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Genetic linkage studies and positional cloning strategies have led with increasing frequency to the identification of molecular defects, often in unforeseen candidate genes or in novel genes with yet unknown biological relevance. Similar advances in understanding the physiological importance of a variety of proteins have been achieved through gene ablation techniques and through the targeted expression of genes using tissue-specific promoters. Since they often provided unanticipated results, these studies using gene manipulation techniques have proved to be of considerable importance for determining the biological role(s) of some known gene products, frequently redirecting the search for abnormalities in these proteins in human or other mammalian disorders. This is illustrated, as outlined in this chapter, by the identification of activating and inactivating PTH/ PTHrP receptor mutations as the cause of two rare genetic disorders in humans.

The actions of amino-terminal PTH (parathyroid hormone) and PTHrP (PTH related peptide) fragments are mediated through the common PTH/PTHrP receptor that belongs to a distinct family of G-proteincoupled receptors, which typically signal through two second-messenger pathways, cyclic adenosine monophosphate, and inositol phosphate. Similar to the widely expressed PTHrP, the mRNA encoding the PTH/PTHrP receptor is found in a large variety of fetal and adult tissues, but its expression is most abundant in three tissues: kidney and bone, where it mediates the calcium-homeostatic endocrine actions of PTH; and the metaphyseal growth plate, where it mediates the autocrine/paracrine actions of locally synthesized PTHrP. In contrast to the common PTH/PTHrP receptor, the closely related PTH-2 receptor is activated only by PTH, but not by PTHrP, and it may have a distinctive primary ligand, the recently described hypothalamic PTH-like molecule. Expression of the PTH-2 receptor is restricted to relatively few tissues and its mRNA levels are typically much less abundant than those encoding the PTH/PTHrP receptor; its biological importance remains to be determined. Perhaps PTH is its ligand in some organs or tissues, and another ligand mediates other biological actions in other (or all) sites.

Endocrine disorders are typically caused by excessive or insufficient production of an otherwise normal hormone. However, in recent years, several endocrine disorders were shown to be caused by other defects, i.e., mutations in peptide hormones, their cognate receptors, G proteins, or downstream effectors (1 ). Although such defects are extremely uncommon and thus provide only a rare diagnostic challenge, their definition at the molecular level often provided important novel insights into the biological significance of the affected protein and/or its functional properties. This is illustrated by the discovery that Jansen’s metaphyseal chondrodysplasia, a rare genetic disorder characterized by short-limbed dwarfism and severe hormone-independent hypercal-cemia, is caused by activating mutations in the parathyroid hormone (PTH) PTH-related peptide (PTHrP) receptor. These findings are likely to have significant implications for understanding the biological importance of the PTH/PTHrP receptor in addition to its role in regulating mineral ion homeostasis.

Two different activating PTH/PTH-related peptide (PTHrP) receptor mutations, H223R and T410P, were recently identified as the most likely cause of Jansen's metaphyseal chondrodysplasia. To assess the functional importance of either amino acid position in the human PTH/PTHrP receptor, H223 and T410 were individually replaced by all other amino acids. At position 223, only arginine and lysine led to agonist-independent cAMP accumulation; all other amino acid substitutions resulted in receptor mutants that lacked constitutive activity or were uninformative due to poor cell surface expression. In contrast, most amino acid substitutions at position 410 conferred constitutive cAMP accumulation and affected PTH/PTHrP receptor expression not at all or only mildly. Mutations corresponding to the H223R or T410P exchange in the human PTH/PTHrP receptor also led to constitutive activity when introduced into the opossum receptor homolog, but showed little or no change in basal cAMP accumulation when introduced into the rat PTH/PTHrP receptor. The PTH/PTHrP receptor residues mutated in Jansen's disease are conserved in all mammalian members of this family of G protein-coupled receptors. However, when the equivalent of either the H223R or the T410P mutation was introduced into several other related receptors, including the PTH2 receptor and the receptors for calcitonin, secretin, GH-releasing hormone, glucagon-like peptide I, and CRH, the resulting mutants failed to induce constitutive activity. These studies suggest that two residues in the human PTH/PTHrP receptor, 223 and 410, have critical roles in signal transduction, but with different sequence constrains.

The Vitamin D receptor (VDR), a member of the nuclear receptor superfamily, mediates the effects of 1,25-dihydroxyvitamin D3 on mineral ion homeostasis. Although the mammalian and avian VDRs have been extensively studied, little is known about the VDR in lower vertebrate species. To address this, we have isolated the Xenopus laevis VDR (xVDR) complementary DNA. Overall, the xVDR shares 79%, 73%, 73%, and 75% identity at the amino acid level with the chicken, mouse, rat, and human VDRs, respectively. The amino acid residues and subdomains important for DNA binding, hormone binding, dimerization, and transactivation are mostly conserved among all VDR species.The xVDR polypeptide can heterodimerize with the mouse retinoid X receptor α, bind to the rat osteocalcin vitamin D response element (VDRE), and induce vitamin D-dependent transactivation in transfected mammalian cells. Northern analysis reveals two xVDR messenger RNA species of 2.2 kb and 1.8 kb in stage 60 Xenopus tissues. In the adult, xVDR expression is detected in many tissues including kidney, intestine, skin, and bone. During Xenopus development, xVDR messenger RNA first appears at developmental stage 13 (preneurulation), increasing to maximum at stages 57–61 (metamorphosis). Our data demonstrate that, in Xenopus, VDR expression is developmentally regulated and that the vitamin D endocrine system is highly conserved during evolution.

An activating mutation of the receptor for parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP) was recently found in a patient with Jansens's metaphyseal chondrodysplasia, a rare form of short-limbed dwarfism associated with hypercalcemia and normal or low serum concentrations of the two hormones. To investigate this and other activating mutations and to refine the classification of this unusual disorder, we analyzed genomic DNA from six additional patients with Jansen's disease.Exons encoding the PTH-PTHrP receptor were amplified by the polymerase chain reaction (PCR), and the products were analyzed by gel electrophoresis or direct nucleotide-sequence analysis. Nucleotide changes were confirmed by restriction-enzyme digestion of genomic DNA or the PCR products.The previously reported mutation, which changes a histidine at position 223 to arginine (H223R), was found in genomic DNA from three of the six patients but not in DNA from their healthy relatives or 45 unrelated normal subjects. A novel missense mutation that changes a threonine in the receptor's sixth membrane-spanning region to proline (T410P) was identified in another patient but not in 62 normal subjects. In two patients with radiologic evidence of Jansen's metaphyseal chondrodysplasia but less severe hypercalcemia, no receptor mutations were detected. In COS-7 cels expressing PTH-PTHrP receptors with the T410P or H223R mutation, basal cyclic AMP accumulation was four to six times higher than in cells expressing wild-type receptors.The expression of constitutively active PTH-PTHrp receptors in kidney, bone, and growth-plate chondrocytes provides a plausible genetic explanation for mineral-ion abnormalities and metaphyseal changes in patients with Jansen's disease.

We studied the effects of parathyroid hormone (PTH) on PTH parathyroid hormone related peptide (PTHrP) receptor mRNA level, PTHrP binding and PTH-stimulated cyclic adenosine monophosphate (cAMP) accumulation in osteoblasts, derived from fetal rat calvariae (ROB). Cells isolated during 10-70 minutes of collagenase treatment were seeded at a density of 25,000 cells/cm2 and cultured for 4 days. These cells show a fast increase in cAMP production after stimulation for 5 minutes with 20 nM bovine parathyroid hormone(1-34) (bPTH(1-34)). When ROB are incubated with bPTH(1-34) (0.04-40nM) for 24 h, a dose-dependent decrease of the PTH/PTHrP receptor mRNA level, PTHrP binding, and PTH-stimulated cAMP accumulation can be observed. Pretreatment of ROB with a high concentration of bPTH(1-34) (40 nM) leads within 15 minutes to a decrease in PTH-stimulated cAMP accumulation. However, it takesor = 3 h before a significant decrease in PTH/PTHrP receptor mRNA level can be observed. Also a significant decrease in PTHrP binding is observed after only 4 h of incubation with bPTH(1-34). Compared with bPTH(1-34), pretreatment of ROB with bPTH(3-34) (40 and 100 nM) for 24 h causes smaller decreases in PTH-stimulated cAMP accumulation, PTHrP binding, and in the PTH/PTHrP receptor mRNA level. We investigated the possible involvement of the protein kinase A signaling pathway in the regulation of the PTH/PTHrP receptor mRNA expression. Both forskolin and (Bu)2cAMP decreased PTHrP binding and PTH/PTHrP mRNA levels. These observations suggest that chronic activation of the PKA signaling pathway may down-regulate PTH/PTHrP receptor expression and thus hormone responsiveness in "normal" osteoblasts. In short, we found that the decrease of the PTH-stimulated cAMP accumulation after long-term pretreatment with bPTH(1-34) is correlated with both PTH/PTHrP receptor mRNA level and PTHrP binding. These data also suggest that the initial desensitization (30 minutes) of PTH-stimulated cAMP responsiveness by pretreatment with a high concentration of bPTH(1-34) (40 nM) is not dependent on the number of available PTH/PTHrP receptors. The protein kinase A signaling pathway is involved in the regulation of the PTH/PTHrP receptor, but, regarding the effect of bPTH(3-34), other signaling systems are also involved.

Most of the bone and kidney-related functions of parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP) are thought to be mediated by the PTH/PTHrP receptor. Recently, a homologous receptor, the PTH-2 receptor, was obtained from rat and human brain cDNA libraries. This receptor displayed the remarkable property of responding potently to PTH, but not to PTHrP. To begin to define residues involved in the ligand specificity of the PTH-2 receptor, we studied the interaction of several PTH/PTHrP hybrid ligands and other related peptide analogs with the human PTH-2 receptor. The results showed that two sites in PTH and PTHrP fully account for the different potencies that the two ligands exhibited with PTH-2 receptors; residue 5 (His in PTHrP and Ile in PTH) determined signaling capability, while residue 23 (Phe in PTHrP and Trp in PTH) determined binding affinity. By changing these two residues of PTHrP to the corresponding residues of PTH, we were able to convert PTHrP into a ligand that avidly bound to the PTH-2 receptor and fully and potently stimulated cAMP formation. Changing residue 23 alone yielded [Trp23]hPTHrP-(1-36), which was an antagonist for the PTH-2 receptor, but a full agonist for the PTH/PTHrP receptor. Residues 5 and 23 in PTH and PTHrP thus play key roles in signaling and binding interactions, respectively, with the PTH-2 receptor. Receptor-selective agonists and antagonists derived from these studies could help to identify the biological role of the PTH-2 receptor and to map specific sites of ligand-receptor interaction.

PTH-related protein activates a G protein-coupled PTH/PTHrP receptor in many cell types and produces diverse biological actions. To study the signal transduction events associated with biological activity of the PTH/PTHrP receptor in vascular smooth muscle, a principal PTHrP-responsive tissue, rat aortic smooth muscle cells (A10) were stably transfected with a plasmid encoding a PTH/PTHrP receptor and tested for ligand binding, PTHrP-(1-34)-induced cAMP levels, inositol phosphate production, and cytosolic calcium transients. Of nineteen G418-resistant lines recovered, all exhibited high affinity binding [approximately dissociation constant (Kd) > 10(-10)) of iodinated [Tyr36]hPTHrP(1-36)NH2 and ligand-induced cAMP accumulation (2- to 100-fold), which was directly proportional to PTH/PTHrP receptor number (range 4 x 10(3) to 7 x 10(7) sites/cell]. PTHrP had no effect on intracellular calcium or inositol phosphate formation in any cell line regardless of receptor number despite the presence of detectable G alpha q). Transient overexpression of individual G alpha q proteins (G alpha q, G alpha 11 or G alpha 14) into PTH/PTHrP receptor-expressing A10 cells conferred the ability of PTHrP to increase intracellular calcium and inositol phosphate formation. Ligand activation of the recombinant PTH/PTHrP receptor elicited appropriate downstream biological effects in A10 cells including inhibition of DNA synthesis and osteopontin messenger RNA (mRNA) expression. Thus, a single PTH/PTHrP receptor, though capable of coupling to different G proteins, signals exclusively through a cAMP-dependent pathway in vascular smooth muscle.

The parathyroid hormone/parathyroid hormone-related peptide receptor belongs to a distinct family of G protein-coupled receptors, the members of which usually signal through at least two second messenger systems, adenylate cyclase and phospholipase C. The parathyroid hormone/ parathyroid hormone-related peptide receptor is most abundantly expressed in bone, kidney and growth-plate chondrocytes, and, at lower levels, in a variety of fetal and adult tissues. To search for human diseases that are caused by parathyroid hormone/parathyroid hormone-related peptide receptor defects, genomic DNA of patients with pseudohypoparathyroidism type Ib and of patients with Jansen's metaphyseal chondrodysplasia was screened for mutations in all coding exons of the receptor gene. Inactivating parathyroid hormone/parathyroid hormone-related peptide receptor mutations were excluded in patients with pseudohypoparathyroidism type Ib. However, a receptor mutation that causes agonist-independent, constitutive cAMP accumulation was identified in a patient with Jansen's metaphyseal chondrodysplasia, a rare form of short-limbed dwarfism associated with hypercalcemia despite normal or low concentrations of parathyroid hormone and parathyroid hormone-related peptide. These findings allow the conclusion to be drawn that parathyroid hormone/parathyroid hormone-related peptide receptors mediate the endocrine actions of parathyroid hormone, which are required for the control of calcium homeostasis and the autocrine-paracrine actions of parathyroid hormone-related peptide, which are required for normal growth-plate development.

Carboxyl-terminal fragments of PTH (C-PTH) appear to have biological properties different from those mediated by the amino-terminal portions of PTH and PTH-related peptide (PTHrP). To characterize a C-PTH receptor that may be involved in mediating these functions, we performed RRAs and affinity cross-linking studies with several clonal cell lines. Radiolabeled recombinant [Leu8,18,Tyr34]human PTH-(1-84)[mutPTH-(1-84) and [Tyr34] human PTH-(19-84)[mutPTH-(19-84) showed little or no specific binding to stably expressed recombinant PTH/PTHrP receptors. However, high affinity binding was observed using osteoblast-like and rat parathyroid (PT-r3) cells. The apparent Kd values were 20-30 nM for PTH-(1-84), mutPTH-(1-84), and mutPTH-(19-84), respectively; 400-800 nM for PTH-(39-84); and more than 5000 nM for PTH-(53-84). [Nle8,18,Tyr34]bovine PTH-(1-34)amide [PTH-(1-34)], PTH-(44-68), PTHrP-(37-74), and PTHrP-(109-141) showed no displacement of either radioligand. C-PTH receptor number was increased up to 2-fold by pretreating ROS 17/2.8 cells with increasing doses of PTH-(1-34), PTH-(1-84), or 8-bromo-cAMP, whereas no change was observed in response to dexamethasone or PTH-(39-84). Cross-linking studies using radiolabeled mutPTH-(1-84) or mutPTH-(19-84) revealed specific labeling of two proteins in ROS 17/2.8 cells that were approximately 40 and 90 kilodaltons in size (including the radioligand of approximately 10 kilodaltons). The intensity of affinity labeling of both proteins was dose dependently inhibited by increasing concentrations of unlabeled PTH-(1-84) and several carboxyl-terminal PTH-(1-84) fragments, but not by PTH-(1-34). Similar studies with PT-r3 cells revealed only a single protein band of about 90 kilodaltons. These data indicate that the carboxyl-terminal portion of PTH-(1-84) binds specifically to a unique receptor/binding protein distinct from the previously isolated PTH/PTHrP receptor.

Calcium-regulated parathyroid hormone release in patients with mild or advanced secondary hyperparathyroidism. Differences in the regulation of parathyroid hormone (PTH) release by calcium are thought to account for excess PTH secretion in patients with secondary hyperparathyroidism (2°HPTH). To determine whether calcium-regulated PTH release varies with the severity of 2°HPTH in patients with end-stage renal disease, dynamic tests of parathyroid gland function were done using the four-parameter model in 26 patients with 2°HPTH documented by bone biopsy. Estimates of the set point did not differ among patients categorized as mild (basal serum PTH < 400 pg/ml), moderate (basal PTH 400 to 600 pg/ml) or severe (basal PTH > 600 pg/ml) 2°HPTH; values were 1.23 ± 0.06 mmol/liter, 1.24 ± 0.06 mmol/liter and 1.23 ± 0.05 mmol/liter, respectively, and none of these set point estimates differed from results obtained in normal volunteers, 1.21 ± 0.02 mmol/liter (NS). The slope of the sigmoidal ionized calcium-PTH curve also did not differ among groups. Set point values did not correspond to basal serum PTH levels, to the maximum serum PTH level observed during hypocalcemia or to the minimum serum PTH level seen during hypercalcemia in patients with 2°HPTH. In contrast, basal serum PTH values were positively correlated with both the maximum serum PTH level observed during hypocalcemia (r = 0.76, P < 0.01), and the minimum serum PTH level attained during calcium infusions (r = 0.78, P < 0.01). Calcium-regulated PTH release does not differ with the degree of 2°HPTH, and set point abnormalities do not account for excess PTH secretion in patients with chronic renal failure as judged by in vivo dynamic tests of parathyroid gland function. The results suggest that variations in parathyroid gland size are the major contributor to excessive PTH secretion in patients with chronic renal failure.

The functional role of the rat parathyroid hormone(PTH)/PTH-related peptide (PTHrP) receptor's carboxyl-terminal region was characterized by comparing the binding and signaling properties of receptors that have 78 and 111 amino acid deletions (R513 and R480, respectively), with those of the 591-amino acid wild-type (WT) receptor. R480 and R513 have 4- and 1.5-fold lower apparent Kd values for rat PTH-(1-34) (rPTH), compared with the WT receptor (WT, 1.81 +/- 0.19 nM; R513, 1.24 +/- 0.12 nM; R480, 0.48 +/- 0.05 nM, mean +/- S.E.). PTH (100 nM)-stimulated cAMP accumulation and polyphosphoinositide hydrolysis both correlated positively with receptor expression. However, whereas PTH-stimulated polyphosphoinositide hydrolysis was indistinguishable among WT and either truncated mutant at comparable levels of expressed receptors, maximal PTH-stimulated cAMP accumulation was 4-6- and 2-3-fold higher in cells expressing R480 and R513, respectively. Furthermore, pretreatment of COS-7 cells with 100 ng/ml of pertussis toxin (PTX) enhanced PTH-stimulated cAMP accumulation in cells expressing the WT receptor, but failed to do so in cells expressing either R480 or R513. Thus, sequences in the PTH/PTHrP receptor's carboxyl-terminal tail lower the affinity of the WT receptor for agonist; directly interact with, or indirectly facilitate the interaction of the receptor with a PTX-sensitive G protein that inhibits adenylyl cyclase; and decrease the efficacy with which the receptor interacts with Gs.

a Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA b Pediatric Nephrology Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA c Pediatric Endocrinology, Horizon Health Network, Saint John, New Brunswick, Canada E2L 4L2 d Pediatric Endocrinology, Baystate Medical Center, Springfield, MA 01199, USA e Bone and Mineral Disorder Clinic, Section of Pediatric Nephrology, The Children's Mercy Hospital and Clinics, University of Missouri at Kansas City, Kansas City, MO 64108, USA

Recent data have shown that PTH down-regulation of its receptor on opossum kidney (OK) cells is not associated with any change in the steady state level of the PTH/PTH-related peptide (PTHrP) receptor messenger RNA. For analysis of down-regulation of the PTH/PTHrP receptor in OK cells, the present work uses a specific receptor anti-serum, SR-2, that is useful for detection and quantification of PTH/PTHrP receptor immunoreactivity on intact cells bearing the opossum PTH/PTHrP receptor. SR-2 specifically binds to COS-7 cells transiently expressing the opossum PTH/PTHrP receptor complementary DNA (OK-O), to LLCPK1 cells stably expressing the recombinant opossum PTH/PTHrP receptor (AOK cells), and to OK cells expressing endogenous PTH/PTHrP receptors, but not to mock-transfected COS-7 cells or untransfected LLCPK1 cells. SR-2 binding was also linearly correlated with PTH binding in COS-7 cells transfected with different amounts of OK-O plasmid DNA. Treatment with PTH (100 nM) for 4 and 6 h did not significantly down-regulate the PTH/PTHrP receptor immunoreactivity, although PTH binding was decreased to 51% and 49% of control, respectively, and PTH-stimulated cAMP accumulation was decreased to 27% and 28% of control, respectively. Treatment with PTH (100 nM) for 24 and 48 h significantly decreased PTH binding to 51% and 60% of control and decreased PTH/PTHrP receptor immunoreactivity to 68% and 58% of control, respectively. Incubation of OK cells with 0.1 nM to 1 microM PTH for 4 h did not down-regulate the PTH/PTHrP receptor immunoreactivity, although PTH binding was decreased dramatically. Scatchard blot analysis revealed that the binding affinity was decreased by 7-fold in OK cells treated with PTH for 4 h without change in receptor number. Conversely, treatment of OK cells with PTH for 24 h resulted in a parallel decrease in both receptor number and receptor immunoreactivity without any change in receptor binding affinity. Treatment of OK cells with dexamethasone (0.1 nM to 1 microM) had no effect on PTH binding or PTH/PTHrP receptor immunoreactivity. Incubation of OK cells with both dexamethasone (1 microM) and PTH (0.1 nM to 1 microM), however, caused a significantly greater down-regulation of both PTH binding and PTH/PTHrP receptor immunoreactivity than in cells treated with PTH alone. These data indicate that during the first 4 h of exposure of OK cells to PTH, PTH/PTHrP receptors remain on the cell surface but have lowered affinity to bind the ligand and that dexamethasone potentiates the effect of PTH on PTH/PTHrP receptor down-regulation in OK cells.

The human PTH/PTH-related peptide (PTH/PTHrP) receptor could be involved in hereditary disorders of PTH or PTHrP action. Knowledge of the gene's chromosomal location would allow studies linking it to specific disease traits. Therefore, we mapped the human PTH/PTHrP receptor gene by polymerase chain reaction of human/rodent somatic cell hybrid panels using oligonucleotide primers designed to amplify a portion of the gene from genomic DNA. The PTH/PTHrP gene was unambiguously assigned to the short arm of human chromosome 3, in the region designated 3p21.1-p24.2. Analysis of a second chromosome 3-specific mapping panel suggests that the gene is located near the 3p21.2-p21.3 boundary. The availability of highly polymorphic markers located in this region will permit exploration of the PTH/PTHrP receptor locus in genetic linkage searches for the causes of bone, calcium, and other potential disorders.

PTH-related protein (PTHrP) is produced in vascular smooth muscle, where it is believed to act as a local vasorelaxant by activating either the classical PTH or a unique PTHrP receptor. We used a newly cloned complementary DNA encoding the rat PTH/PTHrP receptor to study the expression of its messenger RNA (mRNA) in primary aortic vascular smooth muscle cells (VSMC) and in UMR-106 osteoblast-like cells under basal conditions and in response to treatment with agonists. Both cell types expressed a 2.4-kilobase PTH/PTHrP receptor mRNA transcript and exhibited hormone-induced desensitization of PTHrP-(1-34)NH2-stimulated cAMP. In VSMC, angiotensin-II, which induces PTHrP expression, also rapidly (30 min) desensitized the cAMP response and down-regulated (75-90%) receptor mRNA within 1 h. Treatment of cells with phorbol 12-myristate 13-acetate (0.1 microM) mimicked these effects, whereas neither PTHrP-(1-34)NH2, forskolin, nor (Bu)2cAMP altered receptor mRNA expression. By contrast, in UMR-106 cells, PTHrP-(1-34)NH2 induced time- and dose-dependent decreases in receptor mRNA that were preceded by pronounced desensitization (cAMP and ligand binding) of cell surface receptors. These effects were mimicked by (Bu)2cAMP and forskolin, but not by phorbol 12-myristate 13-acetate, suggesting that both receptor mRNA down-regulation and receptor desensitization in UMR cells were mediated through a protein kinase-A pathway. We suggest that VSMC and UMR cells express a common receptor, which is subject to cell-specific regulation. Such diversity in the PTH/PTHrP receptor regulatory mechanisms provides a means for restricting the length and duration of the cellular response to hormone in a cell/tissue-specific manner.

The organization of the PTH/PTHrP receptor gene is highly homologous in three mammalian species, rat, human and mouse. This gene extends over 22 kb and contains at least 15 exons and 14 introns. The most 5' exon we have identified (exon U) is followed by an approximately 1kb intron. The second exon (exon S) encodes the initiator methionine and the putative signal peptide and is followed by the largest intron of this gene (about 11 kb). The amino-terminal extracellular region is encoded by 4 exons (E1, E2, E3 and G); exon G contains all 4 potential glycosylation sites. Membrane-spanning domains 1-4 and portions of their connecting intracellular and extracellular loops are encoded by 4 exons (M1, M2, M3 and M4). The second extracellular loop and portions of 4th and 5th membrane-spanning domains are encoded by one exon, EL2. The 5th membrane-spanning domain and portion of the 3rd intracellular loop are encoded by one exon, M5. The 6th membrane-spanning domain, the 3rd extracellular loop and the proximal part of the 7th membrane-spanning domain are encoded by one single exon (M6/7); the remaining sequence of the 7th membrane-spanning domain is encoded by a short exon, M7. The carboxy-terminal tail of the receptor and the 3' untranslated region are encoded by one single exon, exon T. The 3' untranslated region does not contain the classical polyadenylation signal, AATAAA. Expression in COS-7 cells of a minigene constructed of a 5' rat cDNA fragment (1.3 Kb) ligated in-frame to a 3' genomic fragment at the NsiI site, which is located in exon M6/7 resulted in a transcript that was translated into a functional receptor; it bound PTH and showed PTH-stimulated accumulation of intracellular cAMP. Therefore, the PTH/PTHrP receptor gene contains alternative 3' sequences that allow cleavage and polyadenylation of its transcript.

OBJECTIVE: Our purpose was to study the expression and localization of parathyroid hormone– related protein and expression of the parathyroid hormone/parathyroid hormone– related protein receptor in human umbilical cord. STUDY DESIGN: The expression and localization of parathyroid hormone– related protein and expression of the parathyroid hormone/parathyroid hormone– related protein receptor were studied in isolated tissues from the human umbilical cord by Northern analysis, reverse-transcriptase polymerase chain reaction, Southern gel analysis, and immunolocalization procedures at the light and electron microscopic levels. RESULTS: Parathyroid hormone– related protein was abundantly expressed in the umbilical cord. Immunohistochemical and immunocytochemical techniques confirmed hormone localization in the amnion epithelial layer and in vascular smooth muscle and endothelial cells in vessels from the umbilical cord and placental chorionic plate. Multiplex reverse-transcriptase polymerase chain reaction identified expression of receptor messenger ribonucleic acid in vessels of the umbilical cord; this finding was verified by means of Southern gel analysis of the products of the reverse-transcriptase polymerase chain reaction. CONCLUSION: A parathyroid hormone– related protein paracrine system appears to exist in human umbilical cord. We suggest that it may be involved in the control of fetal placental circulation. (A M J O BSTET G YNECOL 1994; 170:1018-26.)

To study mechanisms controlling the expression of PTH/PTH-related peptide (PTHrP) receptors in ROS 17/2.8 and OK cells, we investigated the regulation of PTH/PTHrP receptor availability and receptor mRNA levels by glucocorticoids and PTH. Treatment of ROS 17/2.8 cells with dexamethasone (1 microM) for 2, 4, and 6 days increased specific binding of PTH to 148 +/- 12%, 203 +/- 10%, and 344 +/- 9% (mean +/- SD), respectively, compared to that in untreated control cells. PTH-stimulated cAMP accumulation also increased with dexamethasone treatment (1 microM) from 230 +/- 15%, 382 +/- 9%, and 820 +/- 9% after 2, 4 and 6 days, respectively, compared to that in untreated cells. Treatment of ROS 17/2.8 cells with [Nle8,Nle18,Tyr34]bovine PTH-(1-34) amide (NlePTH; 100 nM) alone or together with dexamethasone (1 microM), however, markedly decreased PTH binding and PTH-stimulated cAMP accumulation. Northern blot analysis showed that dexamethasone dramatically increased steady state levels of PTH/PTHrP receptor mRNA in a time- and dose-dependent manner, which did not occur when NlePTH (100 nM) was added concomitantly to the cultures. As previously reported, daily NlePTH treatment of ROS 17/2.8 cells reduced PTH/PTHrP receptor availability and PTH-stimulated cAMP accumulation markedly within 2 days, which remained at these low levels during continued PTH treatment. In contrast, the identical treatment reduced steady state levels of PTH/PTHrP receptor mRNA in ROS 17/2.8 transiently and to only a slight extent, which then returned to pretreatment levels. Treatment of OK cells with NlePTH (100 nM) for 1, 2, and 4 days decreased PTH binding to 56 +/- 6%, 44 +/- 4%, and 64 +/- 4% (mean +/- SD) and PTH-stimulated cAMP accumulation to 42 +/- 6%, 19 +/- 4%, and 21 +/- 3% (mean +/- SD), respectively, compared to values in untreated control cells. The same treatment, however, had no significant effect on steady state levels of PTH/PTHrP receptor transcripts. In contrast to its effects in ROS 17/2.8 cells, dexamethasone (1 microM) treatment of OK cells for 1-4 days did not affect PTH binding, nor did it significantly affect steady state levels of PTH/PTHrP receptor mRNA, although the latter was slightly lowered by dexamethasone treatment. PTH-stimulated cAMP accumulation was unchanged after 1-day treatment with dexamethasone and modestly rose to 142 +/- 4% of the control value by day 4 of glucocorticoid exposure.(ABSTRACT TRUNCATED AT 400 WORDS)

PTH/PTH-related peptide (PTHrP) receptor mRNAs are widely distributed in rat tissues. PTH and PTHrP, a peptide responsible for hypercalcemia associated with cancers, bind equivalently to common receptors that initially were cloned from rat bone and opossum renal cell cDNA libraries. In this study we used rat PTH/PTHrP receptor cDNA to probe for receptor expression in different rat tissues by Northern blot analysis. PTH/PTHrP receptor transcripts are highly expressed in PTH target tissues, kidney and bone. Receptor transcripts, however, also are expressed in many other tissues, including aorta, adrenal gland, bladder, brain, cerebellum, breast, heart, ileum, liver, lung, skeletal muscle, ovary, placenta, skin, spleen, stomach, uterus, and testes. The major transcript in most tissues is 2.3-2.5 kilobases in size. At least two larger mRNAs are observed in kidney and liver, and smaller transcripts are found in kidney, skin, and testes. The most abundant testicular transcript is 1.4-1.5 kilobases in size, and it hybridizes with two different cDNA probes that encode portions of the receptor sequence from the putative fourth transmembrane domain to its C-terminal end. It does not hybridize, however, with a probe encoding the first 107 residues of the receptor sequence. Although, PTH/PTHrP receptor mRNAs are highly expressed in kidney and bone, classic PTH targets that are associated with calcium homeostasis, their wide tissue distribution suggests that PTH and/or PTHrP have other physiological roles, particularly in these other tissues. The mechanisms leading to tissue-specific expression of PTH/PTHrP receptor transcripts of different sizes and the functions of these mRNAs remain to be determined.

We characterized cells that express parathyroid hormone/parathyroid hormone related peptide (PTH/PTHrP) receptor mRNA in bones of fetal and postnatal rats by in situ hybridization. During endochondral development of fetal bones, PTH/PTHrP receptor transcripts were highly expressed both in maturing chondrocytes and in osteoblasts in the periosteum and ossification center, but not in fully hypertrophic chondrocytes. Similar to the localization in the fetal bones, PTH/PTHrP receptor mRNA expression was highly localized to maturing chondrocytes in the articular cartilage and growth plate, and to osteoblasts in the femur of young rats. In both young and fetal rats, transcripts for Type X collagen were localized to hypertrophic chondrocytes, mostly between chondrocytes and bone cells both of which express PTH/PTHrP receptor mRNA. Transcripts for PTH/PTHrP receptors and alkaline phosphatase co-localized in the bone of young rats, but they did not co-localize in fetal bones at the early stages of endochondral ossification. These results show that PTH/PTHrP receptor mRNA is expressed in a cell-type and stage-specific manner during skeletal development.

Publisher Summary This chapter discusses the molecular analysis of the parathyroid hormone (PTH) gene and parathyroid hormone-related peptide receptor (PTHrP). The human PTH gene is a single-copy gene that extends over 4200 basepairs of DNA on the short arm of chromosome 11. The gene contains three exons, the first of which is noncoding. The second exon contains the initiator ATG and encodes all of the signal or pre-sequence and most of the short pro-peptide. The third exon encodes the remainder of the pro-sequence, the residues corresponding to the mature hormone, as well as the 3′ noncoding region. PTH/PTHrP receptors from marsupial kidney and from kidney and bone of two mammalian species, human and rat, are highly homologous. PTH and PTHrP bind with equal affinity to similar, perhaps identical, receptors in kidney and bone. PTH/PTHrP receptors stimulate adenylate cyclase with the same efficacy when activated by either ligand or are capable of activating at least two effector molecules, adenylate cyclase and phospholipase C. PTH/PTHrP receptors have significant homology only with the calcitonin and the secretin receptor and little or no similarities with other G protein-linked receptors. These three receptor types represent the first members of a novel family of G protein-linked receptors.

The parathyroid hormone (PTH) story was once fairly simple. PTH raises blood calcium by resorbing bone, conserving urinary calcium, and activating vitamin D in order to increase intestinal calcium absorption. Blood calcium, in turn, decreases PTH secretion. The PTH-calcium homeostatic loop acts to maintain a constant level of blood calcium in the face of varying dietary and skeletal demands. This physiologically appealing story remains a powerful organizing principle for understanding parathyroid function, but has been modified by important observations in the last few years.

ROS17/2.8 cells, a cell line derived from a rat osteosarcoma, have abundant receptors for parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP). A particulate membrane fraction was prepared from these cells and it was solubilized using relatively mild conditions with digitonin (0.25%), a nonionic detergent. When radioligands of both PTH and PTHrP were incubated with this membrane fraction in the absence of any protease inhibitor at 15 degrees C, approximately 75% of these radioligands were degraded within 2 hours. This degradative activity was inhibited more effectively by bacitracin than by any of several other protease inhibitors tested. The digitonin-solubilized PTH/PTHrP receptors were radiolabeled in the presence of bacitracin using radioiodinated [Tyr36]PTHrP(1-36) amide (PTHrP(1-36)) and N-hydroxysuccinimidyl-4-azidobenzoate (HSAB), as cross-linker. When an aliquot of the reaction solution was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography, a broad band was observed that had an apparent molecular size of 90,000 daltons (M(r) = 90 kD). This band was no longer seen when the binding was conducted in the presence of 10(-6) M of unlabeled PTHrP(1-36), and it was decreased in density when binding was conducted in the presence of 10(-6) M of unlabeled [Nle8,18, Tyr34] bovine PTH(1-34) amide (NlePTH). The solubilized receptors retained their capacity to bind the radioligand after partial purification by wheat-germ agglutinin affinity-chromatography. The use of relatively mild detergent conditions thus offers a means to solubilize receptors that retain their capacity to bind PTH and PTHrP.

Parathyroid hormone (PTH), a major regulator of mineral ion metabolism, and PTH-related peptide (PTHrP), which causes hypercalcemia in some cancer patients, stimulate multiple signals (cAMP, inositol phosphates, and calcium) probably by activating common receptors in bone and kidney. Using expression cloning, we have isolated a cDNA clone encoding rat bone PTH/PTHrP receptor from rat osteosarcoma (ROS 17/2.8) cells. The rat bone PTH/PTHrP receptor is 78% identical to the opossum kidney receptor; this identity indicates striking conservation of this receptor across distant mammalian species. Additionally, the rat bone PTH/PTHrP receptor has significant homology to the secretin and calcitonin receptors but not to any other G protein-linked receptor. When expressed in COS cells, a single cDNA clone, expressing either rat bone or opossum kidney PTH/PTHrP receptor, mediates PTH and PTHrP stimulation of both adenylate cyclase and phospholipase C. These properties could explain the diversity of PTH action without the need to postulate other receptor subtypes.

The complementary DNA encoding a 585-amino acid parathyroid hormone-parathyroid hormone-related peptide (PTH-PTHrP) receptor with seven potential membrane-spanning domains was cloned by COS-7 expression using an opossum kidney cell complementary DNA (cDNA) library. The expressed receptor binds PTH and PTHrP with equal affinity, and both ligands equivalently stimulate adenylate cyclase. Striking homology with the calcitonin receptor and lack of homology with other G protein-linked receptors indicate that receptors for these calcium-regulating hormones are related and represent a new family.

A large variety of cellular functions and physiological events are modulated or controlled by hormones and neurotransmitters. The cellular receptors for these molecules can be divided into two general categories: (a) cell surface receptors for peptides, growth factors, and neurotransmitters that are linked to secondary events (some of which are still unknown) (Kahn 1989), and (b) receptors for steroids, dihydrocholecalciferol, and thyroid hormones ultimately allowing interaction with specific DNA sequences and regulation of gene expression (Baulieu and Mester 1989). Modifications in hormone receptors and/or their secondary events have been implicated in a steadily growing number of pathophysiological disorders, thus making detailed knowledge of their primary structure and their functional properties increasingly important. Recent advances in receptor research have led to the isolation, cloning, and expression of multiple receptor proteins, and the subsequent dissection of functionally important domains through site-directed mutagenesis and site-specific monoclonal antibodies (Kris et al. 1985; Strader et al. 1988; Okamura et al. 1989; Cunningham et al. 1989).

FUNCTIONAL ANALYSIS OF HUMAN MUTATIONS IN NAPIIIC REVEALS IMPORTANT RESIDUES FOR SURFACE EXPRESSION AND SODIUM-PHOSPHATE CO-TRANSPORT Clemens Bergwitz1 Graciana Jaureguiberry1, Thomas O Carpenter3, Stuart Forman2, Harald Juppner1 1Endocrine Unit and 2Dept. of Anesthesiology, Massachusetts General Hospital, Boston, MA, United States; 3 Dept. of Pediatrics and Endocrine Unit, Yale School of Medicine, New Haven, CT, United States. We recently reported homozygous and compound heterozygous lossof-function mutations in SLC34A3/NaPi-IIc as the cause for hereditary hypophosphatemic rickets with hypercalciuria (HHRH). To better understand how some of the point mutations reported by us and others (T137M, S138F, S192L, G196R, R468W, del527L, R353L, D237N and A413E) lead to renal phosphate wasting, we generated expression plasmids encoding enhanced green-fluorescence protein (EGFP) concatenated to the N-terminus of wild-type or mutant human NaPi-IIc. After transient transfection in Opossum kidney cells G196R and R468W were not expressed in apical patches (the renal brush border membrane equivalent), nor were these mutants present in plasma membranes of Xenopus oocytes following cRNA injection (50 ng/oocyte). For S138F, S192L, R353L, D237N and A413E oocyte membrane fluorescence was reduced to

Eight children with terminal renal insufficiency on continuous ambulatory peritoneal dialysis were followed for 12 months to evaluate laboratory parameters of mineral ion and bone metabolism. Calcium carbonate (range 47-295 mg/kg body weight per day) was given in combination with low doses of either vitamin D or 1,25(OH2D3. Blood urea nitrogen and serum phosphate concentrations remained well controlled throughout the observation period. A significant increase in serum calcium levels from 2.35 +/- 0.18 to 2.61 +/- 0.22 mmol/l (mean +/- SD) was observed during the first 6 months. Alkaline phosphatase activity and mid-C-regional parathyroid hormone, both indirect parameters of bone metabolism, revealed no evidence of severe secondary hyperparathyroidism. Our data indicate that calcium carbonate may be sufficient to induce relative hypercalcaemia in uraemic children, and thus reduce the risk of developing renal osteodystrophy. Unwanted side-effects of vitamin D preparations, i.e. increased intestinal phosphate absorption and hypercalcaemia after successful renal transplantation, may thus be avoided.

The synthesis, purification, and structural analysis of the major compounds resulting from photoderivatization of [Tyr36]-parathyroid hormone related peptide (1-36)amide [[Tyr36]PTHrP(1-36)amide] are described. The reaction of the synthetic peptide with 4-fluoro-3-nitrophenyl azide under nonaqueous conditions yields three major products (peaks D-1, D-2, and G), which were purified to homogeneity by reverse-phase high-performance liquid chromatography. Subsequent amino acid analysis showed that the peptides of peaks D-1 and G each lack one lysine residue, while the peptide in peak D-2 lacks one alanine residue, suggesting that these residues are chemically modified by photoderivatization. Sequence analysis of the photoderivatized peptides revealed that compounds D-1 and G were derivatized on Lys13 and Lys11, respectively. Compound D-2 was N-blocked, indicating that this compound is derivatized on the alpha-amino function of Ala1. Both Lys residues of D-2 were quantitatively recovered upon sequencing after digestion with endoproteinase Glu-C. Compounds D-2 and G had apparent KdS of 1 X 10(-9) M and 0.6 X 10(-9) M, respectively, for their receptors on ROS 17/2.8 cells, which are identical with or similar to that of the underivatized [Tyr36]PTHrP(1-36)amide. Compound G had the same adenylate cyclase stimulating potency as the underivatized, synthetic [Tyr36]PTHrP(1-36)amide, whereas compound D-2 was only a partial agonist, having about 25% of the maximal cAMP production. Compound D-1, which is modified on Lys13, retained only 2-4% of its receptor binding affinity and biological activity relative to that of its parent compound.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of parathyroid hormone onto B-lymphocytes is detected by the utilization of the labelled antibody membrane assay. The amount of parathyroid hormone bound to the receptor sites was depending on the quantity of cells in the incubation milieu. Each cell line showed typical characteristics in time course of parathyroid hormone binding and maximal receptor capacity. Fragmentation of intact parathyroid hormone, also varying with the cell line tested, was very rapid, even at 24°C. Within 20 min most of the cell lines destroyed 50% of the native hormone in the incubation mixture, indicating a fragmentation rate of up to 2.25 ng/min at 37°C. B max and K D for the different lymphocytes was 5.3–19 · 10 11 M and 1.8–18.5 · 10 11 M, respectively. These values are in the range of reported plasma concentrations and may therefore represent more physiological values for the capacity and affinity of membrane receptors.

Thyroxine (T4), triiodothyronine (T3) and thyroxine-binding globulin (TBG) were determined in healthy individuals ranging in age from newborn to 95 years. T4: 10.25 +/- 1.62 microng/100 ml, T3: 1.62 +/- 0.35 ng/ml and TBG: 1.34 +/- 0.15 mg/100 ml, were found elevated until puberty compared to a middle age group with T4: 7.27 +/- 2.26 microng/100 ml, T3: 1.15 +/- 0.24 ng/ml and TBG: 0.98 +/- 14 mg/100 ml. T4 and T3 followed almost TBG concentration. In old age is dissociation between T4: 5.79 +/- 1.56 microng/100 ml, T3: 0.79 +/- 0.21 ng/ml and TBG: 1.28 +/- 0.15 mg/100 ml was found. Except for old age the ratio T4/TBG and T3/TBG minimized the age dependent variation of T4 and T3 and reduced the coefficient of variance from 26% to 17.7% for T4 and from 26.5 to 25% for T3. Age reduction of T4/TBG is 15% and of T3/TBG 13% respectively more pronounced than for T4 and T3 alone. These data indicate: 1) age related variations of T4 and T3 due to age dependency of TBG, 2) deviation of T4 and T3 values in old age from that expected by their TBG levels and 3) the importance of the routine use of hormone/TBG ratio.

Summary: The physicochemical behaviour of parathyrin on surfaces was investigated. Parathyrin is bound to the wall of polyethylene, polypropylene and flint glass tubes. This adsorptive binding of the hormone, detected by labelled antibodies, is a time-dependent process which is complete within one hour. The addition of plasma decreases but does not prevent adsorption. Coating of the tubes with an anti-N-regional or anti-C-regional antiserum decreases the sensitivity of the detection of 1-34, 53-84 and 1-84 intact parathyrin, when performed in buffer. In the presence of plasma the increase of radiolabelled antibody detecting the parathyrin was more pronounced when tubes were precoated with an anti-parathyrin antibody. The sensitivity of the 1-84 parathyrin assay was also reduced when incubation was carried out in the presence of high concentrations of parathyrinfragments, calcitonin, somatostatin or bovine serum albumin. Assay specificity seems to be related to the specificity of the labelled antibody for the respective hormonal fragment, not to that of the antibody used for coating the tubes. This investigation illustrates the necessity for the careful control of parathyrin assay conditions, e. g. non-specific binding of native parathyrin and radiolabelled tracers to laboratory reaction vessels.