Molecular insights into differentiated ligand recognition of the human parathyroid hormone receptor 2.

The parathyroid hormone receptor 2 (PTH2R) is a class B1 G protein-coupled receptor (GPCR) involved in the regulation of calcium transport, nociception mediation, and wound healing. Naturally occurring mutations in PTH2R were reported to cause hereditary diseases, including syndromic short stature. Here, we report the cryogenic electron microscopy structure of PTH2R bound to its endogenous ligand, tuberoinfundibular peptide (TIP39), and a heterotrimeric Gs protein at a global resolution of 2.8 Å. The structure reveals that TIP39 adopts a unique loop conformation at the N terminus and deeply inserts into the orthosteric ligand-binding pocket in the transmembrane domain. Molecular dynamics simulation and site-directed mutagenesis studies uncover the basis of ligand specificity relative to three PTH2R agonists, TIP39, PTH, and PTH-related peptide. We also compare the action of TIP39 with an antagonist lacking six residues from the peptide N terminus, TIP(7-39), which underscores the indispensable role of the N terminus of TIP39 in PTH2R activation. Additionally, we unveil that a disease-associated mutation G258D significantly diminished cAMP accumulation induced by TIP39. Together, these results not only provide structural insights into ligand specificity and receptor activation of class B1 GPCRs but also offer a foundation to systematically rationalize the available pharmacological data to develop therapies for various disorders associated with PTH2R.

Parathyroid hormone induces adipocyte lipolysis via PKA-mediated phosphorylation of hormone-sensitive lipase.

Parathyroid hormone (PTH) is secreted from the parathyroid glands in response to low plasma calcium levels. Besides its classical actions on bone and kidney, PTH may have other important effects, including metabolic effects, as suggested for instance by increased prevalence of insulin resistance and type 2 diabetes in patients with primary hyperparathyroidism. Moreover, secondary hyperparathyroidism may contribute to the metabolic derangements that characterize states of vitamin D deficiency. PTH has been shown to induce adipose tissue lipolysis, but the details of the lipolytic action of PTH have not been described. Here we used primary mouse adipocytes to show that intact PTH (1-84) as well as the N-terminal fragment (1-37) acutely stimulated lipolysis in a dose-dependent manner, whereas the C-terminal fragment (38-84) was without lipolytic effect. The lipolytic action of PTH was paralleled by phosphorylation of known protein kinase A (PKA) substrates, i.e. hormone-sensitive lipase (HSL) and perilipin. The phosphorylation of HSL in response to PTH occurred at the known PKA sites S563 and S660, but not at the non-PKA site S565. PTH-induced lipolysis, as well as phosphorylation of HSL at S563 and S660, was blocked by both the PKA-inhibitor H89 and the adenylate cyclase inhibitor MDL-12330A, whereas inhibitors of extracellular-regulated kinase (ERK), protein kinase B (PKB), AMP-activated protein kinase (AMPK) and Ca(2+)/calmodulin-dependent protein kinase (CaMK) had little or no effect. Inhibition of phosphodiesterase 4 (PDE4) strongly potentiated the lipolytic action of PTH, whereas inhibition of PDE3 had no effect. Our results show that the lipolytic action of PTH is mediated by the PKA signaling pathway with no or minor contribution of other signaling pathways and, furthermore, that the lipolytic action of PTH is limited by simultaneous activation of PDE4. Knowledge of the signaling pathways involved in the lipolytic action of PTH is important for our understanding of how metabolic derangements develop in states of hyperparathyroidism, including vitamin D deficiency.

Incubation of Fear Is Regulated by TIP39 Peptide Signaling in the Medial Nucleus of the Amygdala.

Fear-related psychopathologies such as post-traumatic stress disorder are characterized by impaired extinction of fearful memories. Recent behavioral evidence suggests that the neuropeptide tuberoinfundibular peptide of 39 residues (TIP39), via its receptor, the parathyroid hormone 2 receptor (PTH2R), modulates fear memory. Here we examined the anatomical and cellular localization of TIP39 signaling that contributes to the increase in fear memory over time following a traumatic event, called fear memory incubation. Contextual freezing, a behavioral sign of fear memory, was significantly greater in PTH2R knock-out than wild-type male mice 2 and 4 weeks after a 2 s 1.5 mA footshock. PTH2R knock-out mice had significantly reduced c-Fos activation in the medial amygdala (MeA) following both footshock and fear recall, but had normal activation in the hypothalamic paraventricular nucleus and the amygdalar central nucleus compared with wild-type. We therefore investigated the contribution of MeA TIP39 signaling to fear incubation. Similar to the effect of global TIP39 signaling loss, blockade of TIP39 signaling in the MeA by lentivirus-mediated expression of a secreted PTH2R antagonist augmented fear incubation. Ablation of MeA PTH2R-expressing neurons also strengthened the fear incubation effect. Using the designer receptor exclusively activated by designer drug pharmacogenetic approach, transient inhibition of MeA PTH2R-expressing neurons before or immediately after the footshock, but not at the time of fear recall, enhanced fear incubation. Collectively, the findings demonstrate that TIP39 signaling within the MeA at the time of an aversive event regulates the increase over time in fear associated with the event context. SIGNIFICANCE STATEMENT: Fear-related psychopathologies such as post-traumatic stress disorder (PTSD) are characterized by excessive responses to trauma-associated cues. Fear responses can increase over time without additional cue exposure or stress. This work shows that modulatory processes within the medial nucleus of the amygdala near the time of a traumatic event influence the strength of fear responses that occur much later. The modulatory processes include signaling by the neuropeptide TIP39 and neurons that express its receptor. These findings will help in the understanding of why traumatic events sometimes have severe psychological consequences. One implication is that targeting neuromodulation in the medial amygdala could potentially help prevent development of PTSD.

PFOA and PFOS are associated with reduced expression of the parathyroid hormone 2 receptor (PTH2R) gene in women.

Little is known about interactions between environmental and genetic risk factors for osteoarthritis (OA). Genetic factors include variation or mutation in genes involved in parathyroid hormone signalling. Exposure to the endocrine disrupting chemicals perfluoro-octanoic acid (PFOA) or perfluorooctane sulfonate (PFOS) have been suggested as potential environmental contributors, although evidence to support this association is conflicting. Here we test the hypothesis that PFOA and PFOS may alter the mRNA expression of genes in the parathyroid signalling cascade to provide evidence on possible pathways between these chemicals and OA. We measured the relationship between PFOA or PFOS serum levels and the in vivo expression of the Parathyroid hormone 1 and 2 genes (PTH, PTH2), Parathyroid hormone 1 and 2 receptor genes (PTH1R, PTH2R) and the parathyroid hormone-like (PTHLH) gene in peripheral blood from a cross-sectional population study designed to assess the potential health effects of these chemicals. We used multivariate linear regression models and found that PFOA or PFOS was inversely correlated with parathyroid hormone 2 receptor (PTH2R) expression (coefficients=-0.43 and -0.32, p=p=0.017 and 0.006 for PFOA and PFOS respectively) in 189 female subjects. The levels of PTH2 transcripts encoding the ligand of PTH2r, were also found to be lower in women with OA (median 2.08) compared with controls (median 3.41, p=0.046). As the parathyroid signalling cascade is a known candidate for osteoarthritis risk and our findings raise the possibility that exposure to these chemicals may contribute to the pathogenesis of OA in some individuals.

Variation in the PTH2R gene is associated with age-related degenerative changes in the lumbar spine.

In the elderly, degenerative changes in the lumbar spine are common, contributing to falsely elevated bone mineral density (BMD) values. The parathyroid hormone (PTH) system plays an important role in the regulation of bone turnover and we explore the hypothesis that polymorphisms (SNPs) within genes in this pathway (PTH, PTHLH, PTH1R and PTH2R) contribute to degenerative manifestations of the spine in elderly women. The study included 1,004 Swedish women aged 75 years from the population-based OPRA cohort who attended follow-up at 5 and 10 years. Lumbar spine BMD was assessed by dual energy X-ray absorptiometry (DXA) and each individual vertebra was evaluated visually on the DXA image for apparent degenerative manifestations. Six SNPs in PTH and 3 SNPs each in PTH1R, PTH2R and PTHLH were analysed. Among women with degenerative manifestations at the lumbar spine, there was an over-representation at baseline of those carrying the PTH2R SNP rs897083 A-allele (p = 0.0021; odds ratio 1.5 95 % CI 1.2-2.0) and across the duration of follow-up (p = 0.0008). No association was observed between degenerative manifestations and variation in the other genes. None of the PTH hormone system genes were associated with vertebral fracture. Variation in the PTH2R gene (Chr2q34, rs897083) may contribute to the age-associated degenerative manifestations that develop at the lumbar spine.

Defective postnatal endochondral bone development by chondrocyte-specific targeted expression of parathyroid hormone type 2 receptor.

The human parathyroid hormone type 2 receptor (PTH2R) is activated by PTH and by tuberoinfundibular peptide of 39 residues (TIP39), the latter likely acting as its natural ligand. Although the receptor is expressed at highest levels in the nervous system, we have observed that both PTH2R and TIP39 are expressed in the newborn mouse growth plate, with the receptor localizing in the resting zone and the ligand TIP39 localizing exclusively in prehypertrophic and hypertrophic chondrocytes. To address the role of PTH2R in postnatal skeletal growth and development, Col2a1-hPTH2R (PTH2R-Tg) transgenic mice were generated. The mice were viable and of nearly normal size at birth. Expression of the transgene in the growth plate was limited to chondrocytes. We found that chondrocyte proliferation was decreased, as determined by in vivo BrdU labeling of proliferating chondrocytes and CDK4 and p21 expression in the growth plate of Col2a1-hPTH2R transgenic mice. Similarly, the differentiation and maturation of chondrocytes was delayed, as characterized by decreased Sox9 expression and weaker immunostaining for the chondrocyte differentiation markers collagen type II and type X and proteoglycans. As well, there was altered expression of Gdf5, Wdr5, and ß-catenin, factors implicated in chondrocyte maturation, proliferation, and differentiation.These effects impacted on the process of endochondral ossification, resulting in delayed formation of the secondary ossification center, and diminished trabecular bone volume. The findings substantiate a role for PTH2R signaling in postnatal growth plate development and subsequent bone mass acquisition.

Genetic variation in the PTH pathway and bone phenotypes in elderly women: evaluation of PTH, PTHLH, PTHR1 and PTHR2 genes.

INTRODUCTION: Parathyroid hormone (PTH) is a key regulator of calcium metabolism. Parathyroid hormone-like hormone (PTHrP) contributes to skeletal development through regulation of chondrocyte proliferation and differentiation during early bone growth. Both PTH and PTHrP act through the same receptor (PTHR1). A second receptor, PTHR2, has been identified although its function is comparatively unknown. PTH hyper-secretion induces bone resorption, whereas intermittent injection of PTH increases bone mass. To explore the effects of genetic variation in the PTH pathway, we have analysed variations in PTH, PTHLH, PTHR1 and PTHR2 in relation to bone mass and fracture incidence in elderly women. MATERIALS AND METHODS: This study includes 1044 elderly women, all 75 years old, from the Malmö Osteoporosis Prospective Risk Assessment study (OPRA). Single nucleotide polymorphisms (SNPs) from 4 genes and derived haplotypes in the PTH signaling pathway were analysed in 745-1005 women; 6 SNPs in the PTH gene and 3 SNPs each in the PTHLH, PTHR1 and PTHR2 genes were investigated in relation to BMD (assessed at baseline), fracture (434 prevalent fractures of all types over lifetime, self-reported and 174 incident fractures up to 7 years, X-ray verified) and serum PTH. RESULTS AND CONCLUSION: Individually, SNPs in the 4 loci did not show any significant association with BMD. Neither were PTHLH, PTHR1 and PTHR2 polymorphisms associated with fracture. Three of 5 common haplotypes, accounting for >98% of alleles at the PTH locus, were identified as independent predictors of fracture. Haplotype 9 (19%) was suggestive of an association with fractures of any type sustained during lifetime (p=0.018), with carriers of one or more copies of the haplotype having the lowest incidence (p=0.006). Haplotypes 1 (13%) and 5 (37%) and 9 were suggestive of an association with fractures sustained between 50 and 75 years (p=0.02, p=0.013 and p=0.034). Carriers of haplotypes 1 and 5 were more likely to suffer a fracture (haplotype 1, p=0.045; haplotype 5, p=0.008). We conclude, that while further genotyping across the gene is recommended, in this cohort of elderly Swedish women, polymorphisms in PTH may contribute to the risk of fracture through mechanisms that are independent of BMD.

Abnormal muscle and hematopoietic gene expression may be important for clinical morbidity in primary hyperparathyroidism.

In primary hyperparathyroidism (PHPT), excess PTH secretion by adenomatous or hyperplastic parathyroid glands leads to elevated serum [Ca(2+)]. Patients present complex symptoms of muscular fatigue, various neuropsychiatric, neuromuscular, and cardiovascular manifestations, and, in advanced disease, kidney stones and metabolic bone disease. Our objective was to characterize changes in muscle and hematopoietic gene expression in patients with reversible mild PHPT after parathyroidectomy and possibly link molecular pathology to symptoms. Global mRNA profiling using Affymetrix gene chips was carried out in biopsies obtained before and 1 yr after parathyroidectomy in seven patients discovered by routine blood [Ca(2+)] screening. The tissue distribution of PTH receptor (PTHR1 and PTHR2) mRNAs were quantitated using real-time RT-PCR in unrelated persons to define PTH target tissues. Of about 10,000 expressed genes, 175 muscle, 169 hematological, and 99 bone-associated mRNAs were affected. Notably, the major part of muscle-related mRNAs was increased whereas hematological mRNAs were predominantly decreased during disease. Functional and molecular network analysis demonstrated major alterations of several tissue characteristic groups of mRNAs as well as those belonging to common cell signaling and major metabolic pathways. PTHR1 and PTHR2 mRNAs were more abundantly expressed in muscle and brain than in hematopoietic cells. We suggest that sustained stimulation of PTH receptors present in brain, muscle, and hematopoietic cells have to be considered as one independent, important cause of molecular disease in PHPT leading to profound alterations in gene expression that may help explain symptoms like muscle fatigue, cardiovascular pathology, and precipitation of psychiatric illness.

Development of a rat parathyroid hormone 2 receptor antagonist.

The parathyroid hormone 2 (PTH2) receptor is a Family B G-protein coupled receptor most highly expressed within the brain. Current evidence suggests that tuberoinfundibular peptide of 39 residues (TIP39) is the PTH2 receptor's endogenous ligand. To facilitate investigation of the physiological function of the PTH2 receptor/TIP39 system, we have developed a novel PTH2 receptor antagonist, by changing several residues within the amino terminal domain of TIP39. Histidine(4), tyrosine(5), tryptophan(6), histidine(7)-TIP39 binds the PTH2 receptor with high affinity, has over 30-fold selectivity for the rat PTH2 receptor over the rat PTH1 receptor and displays no detectable agonist activity. This ligand should be useful for in vivo investigation of PTH2 receptor function.

Strong linkage on 2q33.3 to familial early-onset generalized osteoarthritis and a consideration of two positional candidate genes.

A genomewide screen was performed in four extended families with early-onset generalized osteoarthritis (FOA) without dysplasia. The FOA phenotype within these families shows a dominant Mendelian inheritance pattern and may represent common osteoarthritis (OA) at later ages. An initial locus was confirmed by three additional families and refined by 14 markers to a two-point logarithm of odds score of 6.05 (theta=0.00) for marker D2S155 at chromosome 2q33.3. This locus coincided with the highest multipoint nonparametric linkage score of 4.70 (P-value=0.0013) at marker D2S2358. Haplotype analysis of family members delineated a narrow region with a number of possible positional candidates, of which we investigate here the two most likely ones: PTHR2, encoding parathyroid hormone receptor 2, and FZD5, encoding frizzled receptor 5. For FZD5, we did not observe a segregating variant, however, for PTHR2, a missense variant (A225S) cosegregated with FOA in one family. The frequency of the PTHR2 variant was rare in a population-based sample, aged 55-70 years (N=1228, 0.4%). Of the 11 carriers, 36% showed generalized radiographic OA as compared to 23% in the remaining population. None of the other families that contributed to the linkage revealed a segregating variant. Together, we have identified a locus on chromosome 2q33.3 for FOA. Candidate gene analysis suggested a possible association of a PTHR2 variant with generalized radiographic OA; it is, however, unlikely the major disease gene for the observed linkage to the FOA phenotype.