©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Cloning and Functional Expression of Human Parathyroid Calcium Receptor cDNAs (*)

James E. Garrett (1)(§), Irene V. Capuano (1), Lance G. Hammerland (2), Benjamin C. P. Hung (2), Edward M. Brown (3), Steven C. Hebert (4), Edward F. Nemeth (2), Forrest Fuller (1)

From the (1) Departments of Molecular Biology and (2) Pharmacology, NPS Pharmaceuticals, Salt Lake City, Utah 84108, the (3) Endocrine-Hypertension Division, and the (4) Laboratory of Molecular Physiology and Biophysics, Renal Division, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Parathyroid cells express a cell surface receptor, coupled to the mobilization of intracellular Ca, that is activated by increases in the concentration of extracellular Ca and by a variety of other cations. This ``Ca receptor'' (CaR) serves as the primary physiological regulator of parathyroid hormone secretion. Alterations in the CaR have been proposed to underlie the increases in Ca set-point seen in primary hyperparathyroidism due to parathyroid adenoma. We have isolated human CaR cDNAs from an adenomatous parathyroid gland. The cloned receptor, expressed in Xenopus oocytes, responds to extracellular application of physiologically relevant concentrations of Ca and other CaR agonists. The rank order of potency of CaR agonists displayed by the native receptor (Gd > neomycin B > Ca > Mg) is maintained by the expressed receptor. The nucleotide sequence of the human CaR cDNA predicts a protein of 1078 amino acids with high sequence similarity to a bovine CaR, and displays seven putative membrane-spanning regions common to G protein-coupled receptors. The deduced protein sequence shows potential sites for N-linked glycosylation and phosphorylation by protein kinase C and has a low level of sequence similarity to the metabotropic glutamate receptors. Comparison of the cDNA sequence to that of the normal human CaR gene showed no alteration in the coding region sequence of the CaR in this particular instance of parathyroid adenoma. Human cDNA clones with differing 5`-untranslated regions were isolated, suggesting alternative splicing of the parathyroid CaR mRNA. A rare variant cDNA clone representing a 10 amino acid insertion into the extracellular domain was also isolated. Northern blot analysis of normal and adenomatous parathyroid gland mRNA identified a predominant transcript of 5.4 kilobases, and less abundant transcripts of 10, 4.8 and 4.2 kilobases in RNA from the adenoma. While there is no evidence for alteration of the primary amino acid sequence of the CaR in this adenoma, modulation of CaR biosynthesis through alternative RNA processing may play a role in set-point alterations.


INTRODUCTION

The secretion of parathyroid hormone (PTH)() by the parathyroid gland is regulated by the circulating level of Ca, with elevation of the serum Ca concentration leading to an inhibition of PTH secretion. Functional studies of parathyroid cells have suggested the presence of a cell surface, G protein-coupled ``Ca receptor'' (CaR) capable of detecting physiological changes in the concentration of extracellular Ca and modulating PTH secretion (1, 2) . Increases in the concentration of extracellular Ca lead to increased formation of inositol 1,4,5-trisphosphate (3) and diacylglycerol (4) and a rapid elevation in levels of cytosolic Ca which results from both the mobilization of intracellular Ca and influx of extracellular Ca(1, 2, 5) . In addition, parathyroid cells exposed to high extracellular Ca show an inhibition of adenylate cyclase activity, and this inhibition can be blocked by pretreatment with pertussis toxin (6) . These observations suggest that either a single CaR is capable of effecting both mobilization of intracellular Ca and inhibition of adenylate cyclase, or that isoforms of the CaR, coupled to differing G protein-mediated signaling pathways, exist. Although Ca is the endogenous ligand for the CaR, in vitro a variety of di-, tri-, and polyvalent cations (neomycin, spermine, protamine, La, Gd, Ba, Sr, Mg) have been shown to activate this receptor (7, 8) . Studies on cultured bovine parathyroid cells suggest that the CaR is a cell surface glycoprotein (9) and that protein kinase C activation inhibits CaR signaling (10, 11) .

Alterations in parathyroid cell CaR function have been implicated in at least two different human disease states. In primary hyperparathyroidism (1 HPT), parathyroid cells show an elevated Ca ``set-point'' for PTH suppression (12) , possibly reflecting an alteration in CaR function. Recently, mutations in the CaR gene were identified in kindreds with familial hypocalciuric hypercalcemia (FHH) (13) , an inherited syndrome in which affected individuals display an elevated Ca set-point (14) . A CaR-specific agonist, NPS R-568, has been shown to reduce serum PTH levels in vivo(15) , further demonstrating the role of the CaR in modulating PTH secretion. Altogether, there is compelling evidence that the parathyroid CaR serves a central role in maintaining bodily Ca homeostasis. Cell types in addition to the parathyroid cell are responsive to changes in extracellular Ca, suggesting that the same or a related CaR may modulate the function of a variety of cells. Calcitonin secretion by thyroid C cells (16) and renin secretion by juxtaglomerular cells (17) are well documented examples in which hormone secretion is modulated by extracellular Ca. To facilitate the study of human CaR function in normal and disease states and aid in the identification of novel cell types expressing the CaR, we undertook the isolation of human CaR cDNA clones from an adenomatous parathyroid gland.

A bovine parathyroid cell CaR cDNA was recently isolated by expression cloning in Xenopus oocytes (18) . This clone encoded a novel receptor protein with limited sequence homology to the family of G protein-coupled receptors (GPRs). We have used this bovine clone to demonstrate the presence of CaR-related transcripts in a human adenomatous parathyroid gland removed from an individual diagnosed with primary hyperparathyroidism. Full-length CaR cDNA clones were isolated from a cDNA library prepared from this adenoma, the sequence of the CaR obtained from the adenomatous gland was compared to the sequence of the normal human CaR gene, and the function of the encoded CaR has been examined by expression in Xenopus oocytes. The human parathyroid adenoma CaR cDNA clones encode a CaR, coupled to the mobilization of intracellular Ca, with a pharmacological profile similar to that seen with normal parathyroid cells.


EXPERIMENTAL PROCEDURES

RNA Preparation and Northern Blot Analysis

Human tissue samples were frozen on dry ice and stored at -90° C. Total RNA was prepared by the guanidinium thiocyanate/acid phenol extraction procedure (19) . Poly(A) RNA was obtained by oligo-(dT) cellulose affinity chromatography. RNA was size fractionated on 1.2% agarose/formaldehyde gels and transferred to nylon membranes. The 5.3-kbp cDNA insert of a bovine CaR cDNA clone (pBoPCaR1) was excised from the pSport vector by SalI + NotI digestion, gel-purified, and labeled by random-primed synthesis with [P]dCTP (20) to a specific activity of >10 counts/min/µg. Nylon membranes were hybridized in a solution consisting of 5 SSC (1 SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.2), 400 mM NaPO, 5% SDS, 1 mg/ml bovine serum albumin, 100 µg/ml sonicated salmon testis DNA, and 35% formamide at 42 °C, with BoPCaR1 insert probe at a concentration of 2 10 counts/min/ml. Following hybridization, blots were washed in 2 SSC, 0.5% SDS at 50° (moderate stringency) or 0.1 SSC, 65 °C (high stringency).

cDNA Cloning and DNA Sequence Analysis

Human adenomatous parathyroid mRNA was used in cDNA synthesis (21) using Superscript (Life Technologies, Inc.) reverse transcriptase. cDNA was synthesized with a NotI/dT primer at the 3` end and a EcoRI adaptor at the 5` end. cDNA in the 3-6 kbp size range was gel-isolated and ligated into EcoRI/NotI digested Zap II cloning vector. In vitro packaged phage were plated (440,000 primary recombinants) and screened by standard plaque hybridization techniques (22) , using full-length BoPCaR1 insert probe under the hybridization and moderate stringency wash conditions listed for Northern blot analysis. The same filters were stripped and hybridized with a 780-bp EcoRI fragment probe from the 5` end of the bovine cDNA, to identify human clones with long inserts. Selected hybridizing plaques were taken through secondary and tertiary rounds of purification, and isolated phage clones were used for in vivo rescue of cDNA inserts in the pBluescript SK plasmid vector (23) . Preliminary restriction mapping of cDNA inserts indicated that two classes of inserts were represented, sharing a common central region and differing at the ends. Two cDNA clones, with inserts of 4 and 5 kbp, were selected for sequencing.

DNA sequencing was performed on double-stranded plasmid DNA using the chain termination method (24) with Sequenase enzyme (United States Biochemical Corp., Cleveland, OH). Much of the sequencing was done using ordered deletions across the cDNA inserts (25) , with remaining regions sequenced using specific oligonucleotide primers. All regions were sequenced on both strands. Nucleotide sequences were analyzed with the MacVector (IBI-Kodak, Inc.) package of software, including the most recent release (June, 1994) of the NCBI sequence data bases.

RNA Transcription and Oocyte Expression

The pBluescript vector bearing the CaR insert was linearized by NotI digestion, and capped, sense-strand cRNA was synthesized by T7 RNA polymerase transcription (26) . In vitro transcribed RNA was concentrated by ethanol precipitation, and the size and integrity of the RNA were assessed on denaturing agarose gels. CaR cRNA was injected into Xenopus oocytes (typically 12 ng/oocyte), and oocytes were maintained for 3-5 days prior to assay of CaR function. Two-electrode voltage clamp recording was used to monitor the endogenous Ca-dependent Cl current in response to extracellular application of CaR agonists. Details of oocyte manipulations and electrophysiological procedures were as described previously (27) .


RESULTS

Northern Blot Analysis of Human Parathyroid RNA

An adenomatous parathyroid gland was removed from a 39-year-old male diagnosed with primary hyperparathyroidism. Northern blot analysis of poly(A) RNA prepared from the adenoma, utilizing the bovine CaR cDNA as hybridization probe, identified a prominent transcript of 5.4 kb and more faintly hybridizing bands at 10, 4.8, and 4.2 kb (Fig. 1). All of these hybridizing bands persisted after high stringency washes. Normal parathyroid gland mRNA was prepared from tissue obtained at autopsy from a 65-year-old female. Although the RNA prepared from this post-mortem tissue was somewhat degraded, Northern blot analysis identified a faint CaR transcript at 5.4 kb (Fig. 1), suggesting that this is the primary size of the human CaR transcript in both normal and adenomatous parathyroid glands. Xenopus oocytes expressing the human adenoma mRNA responded to extracellular application of CaR agonists with oscillatory inward Cl currents, indicating that the adenoma RNA encoded a functional CaR (data not shown).


Figure 1: Northern blot analysis of human CaR mRNA in the parathyroid gland. Poly(A) RNA from an adenomatous gland (2 µg, lane A) or a normal gland (3 µg, lane B), hybridized with the bovine CaR cDNA. The blot was hybridized and washed under moderate stringency conditions. The sizes of the hybridizing transcripts are indicated.



Cloning and Sequence of the Human CaR

The human adenoma mRNA was used to construct a cDNA library enriched for inserts in the 3-6 kbp size range. This library (440,000 clones) was screened at moderate stringency using the full-length bovine CaR cDNA as hybridization probe. Approximately 600 plaques (0.14%) in this size-selected library were hybridization-positive. Eighteen clones that hybridized with a probe derived from the 5` end of the bovine CaR clone were plaque-purified and characterized by restriction mapping. Clones could be separated into two classes, with inserts in one class of 4 kbp and in the other 5 kbp. Restriction mapping showed both classes to share a similar central region, with differences at the 5` and 3` ends of the inserts. Clones representing the 4 kbp insert size (phPCaR-4.0) and the 5 kbp insert size (phPCaR-5.2) were completely sequenced. The sequence of the phPCaR-4.0 clone has a large open reading frame encoding a protein of 1078 amino acids (Fig. 2), with 93% identity to the CaR protein predicted by the bovine CaR cDNA. The sequence surrounding the initiation codon of this open reading frame is a good match to the Kozak consensus sequence for translational initiation (28) , and the 5` sequence preceding this start codon contains stop codons in all frames. Hydrophobicity analysis of the predicted human CaR protein shows a cluster of seven hydrophobic regions 20-25 amino acids in length, reminiscent of the seven membrane-spanning regions displayed by all known GPRs. By analogy with the general structure of the GPR superfamily, the human CaR can be proposed to consist of a very large amino-terminal extracellular domain (amino acids 1-612), a central membrane spanning region (amino acids 613-862), and a relatively long carboxyl-terminal intracellular domain (amino acids 863-1078). The extracellular domain contains 11 potential sites of N-linked glycosylation, 10 of which are conserved in the bovine CaR. The bovine CaR contains highly acidic regions in the NH-terminal extracellular domain (amino acids 216-251 and 557-611) and in the second extracellular loop. These clusters of acidic residues have been proposed to serve as sites of low affinity interaction with cationic ligands (18) . All of these acidic amino acids are conserved between the human and bovine CaR proteins. There are five potential sites for protein kinase C phosphorylation located in the intracellular loop regions and the COOH-terminal intracellular domain of the human CaR. Four of these sites are present in the bovine CaR. The human CaR has two potential sites for cAMP-dependent phosphorylation (Ser-899 and Ser-900). Neither of these protein kinase A phosphorylation sites is present in the bovine CaR.


Figure 2: Nucleotide and deduced amino acid sequence of the human parathyroid CaR cDNAs. The sequence of the phPCaR-4.0 clone is presented, along with the sequence of the alternative 5` end and the additional 3` end sequence of the phPCaR-5.2 clone. The putative membrane spanning regions (TM-1 to TM-7) are underlined. The coding region differences in the phPCaR-5.2 clone (10 amino acid insertion at amino acid 536, single codon changes at amino acids 929 and 990) are shown. Polyadenylation signals (AATAAA) are indicated and sites of polyadenylation marked (). Potential sites of N-linked glycosylation (), protein kinase C phosphorylation (), and protein kinase A phosphorylation () are indicated, as are the acidic amino acids (∪) and the cysteine residues that are conserved with the mGluRs (C).



In general, there is a high level of sequence conservation between the bovine and human CaR proteins (Fig. 3). The only region displaying extensive sequence divergence is the COOH-terminal end of the intracellular domain. Of the 74 amino acid differences found between the two species, 43 are located in the region from amino acids 920 to 1078. The few other differences are distributed throughout the remainder of the protein, and many are conservative amino acid substitutions.


Figure 3: Optimized amino acid sequence alignment of human and bovine CaRs with rat mGluR-1. The seven putative transmembrane regions are underlined. Residues showing identity in the three proteins are enclosed in boxes.



The CaR shows limited, but significant homology to the metabotropic glutamate receptors (mGluRs). An alignment of human and bovine parathyroid CaRs to the rat mGluR1 protein is presented (Fig. 3). The proposed structure of the CaR, with large extracellular and intracellular domains and relatively short intracellular loops, is very similar to that proposed for the mGluRs, although overall the human CaR shows only 18-24% identity to the various rat mGluR proteins. Similarity between the CaR and mGluR sequences is maintained throughout the NH-terminal extracellular and membrane-spanning domains, with little conservation seen in the COOH-terminal intracellular domain. A hydrophobic region in the extracellular domain (amino acids 137-160), as well as the first and third intracellular loops, show particularly strong conservation (46, 50, and 75% identities, respectively) between the human CaR and rat mGluR1. Within the extracellular and membrane-spanning domains, the relative positions of 20 cysteine residues are maintained between the mGluRs and both the human and bovine CaRs (Fig. 2, and 3), suggesting conservation of structural conformation. Searches of a protein data base identified a low level of sequence similarity between amino acids 150-430 of the CaR and amino acids 110-370 of mouse and rat N-methyl-D-aspartate receptor subunits (data not shown). Similarity between the corresponding region of rat mGluR1 and the -amino-3-hydroxy-5-methyl-4-isoxazole propionate/kainate receptor subunits has been noted previously (29) .

A second cDNA clone with an insert of 5 kbp (phPCaR-5.2) was sequenced and shown to have several differences from the phPCaR-4.0 clone. In the 5`-untranslated region (UTR), both clones show identical sequence from the start codon to -242 bp, at which point the sequences completely diverge (Fig. 1). The 4.0 sequence extends 132 bp 5` of this point, while the 5.2 sequence extends another 198 bp. In comparing these two human 5`-UTRs to the 5`-UTR of the bovine CaR cDNA, the human and bovine clones show related sequence from the initiation codon to the point at -242 bp after which the bovine clone shows no relationship to either of the two human cDNA sequences. It appears that alternative 5`-UTR exons have been spliced onto a common coding region in the human CaR message. Of 16 full-length cDNA clones analyzed, nine corresponded to the 4.0 clone 5` end and seven to the 5.2 clone 5` end. None of these additional clones had 5` sequence extending significantly further than that presented in Fig. 1.

The 5.2 and 4.0 clones also differ at the 3` ends of the cDNA inserts, again in non-coding sequence. The 4.0 clone terminates in a run of A residues, but this is not preceded by a typical poly(A) addition sequence. The 5.2 clone is identical in sequence with the 4.0 clone at its 3` end, but extends beyond the 4.0 clone 3` terminus another 1138 bp. This extension corresponds to the long 3`-UTR on the bovine CaR cDNA clone. The 5.2 cDNA terminates in a poly(A) tail at its 3` end, and this is preceded by a polyadenylation signal. The 5.2 sequence shows a cluster of A residues at the point where the 4.0 clone terminates. The cDNA synthesis was primed with oligo(dT), and it is possible that the 4.0 poly(A) tail represents priming at this internal cluster of A residues rather than at a true poly(A) tail on a mRNA; the absence of a polyadenylation signal preceding the end of the 4.0 clone would support this conclusion. Of 16 clones analyzed, five had 3` ends corresponding to the 5.2 clone, 10 had 3` ends corresponding to the 4.0 clone, and a single clone had a poly(A) tail beginning at 4001 bp on the 5.2 sequence (Fig. 2). There is a close approximation of the consensus polyadenylation sequence preceding the poly(A) tail on this latter clone, and it would appear that it is a polyadenylation site that is infrequently utilized.

Within the coding sequence the two human clones show three differences. Between amino acids 536 and 537 on the 4.0 sequence, the 5.2 sequence has an insertion of 10 amino acids (30 bp) (Fig. 1). In comparing the human to the bovine CaR in this region, the bovine CaR protein is an exact match to the 4.0 sequence. None of the other cDNA clones analyzed have this 30 bp insertion, and the significance of this cDNA variant remains unknown. Comparison of the 4.0 and 5.2 sequences show two single base pair differences, with each resulting in an amino acid difference. At amino acid 926, the 4.0 cDNA encodes Gln and the 5.2 cDNA encodes Arg; the bovine cDNA encodes Gln at this position. At amino acid 990, the 4.0 cDNA encodes Gly and the 5.2 cDNA encodes Arg; the bovine cDNA encodes Arg at this position. This latter variant has been shown to be a benign polymorphism present in the human population.() The former single base pair variant is most likely an error introduced during cDNA synthesis or cloning procedures. The two cDNAs both give rise to functionally similar proteins following expression in oocytes (see following data), and it appears that these amino acid variations in the phPCaR-5.2 clone have no discernible functional consequence.

Expression of the Human CaR

The functional properties of exogenously expressed Ca mobilizing receptors in Xenopus oocytes can be monitored through the activity of an endogenous Ca-dependent Cl current. Oocytes injected with in vitro transcribed human CaR cRNA respond to extracellular application of Ca (3-10 mM) with large inward Cl currents (Fig. 4A), while water-injected control oocytes show no such response. Although in vitro transcribed RNA from both clones phPCaR-4.0 and 5.2 gave a CaR response following 2-4 days incubation in the oocyte, both human clones routinely gave smaller responses than an equivalent amount of CaR cRNA transcribed from the bovine clone. Deleting the 5`-UTR up to a point 26 bp 5` of the initiation codon gave much stronger (5-10-fold larger Cl current amplitudes) functional responses for the human CaR cRNA (data not shown). Human CaR clones phPCaR-4.0 or 5.2 expressed in oocytes respond to extracellular Ca with half-maximal effective concentrations (EC) of 7.05 ± 0.19 and 7.51 ± 0.12 mM, respectively (Fig. 4B), somewhat elevated from the Ca set point measured in vitro on human parathyroid cells. The bovine CaR, expressed in oocytes under identical conditions, also had a somewhat elevated EC for Ca of 4.5 mM. The EC values of the CaR agonists Gd, neomycin, and Mg determined for the human CaR expressed in oocytes (50 µM, 100 µM, and 12 mM, respectively) are similar to those seen with bovine parathyroid cells (7, 8) . All of these CaR agonists elicit a response in the absence of extracellular Ca, indicating that the activation of the Ca-dependent Cl current is due to release of intracellular Ca stores and not influx of extracellular Ca.


Figure 4: Expression of human CaR cRNA in Xenopus oocytes. A, Inward Cl currents induced by extracellular application of Ca, recorded in a oocyte injected 3 days prior with 10 ng of phPCaR-4.0 cRNA. The time span between application of 3.00 and 5.62 mM Ca was 5 min. B, concentration-response analysis of extracellular Ca activation of phPCaR 4.0 () and 5.2 () clones expressed in oocytes; n = 4 for each data point.




DISCUSSION

We have cloned human parathyroid CaR cDNAs from an adenomatous gland. The encoded CaR protein displays several of the features predicted by functional studies of this novel extracellular cation receptor. When expressed in oocytes, the CaR is activated by known CaR agonists with the same rank order of potency seen in parathyroid cells and is coupled to the mobilization of intracellular Ca. The recent findings that mutations in the CaR gene result in FHH and neonatal severe hyperparathyroidism (13) , and that pharmacological activation of this receptor elicits hypocalcemia (15) , provides further evidence that this cloned CaR protein plays a central role in modulating parathyroid gland function and bodily Ca homeostasis.

The CaR sequence predicts a cluster of seven hydrophobic segments, a feature common to all known GPRs. Based on this feature, the structure of the CaR is proposed to consist of an unusually large NH-terminal extracellular domain, a membrane-spanning domain with seven transmembrane regions, and a relatively large COOH-terminal intracellular domain. Searches of protein and nucleic acid sequence data bases show the CaR to share sequence similarity with the rat mGluRs, but no other known GPRs. Together, the CaR and mGluRs define a novel subfamily within the large group of GPRs. The overall domain structure of the CaR and mGluRs, with large NH-terminal extracellular regions and membrane-spanning domains with relatively short extracellular and intracellular loops, is nearly identical (the proteins can be aligned with very few gaps introduced). Although the sequence similarity between the CaR and mGluRs is low (18-24% identity overall), it extends throughout the extracellular and membrane-spanning domains, with localized regions of identity that are quite high. The putative extracellular domains of both human and bovine CaR contain nine conserved sites for N-linked glycosylation, supporting the extracellular assignment of this region and consistent with previous findings suggesting that the CaR is a glycoprotein (9) . Both the human and bovine CaR proteins contain 20 cysteine residues, located in the NH-terminal extracellular domain and extracellular loops, whose positions are conserved between the CaRs and mGluRs. This observation, coupled with the extensive sequence homology throughout the extracellular domains, suggests that the CaR and mGluRs may share a common structural conformation in this region.

NH-terminal extracellular domain of mGluR1 has been shown to function in ligand selectivity (30) . The extracellular domains of the mGluRs and ionotropic GluRs (iGluRs) share low level sequence homology with each other, and with bacterial periplasmic amino acid-binding proteins (PBPs). The conformation of the ligand-binding sites of mGluRs and iGluRs have been modeled on the known structures of the homologous bacterial PBPs, with the suggestion that the sequence conservation reflects a relationship to a common ancestral amino acid-binding structure (31, 32) . In addition to the homology to mGluRs, the extracellular domain of the CaR protein shows a low level of sequence similarity to an extracellular domain of the N-methyl-D-aspartate receptor (an iGluR). This sequence conservation suggests that the CaR extracellular domain may serve as a ligand interaction site, with structural homology to the ligand-binding domains of the glutamate receptors and PBPs. The ligand for the CaR is not an amino acid, but Ca, and glutamate receptor agonists do not activate the CaR when expressed in oocytes (18) . The CaR functions in ambient Ca concentrations in the low millimolar range, and consistent with this the CaR protein does not contain any of the consensus sequences proposed for high affinity Ca binding (33) . Low affinity Ca binding has been associated with stretches of acidic residues (34) , and the CaR does contain acidic regions in the NH-terminal extracellular domain (amino acids 215-251 and 556-610) and second extracellular loop (ELEDE). These acidic regions are conserved between the human and bovine CaRs, but not in the mGluRs. The CaR extracellular region may represent a ligand-binding domain with a conformation structurally homologous to that of the PBPs/GluRs, but modified for low affinity interaction with cationic compounds.

Activation of protein kinase C decreases the sensitivity of parathyroid cells to regulation by extracellular Ca, and it has been suggested that this effect may result from direct phosphorylation of the CaR (10, 11) . The bovine and human CaRs show potential sites for protein kinase C phosphorylation in the first and third intracellular loops and in the intracellular C-terminal domain, thus providing sites on the CaR consistent with this view. The COOH-terminal intracellular domain is the only region displaying extensive sequence divergence between the human and bovine CaRs, and the human protein has one additional protein kinase C site in this region, as well as potential sites for protein kinase A phosphorylation. The intracellular regions of GPRs are known to function in modulating receptor interactions with cellular signal transduction pathways, and variations in this region raise the possibility of species differences in the coupling of CaR activation to intracellular signaling pathways.

The human CaR cDNA clones were isolated from an adenomatous parathyroid gland removed from a patient with 1° HPT. In 1° HPT, parathyroid cells show an elevation in the Ca concentration at which PTH secretion is half-maximally suppressed (the Ca set-point), and alterations in the CaR have been proposed to underlie this set-point increase (12) , just as a mutation in the CaR has been shown to account for the decreased sensitivity to extracellular Ca seen in one instance of FHH (13) . Somatic mutation leading to alteration of the CaR protein sequence expressed in the adenomatous gland is a possibility, but it is unlikely to be the case in the particular tumor under study. The phPCaR-4.0 and 5.2 clones do have three sequence differences in the coding region, yet extracellular Ca is equipotent in activating either receptor. The EC for Ca activation of both CaR clones expressed in the oocyte is elevated relative to that seen in parathyroid cells, but that is likely a function of expression in the Xenopus system. Elevated EC values for Ca activation of the CaR were seen in oocytes expressing either bovine parathyroid gland mRNA (27) or the pBoPCaR1 cRNA, suggesting that the apparent elevation of the CaR set-point is inherent in this exogenous expression system.

One of the differences between the two cDNA clones sequenced, a 10 amino acid insertion in the NH-terminal extracellular domain of the phPCaR-5.2 clone, arises from alternative splice site selection, and is a rare occurrence in parathyroid mRNA.() The phPCaR-4.0 coding region sequence is an exact match to the normal human CaR gene, except for the change at amino acid 990, and this change is a polymorphism present in the human population and is not associated with parathyroid disease. The sequence difference at amino acid 926 in the phPCaR-5.2 clone is likely due to a cloning artifact, and this clone does give rise to a functional receptor. Thus, two independent CaR cDNA clones from the adenomatous gland do not appear to bear coding region mutations that could account for a set-point increase. A survey of CaR sequences from a collection of adenomatous parathyroid glands will be required to rigorously exclude the occurrence of CaR mutations leading to 1° HPT.

It is possible that the set-point increase characteristic of 1° HPT is not due to an alteration of the primary sequence of the receptor, but reflects a change in the amount of receptor protein relative to the increased mass of the hypercellular adenoma. The severe impairment of parathyroid function in neonatal severe hyperparathyroidism, the homozygous form of FHH, suggests that the levels of functional CaR protein are an important determinant of proper extracellular Ca sensing (13, 30) . A decrease in CaR levels in parathyroid adenomas could account for the observed set-point increases. The parathyroid adenoma examined in this study shows multiple CaR transcripts by Northern blot analysis, and cDNA clones reveal alternative RNA processing of 5`- and 3`-UTRs. The use of alternative promoters, or alternative processing to RNA species that may differ in their stability or the efficiency with which they are translated, are all events which could alter the levels of functional CaR protein expressed in the disease tissue. A third possibility is that CaR levels remain normal in a parathyroid adenoma, but secondary modifications of the receptor protein, or an alteration in the signaling pathways downstream of the receptor, results in the observed set-point increase. For example, increased activity of protein kinase C could result in a decreased sensitivity of the CaR to extracellular Ca. The isolation of these human CaR cDNA clones will facilitate studies aimed at addressing these questions of the involvement of the CaR in the etiology of 1° HPT.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) U20759 and U20760.

§
To whom correspondence should be addressed: NPS Pharmaceuticals, 420 Chipeta Way, Salt Lake City, UT 84108. Tel.: 801-583-4939; Fax: 801-583-4961.

The abbreviations used are: PTH, parathyroid hormone; CaR, calcium receptor; GPR, G protein-coupled receptor; mGluR, metabotropic glutamate receptor; 1° HPT, primary hyperparathyroidism; UTR, untranslated region; kbp, kilobase pair(s); FHH, familial hypocalciuric hypercalcemia; bp, base pair(s); PBP, periplasmic amino acid-binding protein.

J. E. Garrett and H. Heath, III, unpublished observations.

J. E. Garrett, unpublished observations.


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