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CRF is the primary neuroregulator of the function of the hypothalamic-pituitary-adrenal axis. We have recently cloned a mouse CRF receptor (mCRF-R) complementary DNA (cDNA) from an AtT-20 cell cDNA library by polymerase chain reaction. To compare the functions of mCRF-R to those of the human type I and type II CRF receptors (hCRF-RI and hCRF-RII), cDNAs were cloned into the expression vector pcDNA1 and transfected into COS-7 cells. CRF binding and CRF-stimulated cAMP accumulation as well as phosphoinositide hydrolysis were measured. Scatchard analysis of the binding of 125I-labeled [Tyr0]r/hCRF ([125I]CRF) to COS-7 cells expressing mCRF-R and hCRF-RI cDNAs revealed the same apparent Kd (9 nM). In contrast, the apparent binding Kd for hCRF-RII was 20 nM CRF. Maximal stimulatory concentrations (1 microM) of rat/human CRF-(1-41) (r/hCRF) increased cAMP accumulation in COS-7 cells transfected with mCRF-R, hCRF-RI, and hCRF-RII cDNA plasmid (10 micrograms each) from basal values of 8-19 pmol/10(5) cells.15 min to 84 +/- 10, 87 +/- 16, and 45 +/- 16 pmol/10(5) cells.15 min, respectively. The EC50 values of r/hCRF-stimulated cAMP accumulation in COS-7 cells expressing mCRF-R and hCRF-RI cDNAs were similar at 0.4 +/- 0.2 and 0.7 +/- 0.2 nM, respectively. Conversely, the EC50 of r/hCRF-stimulated cAMP accumulation in hCRF-RII-transfected COS-7 cells was 47.5 +/- 18.9 nM. As the level of expression of hCRF-RII was lower than that of hCRF-RI, we compared r/hCRF-stimulated cAMP accumulation in COS-7 cells expressing low and high levels of hCRF-RI. The EC50 for r/hCRF-stimulated cAMP accumulation in COS-7 cells transfected with hCRF-RI did not change when receptor expression was varied by a factor of 1- to 8.4-fold. In contrast, the EC50 for r/hCRF-stimulated cAMP accumulation mediated by hCRF-RII was at least 100-fold higher than that mediated by the hCRF-RI in COS-7 cells, which suggests poor coupling between hCRF-RII and adenylate cyclase. Inositol phosphate (IP) levels were also determined in mCRF-R, hCRF-RI, and hCRF-RII cDNA-transfected COS-7 cells stimulated with increasing concentrations of r/hCRF. r/hCRF-stimulated IPs accumulation was dose dependent in COS-7 cells expressing mCRF-R and hCRF-RI using 100 and 1000 nM r/hCRF. Concentrations of 10 (or less) nM r/hCRF had no effect on IP generation. hCRF-RII did not mediate stimulation of IP even at 1000 nM r/hCRF.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

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.

PTH elicits multiple second messenger signals in target cells. This signaling diversity may reflect coupling of a single species of PTH receptors to multiple effectors, the action of different subtypes of PTH receptors, or both. We recently reported the expression cloning, from rat and opossum cells, of closely related cDNAs encoding receptors for PTH [and PTH-related peptide (PTHRP)]. To determine if these cloned PTH/PTHRP receptors can activate multiple intracellular effectors when present at near-physiological levels in intact target cells, we have stably expressed the rat and opossum PTH/PTHRP receptor cDNAs in LLC-PK1 porcine renal epithelial cells. These cells lack endogenous PTH/PTHRP receptors, but do express abundant calcitonin receptors and many features of a proximal tubular phenotype. Subclones of transfected LLC-PK1 cells exhibited high affinity binding (Kd, 1-5 nM) of [Nle8.18,Tyr34]bovine PTH-(1-34)amide (PTH) and dose-dependent activation by PTH of both cAMP accumulation (EC50, 1 nM) and increased release of cytosolic free calcium from intracellular stores (EC50,or = 20-50 nM) across a wide range of receptor expression. Expressed rat and opossum receptors exhibited similar properties, except for a 5-fold lower binding affinity of the rat receptor for PTH-(7-34). Stimulation by PTH of both cAMP accumulation and elevated cytosolic free calcium was augmented in cells expressing higher numbers of PTH/PTHRP receptors. Like calcitonin, PTH (1-100 nM) reduced the rate of cell proliferation and augmented the rate of inorganic phosphate transport after 24 and 5 h of preincubation, respectively. The growth effect was mimicked by cAMP analogs, forskolin, phorbol esters, and calcium ionophores. Regulation of phosphate transport, however, was mimicked by phorbols, but not by cAMP analogs or forskolin. We conclude that LLC-PK1 cells provide a useful model in which to study the function of cloned PTH/PTHRP receptors. In these cells, a single species of cloned PTH/PTHRP receptors, stably expressed at near-physiological numbers, activates multiple second messenger responses and regulates subsequent biological responses, including at least one (phosphate transport) that is mediated by mechanisms independent of cAMP.

Identical complementary DNAs (cDNAs) that encode a 593-amino acid human PTH (PTH)/PTH-related peptide (PTHrP) receptor were isolated by hybridization techniques from two cDNA libraries which had been constructed from human kidney and human osteoblast-like osteosarcoma cells (SaOS-2). Northern blot analysis of total RNA from human bone- and kidney-derived tissue revealed one single major messenger RNA species of about 2.5 kilobases in both tissues. The human PTH/PTHrP receptor has 91% and 81% identity, respectively, with the previously cloned rat and opossum receptors, indicating a high degree of conservation among mammals. Despite this striking degree of amino-acid conservation, the human PTH/PTHrP receptor has several unique biological properties when transiently expressed in COS-7 cells. The apparent dissociation constants for [Nle8,18,Tyr34] bovine PTH(1-34) amide [bPTH(1-34)] are similar for the human and the rat receptor (approximately 8 vs. approximately 15 nM) whereas [Tyr36]PTHrP(1-36) amide has a slightly lower affinity for the human (15-40 nM) than for the rat receptor (approximately 15 nM). Both ligands stimulate efficiently and with similar efficacy the accumulation of intracellular cAMP. The affinities for the antagonists [Nle8,18,Tyr34] bPTH(3.34) amide [bPTH(3-34)] and in particular for [Nle8,18,Tyr34] bPTH(7-34) amide [bPTH(7-34)] are considerably higher for the human receptor, e.g. approximately 8 nM vs. 30 nM for bPTH(3-34) and approximately 100 nM vs. 5000 nM for bPTH(7-34), respectively. Similar biological findings were previously attributed to differences in species- and/or organ-specific PTH/PTHrP receptors. The expression of the recombinant, highly homologous rat and human receptors in a uniform environment indicate that the moderate differences in the primary receptor structure have profound consequences for the receptor binding affinity of amino-terminally truncated PTH analogs. Furthermore, the molecular cloning of identical cDNAs encoding a human PTH/PTHrP receptor from the two major target organs for PTH, bone and kidney, provides strong evidence for one single PTH/PTHrP receptor in both organs, although additional and/or alternatively spliced receptors cannot be excluded.

The involvement of protein kinase C in normal corticotroph function was studied by analysis of the effects of the phorbol ester derivative phorbol 12-myristate-13-acetate (PMA) and the synthetic diacylglycerol dioctanoylglycerol (DOG) on basal and stimulated ACTH release in cultured rat anterior pituitary cells. Incubation of rat pituitary cells with increasing concentrations of PMA or DOG caused dose-related increases in ACTH release up to 13.4 +/- 2.1- and 10.1 +/- 0.9-fold, respectively, similar to that caused by CRF (9.8 +/- 1.6-fold). Also, stimulation of endogenous diglyceride formation by phospholipase C (100 mU/ml) stimulated ACTH release by 2.5 +/- 0.1-fold. In cells incubated with maximum stimulatory concentrations of CRF (10 nM) or 8-bromo-cAMP (8-Br-cAMP; 5 mM), addition of either 100 microM DOG or 100 nM PMA caused significantly higher ACTH responses than those obtained with CRF, 8-Br-cAMP, DOG, or PMA alone. 8-Br-cAMP (5 mM) and 10 nM CRF significantly increased the effect of 100 nM PMA by 1.4 +/- 0.2- and 1.5 +/- 0.1-fold, respectively. Combinations of 10 nM CRF with either vasopressin (VP) or angiotensin II (AII) increased ACTH secretion to values higher than those produced by CRF, VP, or AII alone. However, addition of maximal stimulatory concentrations of VP or AII (10 nM) did not further increase the effects of either PMA alone or PMA/CRF combinations, indicating that their mechanisms of action may be similar to that of PMA. These results indicate that in addition to the established cAMP-dependent mechanism, stimulation of ACTH release in normal pituitary cells may be elicited by activation of protein kinase C. The evidence also suggests that protein kinase C is involved during stimulation of ACTH release by the cAMP-independent regulators VP and AII and in the synergistic effects of VP and AII with CRF on the corticotroph.