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.

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.

Breakpoint mapping by whole genome sequencing identifies PTH2R gene disruption in a patient with midline craniosynostosis and a de novo balanced chromosomal rearrangement.

BACKGROUND: Craniosynostosis (CRS) is a premature closure of calvarial sutures caused by gene mutation or environmental factors or interaction between the two. Only a small proportion of non-syndromic CRS (NSC) patients have a known genetic cause, and thus, it would be meaningful to search for a causative gene disruption for the development NSC. We applied a whole genome sequencing approach on a 15-month-old boy with sagittal and metopic synostosis to identify a gene responsible for the development of the disease. METHODS AND RESULTS: Conventional chromosome study revealed a complex paracentric inversion involving 2q14.3 and 2q34. Array comparative genomic hybridisation did not show any copy number variation. Multicolour banding analysis was carried out and the breakpoints were refined to 2q14 and 2q34. An intronic break of the PTH2R gene was detected by whole genome sequencing and fluorescence in situ hybridisation analysis confirmed disruption of PTH2R. CONCLUSIONS: We report PTH2R as a gene that is disrupted in NSC. The disruption of the PTH2R gene may cause uncontrolled proliferation and differentiation of chondrocytes, which in turn results in premature closure of sutures. This addition of PTH2R to the list of genes associated with NSC expands our understanding of the development of NSC.

Neuropathic and inflammatory pain are modulated by tuberoinfundibular peptide of 39 residues.

Nociceptive information is modulated by a large number of endogenous signaling agents that change over the course of recovery from injury. This plasticity makes understanding regulatory mechanisms involved in descending inhibition of pain scientifically and clinically important. Neurons that synthesize the neuropeptide TIP39 project to many areas that modulate nociceptive information. These areas are enriched in its receptor, the parathyroid hormone 2 receptor (PTH2R). We previously found that TIP39 affects several acute nociceptive responses, leading us to now investigate its potential role in chronic pain. Following nerve injury, both PTH2R and TIP39 knockout mice developed less tactile and thermal hypersensitivity than controls and returned to baseline sensory thresholds faster. Effects of hindpaw inflammatory injury were similarly decreased in knockout mice. Blockade of α-2 adrenergic receptors increased the tactile and thermal sensitivity of apparently recovered knockout mice, returning it to levels of neuropathic controls. Mice with locus coeruleus (LC) area injection of lentivirus encoding a secreted PTH2R antagonist had a rapid, α-2 reversible, apparent recovery from neuropathic injury similar to the knockout mice. Ablation of LC area glutamatergic neurons led to local PTH2R-ir loss, and barley lectin was transferred from local glutamatergic neurons to GABA interneurons that surround the LC. These results suggest that TIP39 signaling modulates sensory thresholds via effects on glutamatergic transmission to brainstem GABAergic interneurons that innervate noradrenergic neurons. TIP39's normal role may be to inhibit release of hypoalgesic amounts of norepinephrine during chronic pain. The neuropeptide may help maintain central sensitization, which could serve to enhance guarding behavior.

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.

Evidence for the expression of parathyroid hormone 2 receptor in the human brainstem.

BACKGROUND AND PURPOSE: The parathyroid hormone 2 receptor (PTH2R) is a G protein coupled receptor. Pharmacological and anatomical evidence suggests that the recently identified tuberoinfundibular peptide of 39 residues is, and parathyroid hormone and parathyroid hormone-related peptide are not, its endogenous ligand. Initial functional studies suggest that the PTH2R is involved in the regulation of viscerosensory information processing. As a first step towards clinical applications, herein we describe the presence of the PTH2R in the human brainstem. MATERIAL AND METHODS: Total RNA was isolated from postmortem human cortical and brainstem samples for RT-PCR. Good quality RNA, as assessed on formaldehyde gel, was reverse transcribed. The combined cDNA products were used as template in PCR reactions with primer pairs specific for the human PTH2R. In addition, PTH2R immunolabelling was performed on free floating sections of the human medulla oblongata using fluorescent amplification immunochemistry. RESULTS: Specific bands in the RT-PCR experiments and sequencing of PCR products demonstrated the expression of PTH2R mRNA in the human brainstem. A high density of PTH2R-immunoreactive fibers was found in brain regions of the medulla oblongata including the nucleus of the solitary tract, the spinal trigeminal nucleus, and the dorsal reticular nucleus of the medulla. CONCLUSION: Independent demonstration of the presence of PTH2R mRNA and immunoreactivity supports the specific expression of the PTH2R in the human brainstem. The distribution of PTH2R-immunoreactive fibers in viscerosensory brain regions is similar to that reported in mouse and rat suggesting a similar role of the PTH2R in human as in rodents. This finding will have important implications when experimental data obtained on the function of the TIP39-PTH2R neuromodulator system in rodents are to be utilized in human.

Distribution of tuberoinfundibular peptide of 39 residues and its receptor, parathyroid hormone 2 receptor, in the mouse brain.

Tuberoinfundibular peptide of 39 residues (TIP39) was identified as a potent parathyroid hormone 2 receptor (PTH2R) agonist. Existing anatomical data also support the suggestion that TIP39 is the PTH2R's endogenous ligand, but a comprehensive comparison of TIP39 and PTH2R distributions has not been performed. In the present study, we compared the distributions of TIP39 and PTH2R on adjacent mouse brain sections. In addition, we determined the locations of PTH2R-expressing cell bodies by in situ hybridization histochemistry and by labeling beta-galactosidase driven by the PTH2R promoter in knockin mice. An excellent correlation was found between the distributions of TIP39-containing fibers and PTH2R-containing cell bodies and fibers throughout the brain. TIP39 and the PTH2R are abundant in medial prefrontal, insular, and ectorhinal cortices, the lateral septal nucleus, the bed nucleus of the stria terminalis, the fundus striati, the amygdala, the ventral subiculum, the hypothalamus, midline and intralaminar thalamic nuclei, the medial geniculate body, the periaqueductal gray, the ventral tegmental area, the superior and inferior colliculi, the parabrachial nuclei, the locus coeruleus, subcoeruleus and periolivary areas, and the nucleus of the solitary tract. Furthermore, even the subregional distribution of TIP39- and PTH2R-immunoreactive fibers in these regions showed remarkable similarities, providing anatomical evidence that TIP39 may act on the PTH2R. Based on these observations and on previous pharmacological data, we propose that TIP39 is an endogenous ligand of the PTH2R and that they form a neuromodulator system, which is optimally positioned to regulate limbic, endocrine, and auditory brain functions. Published 2007 Wiley-Liss, Inc.

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.

Activation of Parathyroid Hormone 2 Receptor Induces Decorin Expression and Promotes Wound Repair.

In this study, we report that TIP39, a parathyroid hormone ligand family member that was recently identified to be expressed in the skin, can induce decorin expression and enhance wound repair. Topical treatment of mice with TIP39 accelerated wound repair, whereas TIP39-deficient mice had delayed repair that was associated with formation of abnormal collagen bundles. To study the potential mechanism responsible for the action of TIP39 in the dermis, fibroblasts were cultured in three-dimensional collagen gels, a process that results in enhanced decorin expression unless activated to differentiate to adipocytes, whereupon these cells reduce expression of several proteoglycans, including decorin. Small interfering RNA-mediated silencing of parathyroid hormone 2 receptor (PTH2R), the receptor for TIP39, suppressed the expression of extracellular matrix-related genes, including decorin, collagens, fibronectin, and matrix metalloproteases. Skin wounds in TIP39-/- mice had decreased decorin expression, and addition of TIP39 to cultured fibroblasts induced decorin and increased phosphorylation and nuclear translocation of CREB. Fibroblasts differentiated to adipocytes and treated with TIP39 also showed increased decorin and production of chondroitin sulfate. Furthermore, the skin of PTH2R-/- mice showed abnormal extracellular matrix structure, decreased decorin expression, and skin hardness. Thus, the TIP39-PTH2R system appears to be a previously unrecognized mechanism for regulation of extracellular matrix formation and wound repair.

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.

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.

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.

Maternal absence of the parathyroid hormone 2 receptor affects postnatal pup development.

During the lactation period, mothers have a variety of adaptive changes in brain physiology and behaviour that allow them to properly raise their pups. The exact circuitries and mechanisms responsible for these changes are not fully understood. Recent evidence suggests that the neuropeptide tuberoinfundibular peptide of 39 residues (TIP39) and its receptor, the parathyroid hormone 2 receptor (PTH2-R), contribute to these mechanisms. To further investigate this idea, we evaluated the growth rate of pups from dams with a genetically inactivated PTH2-R (PTH2-R-KO), as well as maternal behavioural and neuroendocrine parameters. We observed that PTH2-R-KO-reared pups had a slowed growth rate. This was associated with a reduced volume of milk yielded by PTH2-R-KO dams after 30-min suckling compared to wild-type (WT) dams when pups were returned after 5 h of separation. Our data suggest a reduced sensitivity of PTH2-R-KO dams to pup stimulation. We also observed a significant reduction in suckling-induced c-Fos expression in the paraventricular hypothalamic nucleus and signs of lower prolactin levels in the PTH2-R-KO dams. Our data suggest that the reduced growth rate of PTH2-R-KO-reared pups was likely the result of alterations in the milk-production pathway rather than modifications in behaviour. Although PTH2-R-KO dams showed increased anxiety in the elevated zero-maze test, no differences from WT dams in maternal behaviour were observed. Taken together, our findings suggest the involvement of the TIP39/PTH2-R system in the pathways involved in the successful development of the pups.

TIP39 modulates effects of novelty-induced arousal on memory.

Tuberoinfundibular peptide of 39 residues (TIP39) is a neuropeptide localized to neural circuits subserving emotional processing. Recent work showed that mice with null mutation for the gene coding TIP39 (TIP39-KO mice) display increased susceptibility to environmental provocation. Based on this stressor-dependent phenotype, the neuroanatomical distribution of TIP39, and knowledge that novelty-induced arousal modulates memory functions via noradrenergic activation, we hypothesized that exposure to a novel environment differently affects memory performance of mice with or without TIP39 signaling, potentially by differences in sensitivity of the noradrenergic system. We tested TIP39-KO mice and mice with null mutation of its receptor, the parathyroid hormone 2 receptor (PTH2-R), in tasks of short-term declarative and social memory (object recognition and social recognition tests, respectively), and of working memory (Y-maze test) under conditions of novelty-induced arousal or acclimation to the test conditions. Mice lacking TIP39 signaling showed memory impairment selectively under conditions of novelty-induced arousal. Acute administration of a PTH2-R antagonist in wild-type mice had a similar effect. The restoration of memory functions in TIP39-KO mice after injection of a ß-adrenoreceptor-blocker, propranolol, suggested involvement of the noradrenergic system. Collectively, these results suggest that the TIP39/PTH2-R system modulates the effects of novelty exposure on memory performance, potentially by acting on noradrenergic signaling.

Evolution of the vertebrate pth2 (tip39) gene family and the regulation of PTH type 2 receptor (pth2r) and its endogenous ligand pth2 by hedgehog signaling in zebrafish development.

In mammals, parathyroid hormone (PTH), secreted by parathyroid glands, increases calcium levels in the blood from reservoirs in bone. While mammals have two PTH receptor genes, PTH1R and PTH2R, zebrafish has three receptors, pth1r, pth2r, and pth3r. PTH can activate all three zebrafish Pthrs while PTH2 (alias tuberoinfundibular peptide 39, TIP39) preferentially activates zebrafish and mammalian PTH2Rs. We know little about the roles of the PTH2/PTH2R system in the development of any animal. To determine the roles of PTH2 and PTH2R during vertebrate development, we evaluated their expression patterns in developing zebrafish, observed their phylogenetic and conserved synteny relationships with humans, and described the genomic organization of pth2, pth2r, and pth2r splice variants. Expression studies showed that pth2 is expressed in cells adjacent to the ventral part of the posterior tuberculum in the diencephalon, whereas pth2r is robustly expressed throughout the central nervous system. Otic vesicles express both pth2 and pth2r, but heart expresses only pth2. Analysis of mutants showed that hedgehog (Hh) signaling regulates the expression of pth2 transcripts more than that of nearby gnrh2-expressing cells. Genomic analysis showed that a lizard, chicken, and zebra finch lack a PTH2 gene, which is associated with an inversion breakpoint. Likewise, chickens lack PTH2R, while humans lack PTH3R, a case of reciprocally missing ohnologs (paralogs derived from a genome duplication). The considerable evolutionary conservation in genomic structure, synteny relationships, and expression of zebrafish pth2 and pth2r provides a foundation for exploring the endocrine roles of this system in developing vertebrate embryos.

PTH-receptors regulate norepinephrine release in human heart and kidney.

Recent data suggests that chronic renal failure and hyperparathyroidism are associated with sympathetic overactivity. Since peptide hormones are known to modulate norepinephrine (NE) release by activating prejunctional receptors, this study investigates whether parathyroid hormone fragment (1-34) (hPTH(1-34)) increases neuronal NE release in human heart and kidney. Using specific PTH-receptor agonists and antagonists, this study furthermore highlights functional differences between PTH1 and PTH2 receptors. Human atrial and renal tissues were incubated with [(3)H]-NE and superfused. Three electrical stimulations (5Hz, 1min) induced a stable [(3)H]-NE release which was taken as an index of endogenous NE release. RT-PCR with specific primers for PTH1- and PTH2-receptor was performed in heart and kidney. hPTH(1-34) (0.01-0.1µmol/L) and a stable analog of its second messenger cAMP (8-bromo-cAMP) increased [(3)H]-NE release in human atria. This facilitatory effect of PTH was also observed in human renal cortex. The PTH1-receptor antagonist (D-Trp(12), Tyr(34))-pTH-(7-34) (0.5µmol/L) abolished the effect of hPTH(1-34). This data was verified using isolated perfused mouse kidneys. Tuberoinfundibular peptide of 39 residues (TIP-39) (0.1nmol/L-0.1µmol/L) decreased [(3)H]-NE release in atria. PTH1- and PTH2-receptor expressions were demonstrated in human heart and kidney. Moreover, a splice variant of the PTH2-receptor was detected in human kidney. In conclusion, PTH is able to facilitate NE release in human atria and renal cortex by activation of PTH1-receptors. The highly increased PTH levels that can be observed in chronic renal failure might be one contributor for the elevated sympathetic nerve activity and the associated cardiovascular mortality in patients with end stage renal disease.

The TIP39-PTH2 receptor system: unique peptidergic cell groups in the brainstem and their interactions with central regulatory mechanisms.

Tuberoinfundibular peptide of 39 residues (TIP39) is the recently purified endogenous ligand of the previously orphan G-protein coupled parathyroid hormone 2 receptor (PTH2R). The TIP39-PTH2R system is a unique neuropeptide-receptor system whose localization and functions in the central nervous system are different from any other neuropeptides. TIP39 is expressed in two brain regions, the subparafascicular area in the posterior thalamus, and the medial paralemniscal nucleus in the lateral pons. Subparafascicular TIP39 neurons seem to divide into a medial and a lateral cell population in the periventricular gray of the thalamus, and in the posterior intralaminar complex of the thalamus, respectively. Periventricular thalamic TIP39 neurons project mostly to limbic brain regions, the posterior intralaminar thalamic TIP39 neurons to neuroendocrine brain areas, and the medial paralemniscal TIP39 neurons to auditory and other brainstem regions, and the spinal cord. The widely distributed axon terminals of TIP39 neurons have a similar distribution as the PTH2R-containing neurons, and their fibers, providing the anatomical basis of a neuromodulatory action of TIP39. Initial functional studies implicated the TIP39-PTH2R system in nociceptive information processing in the spinal cord, in the regulation of different hypophysiotropic neurons in the hypothalamus, and in the modulation of affective behaviors. Recently developed novel experimental tools including mice with targeted mutations of the TIP39-PTH2R system and specific antagonists of the PTH2R will further facilitate the identification of the specific roles of TIP39 and the PTH2R.