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H Jüppner

Publications and source records attributed to H Jüppner.

At least 73 records · Page 4Linked to original sources
PubMed1995

Characterization of a novel parathyroid hormone (PTH) receptor with specificity for the carboxyl-terminal region of PTH-(1-84)

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.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1995

Further evidence for a novel receptor for amino-terminal parathyroid hormone-related protein on keratinocytes and squamous carcinoma cell lines.

PTH and PTH-related peptides (PTHrPs) interact with a common PTH/PTHrP receptor (type I), which is expressed in many tissues, including bone and kidney. Amino-terminal PTH and PTHrPs also recognize receptors in several nonclassical PTH target tissues, and in some of these, the signaling mechanisms differ qualitatively from those of the classical type I receptor. In normal keratinocytes and squamous carcinoma cell lines, PTH and PTHrP stimulate a rise in intracellular calcium, but not cAMP, suggesting the existence of an alternate, type II PTH/PTHrP receptor. SqCC/Y1 squamous carcinoma cells stably expressing the type I receptor displayed sensitive intracellular cAMP responses to PTHrP and PTH, indicating that these cells express functional GS proteins and that the type I receptor is capable of signaling through adenylyl cyclase in this cell line. Therefore, the endogenous type II receptor in SqCC/Y1 cells differs from the cloned type I receptor. We next examined whether messenger RNA (mRNA) from keratinocytes and squamous cell lines could hybridize to a human type I PTH/PTHrP receptor complementary DNA [1.9 kilobases (kb)]. No type I receptor mRNA (2.3 kb) was detected in polyadenylated RNA from any of the squamous cell lines. However, squamous cell lines did express several mRNA transcripts that hybridized with the type I receptor probe, yet were smaller (1 and 1.5 kb) or larger (3.5-5 kb) than the cloned receptor mRNA. The predominant mRNA in two squamous carcinoma cell lines and normal keratinocytes was a 1-kb transcript. Northern analysis with five different region-specific probes that span the entire coding region of the human type I receptor was used to map homologous regions within each of the transcripts. Several of the transcripts identified in squamous lines are also present in polyadenylated RNA from SaOS-2 human bone cells, but a unique 1-kb transcript hybridizing to probe 2 (nucleotides 490-870) was observed only in squamous cells. The smaller 1- and 1.5-kb transcripts did not hybridize to probes corresponding to the extreme 5'- and 3'-coding regions of the type I receptor complementary DNA. Ribonuclease protection analysis employing riboprobes that correspond to the five region-specific DNA probes revealed strong RNA signals of the expected size in SaOS-2 cells, but no hybridization with squamous cell RNA. Several smaller, but minor, bands that were unique to squamous cells were observed with riboprobe 2 only, suggesting partial homology of this region with the type I receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗
PubMed1995

Down-regulation of the receptor for parathyroid hormone (PTH) and PTH-related peptide by transforming growth factor-beta in primary fetal rat osteoblasts.

We studied the effects of transforming growth factor-beta 2 (TGF beta 2) on the level of PTH/PTH-related peptide-(PTHrP) receptor messenger RNA (mRNA), PTHrP binding, and PTH-stimulated cAMP accumulation in cultured osteoblasts derived from fetal rat calvariae (ROB). When ROB were pretreated with TGF beta 2 at concentrations ranging from 1-100 pM for 24 h, dose-dependent decreases in the level of PTH/PTHrP receptor mRNA, PTHrP binding, and PTH-stimulated cAMP accumulation were observed. For the PTH/PTHrP receptor mRNA level and PTH-stimulated cAMP accumulation, the half-maximal effective concentration was approximately 4 pM. For the inhibition of PTHrP binding, the half-maximal effective concentration was much higher. A 50% decrease in both PTH/PTHrP receptor mRNA level and PTH-stimulated cAMP accumulation was obtained when ROB were treated with 100 pM TGF beta 2 for 4 h. A comparable decrease in PTHrP binding was only observed after 24 h of incubation with 100 pM TGF beta 2. Actinomycin D induced a rapid decrease in the PTH/PTHrP receptor mRNA level (70% after 4 h), indicating a half-life for the receptor mRNA of 2-3 h. Under the same conditions, PTHrP binding and PTH-stimulated cAMP accumulation did not change. When ROB were treated with cycloheximide for the same period, only a small decrease in PTHrP binding (20%) was observed, suggesting that PTH/PTHrP receptors do not have a rapid turnover. Cycloheximide also reduced PTH-stimulated cAMP production; after coincubation of cycloheximide with TGF beta 2, this inhibition was smaller than that in ROB cultures treated with TGF beta 2 exclusively. From these observations we conclude that TGF beta 2 induces a decrease in steady state levels of PTH/PTHrP receptor mRNA that results in decreased PTHrP receptor binding. The PTH-stimulated cAMP accumulation is at least to some extent independent of the PTH/PTHrP receptor availability. Furthermore, there is a high turnover of PTH/PTHrP receptor mRNA, whereas turnover of the receptor protein is much slower. Finally, protein synthesis is required for TGF beta 2-induced desensitization of cAMP responsiveness to PTH.

Animals↗
PubMed1995

Homolog-scanning mutagenesis of the parathyroid hormone (PTH) receptor reveals PTH-(1-34) binding determinants in the third extracellular loop.

To identify determinants in the rat PTH receptor critical for binding the agonist peptide, PTH-(1-34), we systematically replaced 12 segments (5-33 residues) of the receptor's extracellular surface with the corresponding segments of the homologous rat secretin receptor and screened the resulting mutants in COS-7 cells for altered PTH-(1-34) binding properties. Surface expression of mutant receptors was assessed by the binding of monoclonal antibody 12CA5 to the epitope (HA)-tagged receptors. Of the nine well expressed and therefore informative receptor mutants, four bound radiolabeled PTH-(1-34) at levels that were proportional to the corresponding levels of surface expression, whereas five mutants bound [125I]PTH-(1-34) to levels that were lower than predicted from the cell surface expression levels. These five mutations occurred at the extracellular (EC) end of transmembrane domain 1, the carboxy-terminal portion of the first EC loop, the second EC loop, and the third EC loop. We selected for further fine structure analysis the third EC loop; two specific residues, Trp-437 and Gln-440, were identified at which mutations caused 9- to 16-fold reductions in PTH-(1-34)-binding affinity. The same mutations had little or no effect on the binding affinity of PTH-(3-34). This study provides new information on the location of PTH receptor regions important for high affinity agonist binding and identifies two residues in the third extracellular loop which may contribute to interactions involving the hormone's critical amino terminus.

Amino Acid Sequence↗
PubMed1994

Rapid desensitization of parathyroid hormone dependent adenylate cyclase in perifused human osteosarcoma cells (SaOS-2).

The pulsatile but not the continuous application of parathyroid hormone (PTH) increase bone mass in vivo. To study the effects of intermittent hormonal administration on bone-derived cells in vitro, we established a perifusion system using the human osteosarcoma cell line SaOS-2. Cells were grown in suspension culture attached to collagen beads and were then loaded into a 3 ml syringe for perifusion experiments. The application of PTH(1-34) resulted in a dose-dependent increase of cAMP release by SaOS-2 cells into the effluent medium. Cyclic AMP accumulation was rapidly desensitized by approx. 80% after 30 min of continuous exposure to PTH(1-34) (10(-7) M), while cells remained responsive to forskolin. The recovery of PTH responsiveness required at least 2 h of hormone-free perifusion. Desensitization in the experimental setting was dose-dependent (EC50 = 1 x 10(-10) M PTH(1-34)). Neither 8Br-cAMP (2 x 10(-4) M) nor PMA(1 x 10(-7) M) had an effect on the PTH(1-34)-induced desensitization of the adenylate cyclase. Radioreceptor assays showed that [125I]-[Tyr36]hPTHrP(1-36)amide binding to SaOS-2 cells was decreased by 60-70% by PTH(1-34) (1 x 10(-6) M), bPTH(1-84) (1.8 x 10(-6) M) and bPTH(3-34) (2 x 10(-6) M), whereas 8Br-cAMP (2 x 10(-4) M) had no effect on radioligand binding. PMA (1 x 10(-7) M) appeared to slightly increase [125I]PTHrP binding. This observation is consistent with a small (3-fold) increase in PTH-induced cAMP release as a result of PMA pre-treatment. Receptor internalization was dose-dependent EC50 = 3 x 10(-7) M PTH(1-34)). The maximal effect occurred after 10-30 min and was largely reversible within 2 h. Monensin (3 x 10(-5) M) inhibited the recovery from receptor internalization. We conclude that a perifusion system using SaOS-2 cells is a suitable model to study the effect of discontinuous application of PTH on cAMP release. A rapid, homologous desensitization of PTH(1-34) stimulated cAMP accumulation has been observed that does not appear to involve protein kinase A or C.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1994

The rat, mouse and human genes encoding the receptor for parathyroid hormone and parathyroid hormone-related peptide are highly homologous.

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.

Animals↗
PubMed1994

Differences in binding affinities of human PTH(1-84) do not alter biological potency: a comparison between chemically synthesized hormone, natural and mutant forms.

The purpose of this study was to evaluate receptor binding affinities and biological properties in vitro and in vivo of various recombinant hPTH(1-84) forms representing the natural hormone and a mutagenized hPTH form, [Gln26]hPTH(1-84) (QPTH), after expression in E. coli and Saccharomyces cerevisiae. In LLC-PK1 cells stably transformed with the rat PTH/PTHrP receptor, chemically synthesized hPTH(1-84) and QPTH showed a reduced binding affinity (apparent Kd 18 and 23 nM, respectively) than the recombinant, hPTH(1-84) (apparent Kd 9.5 nM). All recombinant hPTH forms showed a similar potency to stimulate cellular cAMP production (EC50 1.5 nM) and significantly better than chemically synthesized hPTH (EC50 5.7 nM). All hormone forms showed an about equipotent activity in causing elevation in serum calcium, increased excretion of urine phosphate, and cAMP. Thus, the natural recombinant PTH forms showed higher binding affinities and adenylate cyclase activation potencies in LLC-PK1 cells, but the reduced receptor binding affinity exerted by QPTH did not transcend differences in cAMP generation and in vivo biological activities.

Animals↗
PubMed1994

Transforming growth factor-beta1 regulates steady-state PTH/PTHrP receptor mRNA levels and PTHrP binding in ROS 17/2.8 osteosarcoma cells.

The effect of transforming growth factor beta1 (TGF-beta1) on the expression of mRNA for the parathyroid hormone receptor and binding of iodinated parathyroid hormone-related protein in ROS 17/2.8 osteosarcoma cells was evaluated. TGF-beta1 stimulated a 2-7-fold increase in steady state mRNA levels for the parathyroid hormone receptor at a maximal dose of 5 ng/ml, with increased levels of expression at 6 h of TGF-beta1-incubation, and peak levels at 8-24 h. Receptor binding studies revealed a significant increase in PTHrP-specific binding with TGF-beta1 doses as low as 0.5 ng/ml and a 55% increase in numbers of receptors with no alteration in binding affinity with 5.0 ng/ml TGF-beta1. Time course studies indicated that receptor binding was increased at 24 h with peak levels reached at 48 h of treatment. PTH-stimulated cAMP levels were significantly increased in ROS 17/2.8 cells treated with TGF-beta1 (0.5 ng/ml) for 48 h. These data indicate that TGF-beta1 upregulates steady-state mRNA, ligand binding and PTH/PTHrP receptor signaling in rat osteosarcoma cells. The effects of TGF-beta1 on bone may be attributed in part to regulation of the PTH/PTHrP receptor at the molecular level.

Animals↗
PubMed1994

Expression of parathyroid hormone-related protein and its receptor in human umbilical cord: evidence for a paracrine system involving umbilical vessels.

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.

Endothelium, Vascular↗
PubMed1994

Regulation of parathyroid hormone (PTH)/PTH-related peptide receptor messenger ribonucleic acid by glucocorticoids and PTH in ROS 17/2.8 and OK cells.

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)

Animals↗
PubMed1994

The extracellular amino-terminal region of the parathyroid hormone (PTH)/PTH-related peptide receptor determines the binding affinity for carboxyl-terminal fragments of PTH-(1-34).

The recombinant human PTH/PTH-related peptide (PTHrP) receptor, when transiently expressed in COS-7 cells, binds [Nle8,18,Tyr34] bovine PTH-(7-34)amide [PTH-(7-34)], human PTH-(10-34)amide [PTH-(10-34)], and bovine PTH-(15-34)amide [PTH-(15-34)] with at least 50-fold higher affinity than does the rat receptor homolog. In contrast, PTH-(1-34) binding affinities are similar for both receptor homologs. To map those areas of the PTH/PTHrP receptors that determine the binding specificity for carboxyl-terminal fragments of PTH-(1-34), we constructed chimeric rat/human PTH/PTHrP receptors. These bound PTH-(1-34) with normal affinity and, therefore, must have an overall conformation that resembles that of native receptors. Chimeras with the amino-terminal extracellular domain of the human PTH/PTHrP receptor have a considerably higher binding affinity for PTH-(7-34), PTH-(10-34), and PTH-(15-34) than do the reciprocal receptor constructs in which the amino-terminal region is from the rat PTH/PTHrP receptor. The opossum PTH/PTHrP receptor homolog also binds PTH-(7-34) with higher affinity than the rat receptor, and studies of rat/opossum chimeras confirm the importance of the amino-terminal extracellular domain in determining the PTH-(7-34) binding specificity. Mutant rat and human PTH/PTHrP receptors in which either residues 61-105 of the extracellular region or most of the intracellular tail were deleted have PTH-(7-34) binding characteristics indistinguishable from those of either wild-type receptor. These findings indicate that the amino-terminal extracellular region of the PTH/PTHrP receptor contains a domain(s) that largely determines the binding affinity of amino-terminally truncated PTH analogs. This region, therefore, is likely to constitute a site for ligand-receptor interaction.

Animals↗
PubMed1994

Role of protein kinase-A in homologous down-regulation of parathyroid hormone (PTH)/PTH-related peptide receptor messenger ribonucleic acid in human osteoblast-like SaOS-2 cells.

Homologous down-regulation of PTH/PTH-related peptide (PTHrP) receptor expression occurs in several PTH-responsive osteoblastic cell lines, but the mechanisms responsible are not well understood. We have used wild-type SaOS-2 human osteoblastic cells, in which homologous PTH/PTHrP receptor down-regulation occurs within 4 h, and a mutant cAMP-resistant subclone (Ca4A strain), to investigate the mechanisms by which PTH/PTHrP receptor mRNA is regulated. SaOS-2 cells expressed a single 2.2- to 2.5-kilobase transcript of PTH/PTHrP receptor mRNA, as assessed by Northern blot analysis of total RNA with a cDNA probe encoding the human PTH/PTHrP receptor. Homologous down-regulation of this PTH/PTHrP receptor mRNA first became significant when SaOS-2 cells had been treated with human (h) PTH-(1-34) (10(-7) M) for 8-12 h. By 24 h, steady state levels of PTH/PTHrP receptor mRNA were reduced by about 50%. This effect was mimicked by both (Bu)2cAMP (DBcAMP; 0.5 mM) and forskolin (Fsk; 10(-5) M). In contrast, down-regulation of PTH/PTHrP receptor mRNA by hPTH-(1-34), DBcAMP or Fsk was almost completely blocked in cAMP-resistant Ca4A cells. Short term (4-6 h) treatment with hPTH-(1-34), DBcAMP, or Fsk did not reduce steady state levels of PTH/PTHrP receptor mRNA in either SaOS-2 or Ca4A cells, although down-regulation was induced by 4-6 h of treatment with active phorbol esters such as 12-O-tetradecanoyl phorbol-13-acetate (200 nM) or phorbol-12,13-didecanoate (200 nM). Neither thapsigargin (1 microM) nor ionomycin (200 nM), both of which stimulate calcium transients in these cells, altered PTH/PTHrP receptor mRNA expression. Treatment with hPTH-(39-84) and hPTH-(53-84), which do not activate either cAMP-dependent protein kinase or protein kinase-C, but do stimulate 45Ca2+ uptake in these cells, did not alter PTH/PTHrP receptor mRNA expression. In the presence of actinomycin-D (1 microgram/ml), down-regulation of PTH/PTHrP receptor mRNA by hPTH-(1-34) was not observed. Cycloheximide (10 micrograms/ml) did not block down-regulation of PTH/PTHrP receptor mRNA induced by hPTH-(1-34). We conclude that homologous down-regulation of PTH/PTHrP receptor mRNA in SaOS-2 cells occurs later than the decline in functional surface receptors via a mechanism that does not involve enhanced mRNA degradation or new protein synthesis, but is dependent upon cAMP/cAMP-dependent protein kinase.

Blotting, Northern↗
PubMed1994

Determinants of [Arg2]PTH-(1-34) binding and signaling in the transmembrane region of the parathyroid hormone receptor.

Previously, we reported that [Arg2]PTH-(1-34) bound to the rat osteosarcoma cell line, ROS 17/2.8, with 2-fold higher apparent affinity than it did to the opossum kidney cell line, OK, yet the analog was only a weak partial agonist for cAMP stimulation with ROS 17/2.8 cells, whereas it was a full cAMP agonist with OK cells. These results suggested that the rat and opossum PTH receptors differ in a region recognized by the hormone's amino-terminus. In this report we show that the cloned PTH receptors derived from ROS 17/2.8 and OK cells, expressed in COS-7 cells, also displayed altered responses to [Arg2]PTH-(1-34). Thus, [Arg2]PTH-(1-34) bound to the cloned rat PTH receptor with 7-fold higher affinity than it did to the cloned opossum PTH receptor, and in cAMP stimulation assays, it was a much weaker agonist with the rat receptor than it was with the opossum receptor. Studies with rat/opossum PTH receptor chimeras suggested that the membrane-spanning region of the receptor contributed to the different binding and signaling responses to [Arg2]PTH-(1-34). Point mutation analysis identified three sites in or near the extracellular ends of transmembrane domains V and VI, which specifically affected [Arg2]PTH-(1-34) binding and signaling.

Amino Acid Sequence↗
PubMed1994

Role of the extracellular regions of the parathyroid hormone (PTH)/PTH-related peptide receptor in hormone binding.

The PTH/PTH-related peptide receptor is a member of a newly discovered family of G-protein-coupled receptors. Strikingly conserved features among these receptors include the positioning of eight extracellular cysteines and several other residues that are located predominantly within the membrane-embedded region. Deletion mutants or receptors with point mutations of the highly conserved cysteine residues were transiently expressed in COS-7 cells to evaluate PTH binding and PTH-stimulated cAMP production. Deletion of residues 61-105, which are encoded by exon E2 in the PTH/PTH-related peptide receptor gene, did not affect receptor function. An epitope derived from Haemophilus influenza hemagglutinin was, therefore, introduced into this portion of most receptors to allow the independent assessment of cell surface expression. PTH binding capacity was not reduced by the deletion of residues 258-278 in the first extracellular loop. Receptors with deletion of either residues 31-47 in the amino-terminal extension or residues 431-440 in the third extracellular loop failed to bind PTH, although expression of the receptor on the cell surface was only marginally reduced. Most other receptor mutants, including those in which each of the six cysteines in the amino-terminus was replaced by serines, failed to be processed and/or expressed appropriately, whereas the substitution of cysteine-281 or -351 had a less severe effect. The combined replacement of both cysteines concomitantly increased PTH binding and cell surface expression, suggesting the formation of a disulfide bond between these two residues. Our data indicate that residues near the amino-terminus and within the third extracellular loop are necessary for ligand binding, whereas more than 25% of the receptor's extracellular region appears not to be involved.

Amino Acid Sequence↗
PubMed1994

Down-regulation of parathyroid (PTH)/PTH-related peptide receptor immunoreactivity and PTH binding in opossum kidney cells by PTH and dexamethasone.

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.

Animals↗
PubMed1994

Chromosomal localization of the parathyroid hormone/parathyroid hormone-related protein receptor gene to human chromosome 3p21.1-p24.2.

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.

Animals↗