Regulation of inducible and tissue-specific gene expression.

Molecular genetics approaches have been used to identify and characterize cis-acting DNA sequences required for eukaryotic gene regulation. These sequences are modular in nature, consisting of arrays of short (10- to 12-base pair) recognition elements that interact with specific transcription factors. Some transcription factors have been extensively purified and the corresponding genes have been cloned, but the mechanisms by which they promote transcription are not yet understood. Positive and negative regulatory elements that function only in specific cell types or in response to extracellular inducers have been identified. A number of cases of inducible and tissue-specific gene expression involve the activation of preexisting transcription factors, rather than the synthesis of new proteins. This activation may involve covalent modification of the protein or an allosteric change in its structure. The modification of regulatory proteins may play a central role in the mechanisms of eukaryotic gene regulation.

Molecular motors and developmental asymmetry.

The generation of distinct cell fates can require movement of specific molecules or organelles to particular locations within the cell. These subcellular movements are often the jobs of motor proteins. Seemingly disparate developmental processes--determination of right and left in vertebrates, setting up the axes of polarity in insect embryos, mating-type switching in yeast, and coordinated organelle movements in Drosophila--converge in their dependence on motor proteins. The extent of possible regulatory complexity is only beginning to emerge.

Enhanced vascular responses to adrenomedullin in mice overexpressing receptor-activity-modifying protein 2.

Adrenomedullin (AM) levels are elevated in cardiovascular disease, but little is known of the role of specific receptor components. AM acts via the calcitonin receptor-like receptor (CLR) interacting with a receptor-activity-modifying protein (RAMP). The AM1 receptor is composed of CLR and RAMP2, and the calcitonin gene-related peptide (CGRP) receptor of CLR and RAMP1, as determined by molecular and cell-based analysis. This study examines the relevance of RAMP2 in vivo. Transgenic (TG) mice that overexpress RAMP2 in smooth muscle were generated. The role of RAMP2 in the regulation of blood pressure and in vascular function was investigated. Basal blood pressure, acute angiotensin II-raised blood pressure, and cardiovascular properties were similar in wild-type (WT) and TG mice. However, the hypotensive effect of IV AM, unlike CGRP, was enhanced in TG mice (P<0.05), whereas a negative inotropic action was excluded by left-ventricular pressure-volume analysis. In aorta relaxation studies, TG vessels responded in a more sensitive manner to AM (EC50, 8.0+/-1.5 nmol/L) than WT (EC50, 17.9+/-3.6 nmol/L). These responses were attenuated by the AM receptor antagonist, AM(22-52), such that residual responses were identical in all mice. Remaining relaxations were further inhibited by CGRP receptor antagonists, although neither affected AM responses when given alone. Mesenteric and cutaneous resistance vessels were also more sensitive to AM in TG than WT mice. Thus RAMP2 plays a key role in the sensitivity and potency of AM-induced hypotensive responses via the AM1 receptor, providing evidence that this receptor is a selective target for novel therapeutic approaches.

Molecular analysis of the klarsicht gene and its role in nuclear migration within differentiating cells of the Drosophila eye.

BACKGROUND: The temporally regulated, cell-type-specific transport of organelles has great biological significance, yet little is known about the regulation of organelle transport during development. The Drosophila gene klarsicht is required for temporally regulated lipid droplet transport in developing embryos and for the stereotypical nuclear migrations in differentiating cells of the developing eye. Klarsicht is thought to coordinate the function of several molecular motors bound to a single lipid droplet or to facilitate the attachment of dynein to the cargo, but it is not known whether Klarsicht affects motors directly or indirectly. RESULTS: Here, we have cloned the klarsicht gene and shown that it encodes a unique large protein. Drosophila klarsicht null mutants were viable, with obvious defects only in adult eye morphology. Epitope-tagged Klarsicht expressed in the eye from a transgene was perinuclear. In flies carrying transgenes that express markers for microtubule plus and minus ends, microtubules in differentiating cells of the eye were oriented with their plus ends apical and their minus ends at the nucleus. CONCLUSIONS: Drosophila klarsicht null mutants were viable and fertile, demonstrating that klarsicht is essential only for specific motor protein functions. Perinuclear localization of Klarsicht protein indicates that Klarsicht has a direct mechanical role in nuclear migration. Taken together with the finding that the minus ends of the microtubules are associated with the photoreceptor nuclei, the observation that Klarsicht is largely perinuclear supports the idea that Klarsicht associates with dynein, consistent with a model in which Klarsicht assists dynein in 'reeling in' the nucleus.

Human parathyroid hormone. Immunological characterization of antibodies against a glandular extract and the synthetic amino-terminal fragments 1-12 and 1-34 and their use in the determination of immunoreactive hormone in human sera.

Antibodies to a urea-trichloroacetic acid extract [hPTH-(TCA)] of human parathyroid tumors and to the synthetic NH(2)-terminal fragments of human parathyroid hormone hPTH-(1-12) and -(1-34) were developed in goats to characterize immunochemically various PTH preparations and to estimate immunoreactive PTH (iPTH) in human sera. They were quantitated on the basis of their capacity to bind [(131)I]-hPTH-(1-12), [(131)I]hPTH-(1-34) or [(131)I]bovine PTH (bPTH-(1-84)). The quality of the antibodies was assessed by reference to inhibition of their interaction with labeled peptides by synthetic hPTH comprising 34 NH(2)-terminal amino acid residues or fragments thereof [hPTH-(1-12), -(13-34), -(18-34), -(25-34), -(18-24)] or by the Sephadex G-100-purified full-length peptide hPTH-(1-84) [hPTH-(1-84)G-100]. The synthetic peptides used in this work correspond in their structure to the NH(2)-terminal amino acid sequence 1-34, as elucidated by Brewer and collaborators (1972. Proc. Natl. Acad. Sci. U. S. A.69: 3583-3588). Inhibition studies were also carried out with bPTH-(1-34) and bPTH-(1-84). Anti-hPTH-(TCA) exhibited specificities directed to determinants in the COOH-terminal and NH(2)-terminal part of hPTH-(1-84) and exhibited cross-reactivity with bPTH-(1-84). Anti-hPTH-(1-34), on the other hand, showed immunological specificities mainly directed to antigenic determinants located in the COOH-terminal half of hPTH-(1-34). In addition, some reactivity with the NH(2)-terminal hPTH-(1-12) and with the extractive full-length peptides of human and bovine origin was observed. Antibodies to hPTH-(1-12) cross-reacted with hPTH-(1-34) and -(1-84)G-100.IPTH WAS RADIOIMMUNOLOGICALLY DETERMINED IN HUMAN SERA BY THE FOLLOWING SYSTEMS: (a) [(131)I]bPTH-(1-84), anti-hPTH-(TCA) and hPTH-(1-84)G-100 as standard; (b) [(131)I]hPTH-(1-34), anti-hPTH-(1-34) and hPTH-(1-34) as standard. With system (a), COOH-terminal fragments of hPTH-(1-84) having a molecular weight of approximately 7,000 were detected, and there was an almost total discrimination of serum iPTH levels in normal and in hyperparathyroid subjects. With system (b), on the other hand, several molecular species of iPTH were detected, including a component larger than hPTH-(1-84) and others similar to hPTH-(1-84) and to a fragment co-eluting with the NH(2)-terminal fragment hPTH-(1-34). When serum iPTH was assayed in system (b), there was a large overlap of iPTH levels in control subjects and in patients with primary hyperparathyroidism.

Acute parathyroid hormone response to epinephrine in vivo.

The acute effects of epinephrine, norepinephrine, and isoproterenol on the plasma immunoreactive parathyroid hormone (iPTH) response were studied in 13 550-600 kg cows. Catecholamines were infused for 7.0 min. During epinephrine infusions at 0.08 mumol/min iPTH increased from 0.48+/-0.12 (mean+/-SE, ng/ml) to 1.09+/-0.18 ng/ml (P < 0.02). Small increases in plasma free fatty acids and glucose could be detected with 0.08 mumol/min epinephrine; the iPTH response to epinephrine was as sensitive as the free fatty acid and glucose responses and possibly of physiological importance. Plasma calcium (total and ionized) and magnesium did not change. The responses were more pronounced at 0.8 mumol/min epinephrine with a mean iPTH increase from 0.49+/-0.16 ng/ml to 1.74+/-0.35 ng/ml (P < 0.01). Small decreases in plasma calcium occurred at 0.8 mumol/min epinephrine, but the plasma magnesium remained unchanged. However, when the plasma calcium was lowered with ethylene glycol bis(beta-aminoethyl ether)-N, N'-tetraacetic acid (EGTA), a much more pronounced lowering of the plasma calcium was required to produce comparable increases of the plasma iPTH concentrations than when epinephrine was infused. It appears that epinephrine has a direct effect on the release of iPTH from the parathyroid glands. Simultaneous infusions of calcium and epinephrine suppressed the stimulation by epinephrine. This points towards a common mechanism of the regulation of parathyroid hormone secretion caused by decreases in the extracellular calcium concentration and/or alterations in the distribution of calcium within parathyroid cells following the administration of epinephrine. The iPTH response to epinephrine was suppressed in the presence of propranolol. Isoproterenol was less active in raising iPTH than epinephrine, and norepinephrine was the least active. The stimulation by isoproterenol and the suppression by propranolol suggest beta adrenergic receptor sites within the parathyroid glands.

Suppressive effects of 24,25-dihydroxycholecalciferol on bone resorption induced by acute bilateral nephrectomy in rats.

The possible suppressive effects of 24,25-dihydroxycholecalciferol on secondary hyperparathyroidism and increased bone resorption were investigated in adult rats raised on a diet normal in calcium, phosphorus, and vitamin D, and subjected to acute bilateral nephrectomy. The animals had received subcutaneous radiocalcium 4 wk before the experiment. 5 h after nephrectomy an increase in serum total calcium, (45)Ca-specific activity, citrate, phosphorus, and magnesium concentrations were observed. Serum immunoreactive parathyroid hormone increased, while serum calcitonin decreased. The osteoclast count in the tibial metaphyses was augmented. The biochemical and histological changes observed were partly parathyroid hormone and calcitonin independent, as they also occurred in parathyroidectomized hypocalcemic rats. Pretreatment with 650 pmol of 24,25-dihydroxycholecalciferol 16 h before nephrectomy prevented bone calcium mobilization and diminished the rise in serum total calcium and citrate both in parathyroid-intact and in parathyroidectomized animals. In parathyroid-intact rats, serum immunoreactive parathyroid hormone and calcitonin remained normal in spite of the fall in serum-ionized calcium, and the number of osteoclasts did not increase. In parathyroidectomized rats, 24,25-dihydroxycholecalciferol did not prevent the postnephrectomy rise in the osteoclast count. This latter observation suggests that this metabolite exerts its effect on bone either by acting on cells other than osteoclasts, i.e., the osteocytes, or by inhibiting cell activity. At equimolar dosage 1,25-dihydroxycholecalciferol had a potent stimulatory effect on bone resorption. This effect of 1,25-dihydroxycholecalciferol was partly blocked by the simultaneous administration of 24,25-dihydroxycholecalciferol. The potential clinical significance of these observations remains to be determined.

Parathyroid hormone responses to catecholamines and to changes of extracellular calcium in cows.

Modifications of the plasma level of immunoreactive parathyroid hormone (PTH) in cattle were induced by changes of the plasma concentrations of epinephrine, isoproterenol, or calcium. During abrupt hypocalcemia, PTH, obtained by infusions with ethylene glycol-bis (beta-aminoethylether) N, N'-tetraacetate (EGTA), increased during the first 4-8 min. After a transient decline, the hormone levels rose again and remained elevated. Infusions of calcium suppressed the hypocalcemia-induced augmentation of PTH levels within a few minutes. Prolonged epinephrine (and isoproterenol) infusions also rapidly increased PTH levels, however, in this case, they returned to basal concentrations after 50-60 min. Additional epinephrine infusions could not further raise PTH values. Moreover, three short-lasting infusions of epinephrine (7 min each), given at 30-min intervals, increased PTH levels to the same extent, whereas additional infusions were much less effective. The PTH response to epinephrine was completely restored, when the interval after a prolonged epinephrine infusion had been prolonged to > 100 min. During moderate hypocalcemia, occurring at the end of EGTA infusions and lasting for 90 min, the PTH response to a short-lasting epinephrine infusion (7 min) was more pronounced than in normocalcemic animals. During severe hypocalcemia, in which superimposed short-lasting infusions of EGTA (7 min) led to an additional abrupt fall of plasma calcium concentrations but not to a corresponding additional rise of the PTH levels, epinephrine rapidly and further increased PTH concentrations. On the other hand, at the end of prolonged infusions of epinephrine, when additional infusions of epinephrine were ineffective in raising PTH levels, EGTA-induced hypocalcemia consistently increased PTH concentrations. The EGTA-induced augmentation of PTH levels was enhanced by epinephrine and isoproterenol but not by propranolol. The present findings indicate, that variations of the extracellular calcium concentrations and beta-adrenergic agonists modify PTH levels by two different and independent mechanisms. On the other hand, it appears that the magnitude of change of the PTH levels to either stimulus is partially modulated by exposure to the other.

Calcitonin gene-related peptide is a stimulator of renin secretion.

Calcitonin gene-related peptide (CGRP) was found to stimulate renin secretion in vivo in normal human volunteers. Moreover, CGRP stimulated the release of renin in vitro from isolated rat renal juxtaglomerular cells (half-maximal effective concentration [EC50] 100 nM) concomitant with stimulation of cAMP production (EC50 60 nM). Immunoreactive CGRP was recognized in rat renal cortical nerve fibers, and intact rat CGRP was identified in extracts of the rat renal cortex. Because CGRP containing sensory nerve fibers are seen in the region of the juxtaglomerular apparatus, it would seem that the release of CGRP from these afferent nerves may be involved in the physiological control of renin secretion.

A new bioactive form of human calcitonin.

Distinct immunoreactive forms of calcitonin (CT), extracted with 2 M acetic acid from two pancreatic tumors, were characterized and identified by gel permeation chromatography and by reverse-phase high-performance liquid chromatography. The extracted CT forms were compared to CT obtained from medullary thyroid carcinoma and from normal thyroid glands, and were, furthermore, analyzed in a rat hypocalcemic bioassay. On gel filtration analysis, two broad peaks coeluting with synthetic human CT-(1-32) and extracted dimeric CT, respectively, were found in variable amounts. An acetonitrile gradient high-performance liquid chromatography system revealed two to three predominant CT peaks. Biologically active monomeric and dimeric CT and the biologically inactive sulfoxide form of human CT-(1-32) have been identified. Moreover, we have detected for the first time a new biologically active CT-like component which was most prominently recognized in a benign pancreatic tumor.

Comparison of parathyroid hormone receptors in rat osteosarcoma cells and kidney.

Parathyroid hormone/parathyroid-hormone-related peptide (PTH/PTHrP) receptors have been characterized with chicken parathyroid hormone related protein [Tyr36]chPTHrP(1-36)amide (chPTHrP(1-36)) as radioligand in rat UMR-106 osteosarcoma (UMR) cells and in rat renal cortical membranes (RCM). Binding of 125 pM [125I][Tyr36]chPTHrP(1-36) was displaced by chPTHrP(1-36) with ID50 values of 2.6 +/- 0.22 nM (mean +/- S.E.) and 0.9 +/- 0.03 nM in UMR cells and RCM, respectively. ID50 values in membranes from UMR cells and RCM were the same in the presence and absence of 10 microM guanosine-5'-O-(3-thiotriphosphate). Rat [Nle8,18] PTH(1-34) was 5-fold more potent than chPTHrP(1-36) in RCM, but not in UMR cells. Hill coefficients derived from binding inhibition were 0.93 and 0.35 in UMR and RCM, respectively. For affinity labeling, N-hydroxysuccinimidyl-4-azidobenzoate-modified [125I]chPTHrP(1-36) was used. Specifically-labeled PTH/PTHrP-binding proteins had a molecular mass of 83 kDa in UMR cells and RCM. Treatment with N-endoglycosidases lowered the molecular mass of chPTHrP binding proteins to 54 kDa in UMR and RCM. In conclusion, skeletal UMR-106 cells and renal cortical membranes of the rat reveal PTH/PTHrP receptors with no apparent tissue specific differences in molecular mass of the polypeptide backbone and polysaccharide chains. Higher affinity of rat PTH(1-34) binding and lower Hill coefficients in kidney compared to bone are consistent with tissue specific receptor-ligand interactions.

Identity of calcitonin extracted from normal human thyroid glands with synthetic human calcitonin-(1-32).

Calcitonin in human thyroid glands obtained at autopsy from normal subjects were extracted with 2 M acetic acid. The extracts were additionally purified by adsorption to Sep-Pak C18 cartridges, and calcitonin was identified after gel filtration analysis, reverse-phase high-performance liquid chromatography (HPLC), thin-layer chromatography and isoelectric focusing. The purification steps were monitored by radioimmunoassay, and partially purified calcitonin was used for biological and physicochemical comparison with synthetic human calcitonin-(1-32) and its Met8-sulfoxide form. On gel filtration analysis a predominant peak coeluted with the synthetic hormone, and on HPLC two discrete peaks with the retention times of monomeric and dimeric human calcitonin were found. Thin-layer chromatography allowed the detection of two peaks with the Rf of human calcitonin-(1-32) and of its sulfoxide, respectively. The pI (7.9) of the predominant peaks of synthetic calcitonin were identical. Our findings provide strong evidence that the predominant forms of human calcitonin extracted from normal thyroid glands correspond to synthetic calcitonin-(1-32) and to dimeric calcitonin.

Separation of inhibitory activity from biologically active parathyroid hormone in patients with pseudohypoparathyroidism type I.

Patients with pseudohypoparathyroidism type I have the symptoms of hypoparathyroidism despite elevated levels of immunoreactive parathyroid hormone (PTH). However, the circulating levels of bioactive PTH, as measured in a cytochemical bioassay, are generally within the normal range suggesting that the high levels of immunoreactive PTH are either due to the presence of biologically inactive fragments of parathyroid hormone or to the presence of an 'inhibitor' of PTH bioactivity. Gel-permeation chromatography has been used to fractionate plasma from patients with pseudohypoparathyroidism type I and revealed the presence of high levels of bioactive PTH and of an 'inhibitor'. This inhibitory activity was absent or much lower in plasma from control subjects. These results indicate, therefore, that in pseudohypoparathyroidism type I the expression of the biological activity of PTH at the level of the kidney is affected by the presence of a circulating inhibitor which can be separated from intact PTH by gel-permeation chromatography.

In vivo Structure/Function analysis of the Drosophila fat facets deubiquitinating enzyme gene.

The Drosophila Fat facets protein is a deubiquitinating enzyme required for patterning the developing compound eye. Ubiquitin, a 76-amino-acid polypeptide, serves as a tag to direct proteins to the proteasome, a protein degradation complex. Deubiquitinating enzymes are a large group of proteins that cleave ubiquitin-protein bonds. Fat facets belongs to a class of deubiquitinating enzymes called Ubps that share a conserved catalytic domain. Fat facets is unique among them in its large size and also because Fat facets is thought to deubiquitinate a specific substrate thereby preventing its proteolysis. Here we asked which portions of the Fat facets protein are essential for its function. P-element constructs that express partial Fat facets proteins were tested for function. In addition, the DNA sequences of 12 mutant fat facets alleles were determined. Finally, regions of amino acid sequence similarity in 18 Drosophila Ubps revealed by the Genome Project were identified. The results indicate functions for specific conserved amino acids in the catalytic region of Fat facets and also indicate that regions of the protein both N- and C-terminal to the catalytic region are required for Fat facets function.

Genetic analysis of the Drosophila DNAprim gene. The function of the 60-kd primase subunit of DNA polymerase opposes the fat facets signaling pathway in the developing eye.

The Drosophila DNAprim gene encodes the large subunit (60 kD) of DNA primase, the part of DNA polymerase alpha that synthesizes RNA primers during DNA replication. The precise function of the 60-kD subunit is unknown. In a mutagenesis screen for suppressors of the fat facets (faf) mutant eye phenotype, we identified mutations in DNAprim. The faf gene encodes a deubiquitinating enzyme required specifically for patterning the compound eye. The DNA sequences of four DNAprim alleles were determined and these define essential protein domains. We show that while flies lacking DNAprim activity are lethal, flies with reduced DNAprim activity display morphological defects in their eyes, and unlike faf mutants, cell cycle abnormalities in larval eye discs. Mechanisms by which DNA primase levels might influence the faf-dependent cell communication pathway are discussed.

Improved cardiac performance with human calcitonin gene related peptide in patients with congestive heart failure.

STUDY OBJECTIVE: The aim of the study was to assess the cardiovascular effects of human calcitonin gene related peptide (CGRP) in patients with congestive heart failure. DESIGN: The effects of CGRP II (or beta), 12.5 micrograms.h-1, given by intravenous infusion for 24 h to digitalised patients with congestive heart failure, were assessed by measurement of cardiac functional indices. PATIENTS: Five patients (four female) were studied. Age was 73-82 years. Three were in New York Heart Association phase III and two in phase IV. MEASUREMENTS AND MAIN RESULTS: The pre-ejection period to left ventricular ejection time ratio and the QT distance adjusted for heart rate were lowered by 21% and 4% respectively. The left ventricular shortening index was raised by 43%. The arterial pressure and heart rate did not change consistently. CONCLUSION: Calcitonin gene related peptide improves myocardial contractility in patients with congestive heart failure. This is the first time this has been shown.

Effect of dexamethasone on parathyroid hormone (PTH) and PTH-related protein regulated phosphate uptake in opossum kidney cells.

Effects of dexamethasone (DEX) on receptor binding of bovine PTH (1-34) [bPTH (1-34)] and human PTH-related protein (1-34) [hPTHrP(1-34)] and cAMP accumulation, and on the inhibition of Na(+)-dependent phosphate uptake were studied in opossum kidney (OK) cells. Maximal specific binding of [125I]hPTHrP(1-34) was reduced by 40% in cells treated with 100 nM DEX, but half-maximal inhibition of binding of [125I]hPTHrP(1-34) by bPTH(1-34) or hPTHrP(1-34) was unaffected by DEX (0.5 nM and 1.4 nM, in the absence and presence of DEX, respectively). Moreover, the EC50 of bPTH(1-34) and hPTHrP(1-34)-evoked cAMP accumulation ranged from 3-7 nM and was the same with and without DEX. The EC50 of the inhibition of phosphate uptake by bPTH(1-34) and hPTHrP(1-34) ranged from 0.2-0.5 nM. Treatment with 100 nM DEX increased phosphate uptake 1.5-fold (EC50 3 nM), and the inhibition of phosphate uptake by bPTH(1-34) and hPTHrP(1-34) was suppressed. The inhibition of phosphate uptake by forskolin was also suppressed in cells treated with 100 nM DEX. DEX, on the other hand, enhanced forskolin-stimulated cAMP accumulation 1.6- to 1.8-fold. 12-O-Tetradecanoylphorbol-13-acetate did not affect cAMP production, but phosphate uptake was inhibited both in the absence and the presence of 100 nM DEX (EC50 3 nM). In conclusion, treatment of OK cells with DEX only minimally affected PTH receptor binding, and cAMP accumulation evoked by bPTH(1-34) and hPTHrP(1-34) remained unaltered. The inhibition of phosphate uptake by PTH, however, was suppressed.

Calcium-regulated biosynthesis of the parathyroid secretory protein, proparathyroid hormone, and parathyroid hormone in dispersed bovine parathyroid cells.

Dispersed bovine parathyroid cells were incubated in vitro with 3H-labeled amino acids for 30--120 min. Analysis of cell extracts by sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis revealed a calcium-regulated incorporation of 3H-labeled amino acids into three major peaks, one with a molecular weight (Mr) of 70,000 and two peaks in the 10,000 Mr region, and a minor 5,000 Mr peak. Furthermore, [3H]mannose was incorporated into the 70,000 Mr peak, which corresponds to the parathyroid secretory protein (PSP) recognized as a glycoprotein. The two peaks in the 10,000 Mr region had the electrophoretic mobility of [3H]bovine proparathyroid hormone (ProPTH) and [3H]bovine PTH (1--84), respectively. Immunoreactive PTH was only detected in a peak comigrating with [3H]PTH-(1--84). The nonimmunoreactive 5,000 Mr peptide was not further identified. The incorporation of radioactive amino acids into PSP, ProPTH, PTH-(1--84), and the 5000-daltons peptide was inversely related to the calcium concentration in the incubation medium. The incorporation of radioactive mannose into PSP was also greater at low extracellular calcium concentrations. Our findings provide evidence for an inverse relationship between the extracellular calcium concentration and the formation of PSP, ProPTH, PTH-(1--84), and a yet to be identified 5000-dalton peptide in dispersed bovine parathyroid cells.

Calcitonin inhibits phosphate uptake in opossum kidney cells stably transfected with a porcine calcitonin receptor.

Calcitonin (CT), and PTH and PTH-related protein (PTHrP) stimulated urinary excretion of phosphate is brought about through inhibition of Na/P04 cotransport in proximal renal tubules. PTH/PTHrP receptors linked to inhibition of phosphate uptake have been characterized in a renal tubular cell line from the American opossum (OK). Specific binding of [125I]salmon CT (sCT) to OK cells was not recognized, but 1 microM sCT stimulated cAMP accumulation 10-fold and reduced phosphate uptake by 7 +/- 2% (P < 0.05). The responses were amplified in OK cells stably transfected with a cloned CT receptor from a porcine LLC-PK1 kidney cell line. The transfected cells expressed 20,000 CT receptors per cell with a Kd of 0.05 nM and an EC50 of cAMP accumulation of 6.2 nM; maximal cAMP stimulation in response to 1 microM sCT was 259-fold (P < 0.01). Phosphate uptake was inhibited by 35 +/- 4% in response to 1 microM sCT and by 34 +/- 3% to 1 microM chicken PTHrP(1-36) (P < 0.01). Half-maximal inhibition was obtained with 0.63 +/- 0.30 nM sCT and with 1.39 +/- 0.67 nM chicken PTHrP(1-36). The inhibition of [125I]sCT binding by nonlabeled human amylin required about 5000-fold higher concentrations than those of sCT, and human calcitonin gene-related peptide-I (CGRP) at up to 1 microM did not affect [125I]sCT binding. The rank order of potencies with respect to stimulation of cAMP accumulation and inhibition of phosphate transport of sCT, amylin and CGRP was the same. This is the first report linking a cloned CT receptor to inhibition of phosphate transport in a renal proximal tubular cell.

A receptor activity modifying protein (RAMP)2-dependent adrenomedullin receptor is a calcitonin gene-related peptide receptor when coexpressed with human RAMP1.

Adrenomedullin (ADM) and alpha- and beta-calcitonin (CT) gene-related peptide (alpha-, betaCGRP) are structurally related vasodilatory peptides with homology to CT and amylin. An originally orphan human CT receptor-like receptor (hCRLR) is a Gs protein-coupled CGRP or ADM receptor when coexpressed with recently identified human single transmembrane domain receptor activity modifying proteins 1 (hRAMP1) or -2 (hRAMP2), respectively. Here, the function of the rat CRLR homologue (rCRLR) has been investigated in rat osteoblast-like UMR-106 cells and in COS-7 cells, in the absence and presence of hRAMP1 and -2 and combinations thereof. Transient expression of rCRLR in UMR-106 cells revealed an ADM receptor, and [125I]rat (r) ADM binding was enhanced with hRAMP2 and inhibited by 50% when hRAMP1 was coexpressed. Detectable [125I]h alphaCGRP binding required the presence of hRAMP1, and the expression of CGRP binding sites was unaffected by coexpressed hRAMP2. Specificity of ADM binding sites in [125I]rADM binding inhibition experiments was reflected by an over 1000-fold higher potency of rADM [half-maximal effective concentration = 0.19 +/- 0.05 nM (mean +/- SEM, n = 4)], compared with r alphaCGRP and r betaGRP, to induce a cAMP-responsive luciferase reporting gene (CRE-luc). In rCRLR and hRAMP1 cotransfected cells, expressing predominantly CGRP binding sites, r betaCGRP, r alphaCGRP, and rADM induced CRE-luc with half-maximal effective concentration of 0.27 +/- 0.17 nM, 0.37 +/- 0.27 nM, and 1.4 +/- 0.9 nM, respectively. In COS-7 cells, the results were comparable, but rCRLR required coexpressed hRAMP2 for ADM receptor function. This is consistent with higher levels of endogenous RAMP2 encoding messenger RNA in UMR-106, compared with COS-7 cells. In conclusion, the recognition of RAMP1 and -2 as mediators of CRLR expression as a CGRP or ADM receptor has been extended, with evidence that endogenous RAMP2 is sufficient to reveal an ADM receptor in UMR-106 cells. Inhibition of RAMP2-evoked ADM receptor expression by RAMP1 and generation of a CGRP receptor is consistent with competitive interactions of the different RAMPs with rCRLR.