Expression, purification, and biochemical characterization of the amino-terminal extracellular domain of the human calcium receptor.

We purified the extracellular domain (ECD) of the human calcium receptor (hCaR) from the medium of HEK-293 cells stably transfected with a hCaR cDNA containing an isoleucine 599 nonsense mutation. A combination of lectin, anion exchange, and gel permeation chromatography yielded milligram quantities of >95% pure protein from 15 liters of starting culture medium. The purified ECD ran as an approximately 78-kDa protein on SDS-polyacrylamide gel electrophoresis and was found to be a disulfide-linked dimer. Its NH2-terminal sequence, carbohydrate content, and CD spectrum were defined. Tryptic proteolysis studies showed two major sites accessible to cleavage. These studies provide new insights into the structure of the hCaR ECD. Availability of purified ECD protein should permit further structural studies to help define the mechanism of Ca2+ activation of this G protein-coupled receptor.

Expression of an extracellular calcium-sensing receptor in rat stomach.

BACKGROUND & AIMS: Circulating levels of Ca2+ can influence secretory functions and myoelectrical properties of the stomach. A Ca2+-sensing receptor (CaR) has recently been identified in tissues that regulate systemic Ca2+ homeostasis. The aim of this study was to evaluate expression of CaR in the stomach of the rat. METHODS: In forestomach and glandular stomach, reverse-transcription polymerase chain reaction was used to amplify a 380-base pair product, which is 99% homologous with transcripts obtained in parathyroid and kidney. RESULTS: Northern analysis of gastric mucosal polyA+ RNA revealed 7. 5- and 4.1-kilobase transcripts, similar to those obtained in rat parathyroid and kidney. Immunohistochemistry revealed CaR expression in regions of the submucosal plexus and myenteric neurons. In sections of intact tissue, preparations of primary culture surface cells and surgically dissected gastric glands, staining was observed consistently in epithelial cells of the gastric glands and in gastric surface cells. In parietal cells in isolated gastric glands, intracellular levels of Ca2+ responded to conditions that are known to activate CaR. CONCLUSIONS: These are the first reported observations that CaR is expressed in different epithelial cells of mammalian gastric mucosa and its enteric nerve regions. The effects of extracellular Ca2+ on gastric function may be attributable to activation of CaR.

Ca2+ receptor mRNA and protein increase in the rat parathyroid gland with advancing age.

Parathyroid hormone (PTH) release is regulated by extracellular calcium through a Ca2+ receptor (CaR) located on the surface of the parathyroid cell. With advancing age, the serum concentration of PTH increases, and evidence suggests that the calcium set-point for PTH release may also increase. To determine whether these changes are linked to a change in CaR expression, we quantitated mRNA and protein for the receptor in parathyroid glands of 6-week-, 6-month- and 24-month-old rats. Thyroid and kidney tissue were also studied. Between 6 weeks and 24 months of age, CaR mRNA in the parathyroid gland increased 11.4- and 3.3-fold as measured by competitive reverse transcription PCR and solution hybridization assays respectively. Message levels for the receptor also increased in the thyroid but not in the kidney. Coincident with the increase in message levels, receptor protein concentration in the parathyroid increased 7-fold between 6 weeks and 24 months of age. These results suggest that the altered relationship between extracellular calcium and PTH release observed in aging is associated with dramatic changes in CaR metabolism. That PTH secretion is increased despite increased receptor concentration suggests that aging may impair calcium binding or coupling between the CaR and down-stream effector elements in the pathway regulating PTH release.

N-linked glycosylation of the human Ca2+ receptor is essential for its expression at the cell surface.

The human Ca2+ receptor (hCaR) is a member of the superfamily of G protein-coupled receptors. Its large (approximately 600 residue) amino-terminal extracellular domain contains 9 potential N-linked glycosylation sites. Immunoblot of cell membranes derived from HEK-293 cells, stably transfected with the hCaR, showed two major immunoreactive bands of approximately 150 and 130 kDa, respectively. Complete digestion of the membranes with PN-glycosidase F yielded a single major immunoreactive band of approximately 115 kDa, confirming the presence of N-linked glycosylation. Treatment of these cells with tunicamycin, which blocks N-linked glycosylation, inhibited signal transduction in response to Ca2+. Flow cytometric analysis showed decreased expression of the hCaR on the cell membrane in tunicamycin-treated cells. Immunoblot of tunicamycin-treated cells showed a reduction in the amount of the 150-kDa band and conversion of the 130-kDa band to the presumptively nonglycosylated 115-kDa form. Tunicamycin treatment of cells, transfected with a mutant hCaR complementary DNA containing a nonsense codon at position 599 preceding the 1st transmembrane domain, blocked the secretion of a 95-kDa protein, representing the amino-terminal extracellular domain, into the medium. These results demonstrate that N-linked glycosylation is required for normal expression of the hCaR at the cell surface.

Localization of calcium receptor mRNA in the adult rat central nervous system by in situ hybridization.

The capacity to sense changes in the concentrations of extracellular ions is an important function in several cell types. For example, hormone secretion by parathyroid cells and thyroid C-cells is primarily regulated by the level of extracellular ionized calcium (Ca2+). The G-protein-coupled receptor that mediates the parathyroid cell response to Ca2+ has been cloned and we have used in situ hybridization to map calcium receptor (CaR) mRNA expression in the adult rat brain. Cells expressing CaR mRNA were present in many areas of the brain suggesting that a variety of cell types express the CaR. Particularly high numbers of CaR expressing cells were found in regions associated with the regulation of fluid and mineral homeostasis, most notably the subfornical organ. These data suggest that the capacity to detect changes in extracellular Ca2+ concentrations may have important functional consequences in several neural systems.

Monoclonal antibodies against synthetic peptides corresponding to the extracellular domain of the human Ca2+ receptor: characterization and use in studying concanavalin A inhibition.

We generated monoclonal antibodies against two synthetic peptides corresponding to residues 214-235 (ADD) and 374-391 (LRG) of the human Ca2+ receptor (hCaR) extracellular domain (ECD). Although both antibodies reacted well with their respective immunizing peptides on peptide-based enzyme linked immunosorbent assay, ADD was much more strongly reactive with the hCaR than LRG in assays such as immunoblots done under denaturing conditions. The opposite pattern was seen in flow cytometry analysis of the native receptor stably expressed in transfected 293 cells. We speculate that the ADD epitope is unexposed in the native receptor while the reverse is true for the LRG epitope. The ability to measure cell surface expression of the hCaR under native conditions using flow cytometry with the LRG monoclonal allowed us to study the basis for Concanavalin A (Con A) inhibition of CaR activation by Ca2+. Our studies show that Con A inhibition is partially accounted for by receptor internalization but, additionally, Con A may prevent Ca2+ stimulation directly by binding to carbohydrate residues in the receptor ECD.

Calcitonin-secreting cells of the thyroid express an extracellular calcium receptor gene.

Calcitonin (CT) secretion by parafollicular cells of the thyroid (C cells) is regulated by small changes in the concentration of extracellular calcium ([Ca2+]e). Elevation of [Ca2+]e elicits a rise in the C cell cytoplasmic calcium concentration and stimulates CT release. The molecular entity through which C cells detect changes in [Ca2+]e and modulate hormone secretion is unknown. Recently, an extracellular calcium-sensing receptor (CaR) complementary DNA was isolated from bovine parathyroid gland. To assess whether parathyroid cells and C cells use similar mechanisms to detect changes in ambient Ca2+, rat, human, and sheep C cells were examined for expression of the parathyroid CaR or a related receptor isoform. Reverse transcription-polymerase chain reaction analysis identified CaR transcripts in rat and human thyroid gland. Northern blot analysis demonstrated CaR messenger RNA (mRNA) in rat thyroid gland, a human medullary thyroid carcinoma (MTC) isolate, and a highly enriched preparation of sheep C cells. Rat MTC 44-2 cells, a cell line responsive to changes in [Ca2+]e, express abundant levels of CaR mRNA. Human TT cells, a C cell line lacking the extracellular calcium-sensing function, have undetectable levels of CaR mRNA by Northern blot analysis. Western blot analysis, using antiserum specific to the parathyroid CaR, detected CaR protein in rMTC 44-2, but not TT cells. Immunostaining of both dispersed sheep C cells and rat thyroid gland sections identified C cell-specific expression of the CaR protein, and in situ hybridization analysis confirmed the C cell-specific expression of CaR mRNA in the intact rat thyroid. The nucleotide sequence of the coding region of the rMTC 44-2 CaR transcripts was found to encode the same CaR protein as that expressed in the parathyroid and kidney. The results demonstrate that C cells express the same extracellular calcium-sensing receptor that is found in parathyroid and kidney, and the presence of this receptor protein in C cell lines correlates with the extracellular calcium-sensing function. This CaR is likely to represent the primary molecular entity through which C cells detect changes in [Ca2+]e and control CT release, suggesting that activation of the same receptor can either stimulate or inhibit hormone secretion in different cell types.

Calcium receptor messenger ribonucleic acid levels in the parathyroid glands and kidney of vitamin D-deficient rats are not regulated by plasma calcium or 1,25-dihydroxyvitamin D3.

The level of extracellular ionized calcium ([Ca2+]o) is the primary physiological regulator of PTH secretion. Complementary DNAs encoding the calcium receptor (CaR) protein that mediates this response have been cloned from bovine and human parathyroid glands. This protein is a seven-transmembrane, G-protein-coupled receptor linked to the mobilization of intracellular Ca2+ in response to increases in [Ca2+]o. More recently, a rat kidney CaR has been cloned and shown to be 92% identical at the amino acid level to the bovine parathyroid CaR. Homologous or heterologous regulation of the expression and/or function of a variety of G-protein-coupled receptors has been documented in numerous cell types. Therefore, we determined whether [Ca2+]o and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], major regulators of PTH synthesis and secretion, affect CaR gene expression in parathyroid gland and kidney in rats. CaR messenger RNA (mRNA) levels were quantified in pairs of parathyroid glands and single kidneys from individual animals using a solution hybridization assay. The effects of Ca2+ and 1,25-(OH)2D3 on CaR gene expression were assessed independently in vitamin D-deficient (-D) rats. A wide range of plasma Ca2+ levels (0.7-1.9 mM) was produced by supplementing -D diets with varying amounts of calcium and by infusing CaCl2 i.v. for 7 days using osmotic minipumps. There was no correlation between plasma Ca2+ levels and steady state CaR mRNA levels in parathyroid gland (r = -0.18) or kidney (r = 0.25). In another group of -D rats, 1,25-(OH)2D3 was infused sc at 25 and 275 ng/kg.day for 10-12 days. Dietary calcium was adjusted to maintain normocalcemia in some of the groups. No effect of 1,25-(OH)2D3 administration on CaR mRNA levels occurred in parathyroid glands or kidney regardless of the resultant plasma Ca2+ or 1,25-(OH)2D3 levels. In conclusion, neither parathyroid gland nor kidney CaR mRNA levels are regulated by plasma Ca2+ and 1,25-(OH)2D3 levels in the experimental models examined here.

Parathyroid gland calcium receptor mRNA levels are unaffected by chronic renal insufficiency or low dietary calcium in rats.

Extracellular ionized calcium (Ca(2+)) is the primary physiological regulator of parathyroid hormone (PTH) secretion and the G protein-coupled receptor (CaR) that mediates this response has been cloned from bovine and human parathyroid glands. The Ca(2+) set-point for the regulation of PTH secretion is right-shifted in primary hyperparathyroidism (1°HPT), but whether there is a similar shift in 2°HPT is unclear. Additionally, the molecular defects associated with such changes in the set-point remain uncharacterized. These experiments were designed to determine (1) if changes in set-point occur in rats with 2°HPT induced by chronic renal insufficiency (CRI) or dietary Ca deficiency, and (2) whether any changes in set-point are mirrored by changes in steady-state mRNA levels for the parathyroid CaR. CaR mRNA levels were quantified in pairs of glands from individual rats using a solution hybridization assay. Blood urea nitrogen and PTH levels were ∼ 4-fold higher in rats with CRI induced by 5/6 nephrectomy 7 weeks earlier. Rats with CRI were also significantly hypocalcemic and hyperphosphatemic. The setpoint was unchanged in CRI rats and CaR mRNA levels were also unaffected. Normal rats fed a 0.02% Ca diet for 6 weeks were markedly hypocalcemic, and had 10- and 15-fold increases in plasma PTH and 1,25-dihydroxyvitamin D(3) levels, respectively. Technical problems prevented assessment of the set-point in these animals, but parathyroid gland CaR mRNA levels were identical in both dietary groups. Thus, neither alterations in mRNA levels for the CaR nor changes in the set-point play demonstrable roles in the pathogenesis of 2°HPT in these models.

Promoter for the regulatory type I beta subunit of the 3',5'-cyclic adenosine monophosphate-dependent protein kinase directs transgene expression in the central nervous system.

Cyclic AMP-dependent protein kinase (cAPK) modulates synaptic transmission and influences memory and learning. Among the various isoforms of regulatory and catalytic subunits that comprise mammalian cAPK, only the regulatory type I beta (RI beta) subunit is unique to nervous tissue. The requirement for RI beta in neurons is presently unknown. Previous studies demonstrate that holoenzyme containing RI beta activates at lower concentrations of cAMP compared to other forms of cAPK. Thus, neurons that induce RI beta expression may become more sensitive to subsequent hormonal signals and maintain more long-term phosphorylation events. To further elucidate the function of this novel protein, we have begun to investigate its gene. Here we report the isolation of the mouse RI beta promoter as determined by S1 nuclease analysis and transgenic mouse expression. A beta-galactosidase fusion gene containing 1.5 kilobases of 5'-nontranscribed RI beta DNA and 2 kilobases of intron 1 was expressed preferentially in the cortex and hippocampus of the brain and within the spinal cord. In addition to mimicking the location of endogenous RI beta expression, the transgene was activated at a similar time (embryonic day 11.5) during mouse fetal development. Isolation of the RI beta promoter will help identify the elements that direct transcription in a subset of neurons and illuminate the physiological conditions that may regulate RI beta expression. This promoter can also be used to target the expression of wild type and mutant cAPK subunit genes in order to investigate synaptic plasticity in animals.

Regulation of Cl- transport in T84 cell clones expressing a mutant regulatory subunit of cAMP-dependent protein kinase.

Cl- channels in the apical membranes of salt-secreting epithelia are activated by both cAMP and Ca2+ second-messenger systems, and dysfunctions in their hormonal regulation have been demonstrated in patients with cystic fibrosis. We have transfected the epithelial cell line T84 with an expression vector containing a mutant form of the regulatory subunit of the cAMP-dependent protein kinase. Stable transformants that express this construct have reduced basal cAMP-dependent protein kinase activity and do not increase kinase activity beyond the basal level of control cells in response to cAMP. Forskolin, vasoactive intestinal peptide, and prostaglandin E2 each stimulate intracellular cAMP accumulation in both mutant and control clones; however, the activation of Cl- channels in response to elevated cAMP is blocked in mutant clones, indicating direct involvement of the cAMP-dependent protein kinase. In contrast, Ca2+ ionophores retain their ability to activate the Cl- channel in T84 cells expressing the mutant regulatory subunit, suggesting that activation of the channel by means of Ca2+ does not require the participation of cAMP-dependent protein kinase activity. These clones will be useful for further studies of the interactions between the cAMP- and Ca2(+)-dependent regulatory pathways in salt-secreting epithelial cells. They can also be used to identify the mediators of Ca2(+)-dependent Cl- channel activation in isolation from interactions with the cAMP second-messenger pathway.

The effect of hypophysectomy and growth hormone administration on pre-prosomatostatin messenger ribonucleic acid in the periventricular nucleus of the rat hypothalamus.

Physiological evidence suggests that hypothalamic somatostatin (SS) inhibits pituitary GH release and that GH acts through a short-loop feedback mechanism to stimulate SS secretion. The feedback action of GH could be mediated by an effect on SS synthesis, secretion, or both. We hypothesized that GH acts to regulate the expression of the SS gene and that changes in the level of circulating GH would result in corresponding changes in SS mRNA in cells of the periventricular nucleus (PeN) of the hypothalamus. To test this hypothesis we measured the effect of hypophysectomy (HPX) and HPX with bovine GH (bGH) replacement on SS mRNA signal levels in cells of the PeN of the rat brain. We report that HPX male rats treated with bGH have significantly higher PeN SS mRNA signal than their vehicle-treated controls (P less than 0.05) and that bGH administration to sham-HPX rats results in elevated PeN SS mRNA signal levels compared to those in sham-HPX rats treated with vehicle (P less than 0.05). These observations suggest that GH participates in the regulation of its own secretion by influencing the expression of the SS gene and that one mechanism of short-loop pituitary feedback may involve the modulation of neuropeptide gene expression.

Reduced preprosomatostatin messenger ribonucleic acid in the periventricular nucleus of hypophysectomized rats determined by quantitative in situ hybridization.

Physiological evidence suggests that somatostatin (SS) inhibits the release of GH and TSH from the anterior pituitary and that elements of these two systems may feed back to regulate hypothalamic SS release or synthesis. Hypophysectomy reduces hypothalamic SS content in rats, an effect that may be attributable to a change in SS synthesis, storage, or release. We tested the hypothesis that hypophysectomy would reduce hypothalamic SS synthetic capacity, as reflected by a reduction in SS mRNA levels. Using in situ hybridization and a computerized image analysis system, we measured SS mRNA signal levels over individual cells in the periventricular nucleus of hypophysectomized and intact male rats. SS mRNA signal levels were 45.1% lower in hypophysectomized rats compared to those in intact controls (P less than 0.05). These results demonstrate that SS synthetic capacity in the periventricular nucleus is influenced by the presence of the pituitary, and this system may represent one example of the regulation of central nervous system neuropeptide gene expression by a circulating pituitary hormone.