high doses of active vitamin D and calcium can contribute to development of nephrocalcinosis and chronic kidney disease., (© 2015 American Society for Bone and Mineral Research.)

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) are two related peptides that control calcium/phosphate homeostasis and bone development, respectively, through activation of the PTH/PTHrP receptor (PTH1R), a class B G protein-coupled receptor. Both peptides hold clinical interest for their capacities to stimulate bone formation. PTH and PTHrP display different selectivity for two distinct PTH1R conformations, but how their binding to the receptor differs is unclear. The high resolution crystal structure of PTHrP bound to the extracellular domain (ECD) of PTH1R reveals that PTHrP binds as an amphipathic alpha-helix to the same hydrophobic groove in the ECD as occupied by PTH, but in contrast to a straight, continuous PTH helix, the PTHrP helix is gently curved and C-terminally "unwound." The receptor accommodates the altered binding modes by shifting the side chain conformations of two residues within the binding groove: Leu-41 and Ile-115, the former acting as a rotamer toggle switch to accommodate PTH/PTHrP sequence divergence, and the latter adapting to the PTHrP curvature. Binding studies performed with PTH/PTHrP hybrid ligands having reciprocal exchanges of residues involved in different contacts confirmed functional consequences for the altered interactions and enabled the design of altered PTH and PTHrP peptides that adopt the ECD-binding mode of the opposite peptide. Hybrid peptides that bound the ECD poorly were selective for the G protein-coupled PTH1R conformation. These results establish a molecular model for better understanding of how two biologically distinct ligands can act through a single receptor and provide a template for designing better PTH/PTHrP therapeutics.

PTH and PTHrP use the same G protein-coupled receptor, the PTH/PTHrP receptor (PTHR), to mediate their distinct biological actions. The extent to which the mechanisms by which the two ligands bind to the PTHR differ is unclear. We examined this question using several pharmacological and biophysical approaches. Kinetic dissociation and equilibrium binding assays revealed that the binding of [(125)I]PTHrP(1-36) to the PTHR was more sensitive to GTPgammaS (added to functionally uncouple PTHR-G protein complexes) than was the binding of [(125)I]PTH(1-34) ( approximately 75% maximal inhibition vs. approximately 20%). Fluorescence resonance energy transfer-based kinetic analyses revealed that PTHrP(1-36) bound to the PTHR more slowly and dissociated from it more rapidly than did PTH(1-34). The cAMP signaling response capacity of PTHrP(1-36) in cells decayed more rapidly than did that of PTH(1-34) (t(1/2) = approximately 1 vs. approximately 2 h). Divergent residue 5 in the ligand, Ile in PTH and His in PTHrP, was identified as a key determinant of the altered receptor-interaction responses exhibited by the two peptides. We conclude that whereas PTH and PTHrP bind similarly to the G protein-coupled PTHR conformation (RG), PTH has a greater capacity to bind to the G protein-uncoupled conformation (R(0)) and, hence, can produce cumulatively greater signaling responses (via R(0)-->RG isomerization) than can PTHrP. Such conformational selectivity may relate to the distinct modes by which PTH and PTHrP act biologically, endocrine vs. paracrine, and may help explain reported differences in the effects that the ligands have on calcium and bone metabolism when administered to humans.

Parathyroid hormone (PTH) has a central role in the regulation of serum calcium and phosphate, while parathyroid hormone-related peptide (PTHrP) has important developmental roles. Both peptides signal through the same receptor, the PTH/PTHrP receptor (a class B G-protein-coupled receptor). The different biological effects of these ligands result from their modes of regulation and secretion, endocrine vs. paracrine/autocrine. The importance of PTH and PTHrP is evident by the variety of clinical syndromes caused by deficiency or excess production of either peptide, and the demonstration that intermittent injection of PTH increases bone mass, and thus provides a means to treat osteoporosis. This, in turn, has triggered increased interest in understanding the mechanisms of PTH/PTHrP receptor action and the search for smaller peptide or non-peptide agonists that have efficacy at this receptor when administered non-parenterally.

Recent functional studies have suggested that position 19 in PTH interacts with the portion of the PTH-1 receptor (P1R) that contains the extracellular loops and seven transmembrance helices (TMs) (the J domain). We tested this hypothesis using the photoaffinity cross-linking approach. A PTHrP(1-36) analog and a conformationally constrained PTH(1-21) analog, each containing para-benzoyl-l-phenylalanine (Bpa) at position 19, each cross-linked efficiently to the P1R expressed in COS-7 cells, and digestive mapping analysis localized the cross-linked site to the interval (Leu232-Lys240) at the extracellular end of TM2. Point mutation analysis identified Ala234, Val235, and Lys240 as determinants of cross-linking efficiency, and the Lys240-->Ala mutation selectively impaired the binding of PTH(1-21) and PTH(1-19) analogs, relative to that of PTH(1-15) analogs. The findings support the hypothesis that residue 19 of the receptor-bound ligand contacts, or is close to, the P1R J domain-specifically, Lys240 at the extracellular end of TM2. The findings also support a molecular model in which the 1-21 region of PTH binds to the extracellular face of the P1R J domain as an alpha-helix.

The carboxyl-terminal portions of parathyroid hormone (PTH)-(1--34) and PTH-related peptide (PTHrP)-(1-36) are critical for high affinity binding to the PTH/PTHrP receptor (P1R), but the mechanism of receptor interaction for this domain is largely unknown. To identify interaction sites between the carboxyl-terminal region of PTHrP-(1--36) and the P1R, we prepared analogs of [I(5),W(23),Y(36)]PTHrP-(1--36)-amide with individual p-benzoyl-l-phenylalanine (Bpa) substitutions at positions 22--35. When tested with LLC-PK(1) cells stably transfected with human P1R (hP1R), the apparent binding affinity and the EC(50) of agonist-stimulated cAMP accumulation for each analog was, with the exception of the Bpa(24)-substituted analog, similar to that of the parent compound. The radiolabeled Bpa(23)-, Bpa(27)-, Bpa(28)-, and Bpa(33)-substituted compounds affinity-labeled the hP1R sufficiently well to permit subsequent mapping of the cross-linked receptor region. Each of these peptides cross-linked to the amino-terminal extracellular domain of the P1R: [I(5),Bpa(23),Y(36)]PTHrP-(1-36)-amide cross-linked to the extreme end of this domain (residues 33-63); [I(5),W(23),Bpa(27),Y(36)]PTHrP-(1--36)-amide cross-linked to residues 96--102; [I(5),W(23),Bpa(28),Y(36)]PTHrP-(1--36)- amide cross-linked to residues 64--95; and [I(5),W(23), Bpa(33),Y(36)]PTHrP-(1--36)-amide cross-linked to residues 151-172. These data thus predict that residues 23, 27, 28, and 33 of native PTHrP are each near to different regions of the amino-terminal extracellular receptor domain of the P1R. This information helps define sites of proximity between several ligand residues and this large receptor domain, which so far has been largely excluded from models of the hormone-receptor complex.

The N-terminal regions of PTH and PTH-related peptide (PTHrP) are involved in receptor-mediated signaling and subtype selectivity. To better understand the molecular basis for these processes, we first prepared a series of [I5,W23,Y36]-PTHrP(1-36)NH2 analogs having stepwise deletions of residues 1-4 and characterized them with the human (h)PTH-1 and hPTH-2 receptor subtypes stably transfected in LLC-PK1 cells. Deletions beyond residue 2 caused progressive and severe losses in cAMP-signaling efficacy without dramatically diminishing receptor-binding affinity; consistent with this, [I5,W23]-PTHrP(5-36) was a potent antagonist for both PTH receptor subtypes. We then prepared and characterized photolabile analogs of [I5,W23,Y36]-PTHrP(1-36)NH2 that were singly modified with parabenzoyl-L-phenylalanine (Bpa) along the first six residues. These full-length analogs exhibited receptor subtype-selective agonism, antagonism, and photochemical cross-linking profiles. In particular, the [Bpa2]- and [Bpa4]-substituted analogs selectively antagonized and preferentially cross-linked to the PTH-1 receptor and PTH-2 receptor, respectively. These results demonstrate that the 1-5 region of [I5,W23]-PTHrP(1-36) is critical for activating the PTH-1 and PTH-2 receptors and suggest that the individual residues in this region play distinct roles in modulating the activation states of the two receptors. The cross-linking of both agonist and antagonist ligands to these PTH receptors lays the groundwork for identifying critical signaling determinants in the ligand binding pocket of the receptor.

Low resolution mutational studies have indicated that the amino-terminal extracellular domain of the rat parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor (rP1R) interacts with the carboxyl-terminal portion of PTH-(1-34) or PTHrP-(1-36). To further define ligand-receptor interactions, we prepared a fully functional photoreactive analog of PTHrP, [Ile5,Bpa23,Tyr36]PTHrP-(1-36)-amide ([Bpa23]PTHrP, where Bpa is p-benzoyl-L-phenylalanine). Upon photolysis, radioiodinated [Bpa23]PTHrP covalently and specifically bound to the rP1R. CNBr cleavage of the broad approximately 80-kDa complex yielded a radiolabeled approximately 9-kDa non-glycosylated protein band that could potentially be assigned to rP1R residues 23-63, Tyr23 being the presumed amino-terminus of the receptor. This assignment was confirmed using a mutant rP1R (rP1R-M63I) that yielded, upon photoligand binding and CNBr digestion, a broad protein band of approximately 46 kDa, which was reduced to a sharp band of approximately 20 kDa upon deglycosylation. CNBr digestion of complexes formed with two additional rP1R double mutants (rP1R-M63I/L40M and rP1R-M63I/L41M) yielded non-glycosylated protein bands that were approximately 6 kDa in size, indicating that [Bpa23]PTHrP cross-links to amino acids 23-40 of the rP1R. This segment overlaps a receptor region previously identified by deletion mapping to be important for ligand binding. Alanine scanning of this region revealed two residues, Thr33 and Gln37, as being functionally involved in ligand binding. Thus, the convergence of photoaffinity cross-linking and mutational data demonstrates that the extreme amino-terminus of the rP1R participates in ligand binding.