Gs/Gi Regulation of Bone Cell Differentiation: Review and Insights from Engineered Receptors.

G-protein coupled receptors (GPCRs) and their ligands are critical for normal osteoblast formation and function. GPCRs mediate a wide variety of biological processes and are activated by multiple types of extracellular signals, ranging from photons to small molecules to peptides. GPCRs signal through a select number of canonical pathways: the Gs and Gi pathways increase or decrease intracellular cAMP levels, respectively, by acting on adenylate cyclase, while the Gq pathway increases intracellular calcium by activating phospholipase C. In addition, non-canonical GPCR pathways such as ß-arrestin activation are important for osteoblast function. Since many cells express multiple GPCRs, and each individual GPCR may activate multiple signaling pathways, the resulting combinatorial signal provides a mechanism for regulating complex biological processes and effector functions. However, the wide variety of GPCRs, the possibility of multiple receptors acting with signaling redundancy, and the possibility of an individual GPCR activating multiple signaling pathways, also pose challenges for elucidating the role of a particular GPCR. Here, we briefly review the roles of Gs and Gi GPCR signaling in osteoblast function. We describe the successful application of a strategy for directly manipulating the Gs and Gi pathways using engineered receptors. These powerful tools will allow further elucidation of the roles of GPCR signaling in specific lineages of osteoblastic cells, as well as in non-osteoblast cells, all of which remain critical areas of active research.

Mineral composition is altered by osteoblast expression of an engineered G(s)-coupled receptor.

Activation of the G(s) G protein-coupled receptor Rs1 in osteoblasts increases bone mineral density by 5- to 15-fold in mice and recapitulates histologic aspects of fibrous dysplasia of the bone. However, the effects of constitutive G(s) signaling on bone tissue quality are not known. The goal of this study was to determine bone tissue quality in mice resulting from osteoblast-specific constitutive G(s) activation, by the complementary techniques of FTIR spectroscopy and synchrotron radiation micro-computed tomography (SRµCT). Col1(2.3)-tTA/TetO-Rs1 double transgenic (DT) mice, which showed osteoblast-specific constitutive G(s) signaling activity by the Rs1 receptor, were created. Femora and calvariae of DT and wild-type (WT) mice (6 and 15 weeks old) were analyzed by FTIR spectroscopy. WT and DT femora (3 and 9 weeks old) were imaged by SRµCT. Mineral-to-matrix ratio was 25% lower (P = 0.010), carbonate-to-phosphate ratio was 20% higher (P = 0.025), crystallinity was 4% lower (P = 0.004), and cross-link ratio was 11% lower (P = 0.025) in 6-week DT bone. Differences persisted in 15-week animals. Quantitative SRµCT analysis revealed substantial differences in mean values and heterogeneity of tissue mineral density (TMD). TMD values were 1,156 ± 100 and 711 ± 251 mg/cm(3) (mean ± SD) in WT and DT femoral diaphyses, respectively, at 3 weeks. Similar differences were found in 9-week animals. These results demonstrate that continuous G(s) activation in murine osteoblasts leads to deposition of immature bone tissue with reduced mineralization. Our findings suggest that bone tissue quality may be an important contributor to increased fracture risk in fibrous dysplasia patients.

Conditional expression of a Gi-coupled receptor in osteoblasts results in trabecular osteopenia.

G protein-coupled receptors (GPCRs) coupled to activation of Gs, such as the PTH1 receptor (PTH1R), have long been known to regulate skeletal function and homeostasis. However, the role of GPCRs coupled to other G proteins such as Gi is not well established. We used the tet-off system to regulate the expression of an activated Gi-coupled GPCR (Ro1) in osteoblasts in vivo. Skeletal phenotypes were assessed in mice expressing Ro1 from conception, from late stages of embryogenesis, and after weaning. Long bones were assessed histologically and by microcomputed tomography. Expression of Ro1 from conception resulted in neonatal lethality that was associated with reduced bone mineralization. Expression of Ro1 starting at late embryogenesis resulted in a severe trabecular bone deficit at 12 wk of age (>51% reduction in trabecular bone volume fraction in the proximal tibia compared with sex-matched control littermates; n = 11; P < 0.01). Ro1 expression for 8 wk beginning at 4 wk of age resulted in a more than 20% reduction in trabecular bone volume fraction compared with sex-matched control littermates (n = 16; P < 0.01). Bone histomorphometry revealed that Ro1 expression is associated with reduced rates of bone formation and mineral apposition without a significant change in osteoblast or osteoclast surface. Our results indicate that signaling by a Gi-coupled GPCR in osteoblasts leads to osteopenia resulting from a reduction in trabecular bone formation. The severity of the phenotype is related to the timing and duration of Ro1 expression during growth and development. The skeletal phenotype in Ro1 mice bears some similarity to that produced by knockout of Gs-alpha expression in osteoblasts and thus may be due at least in part to Gi-mediated inhibition of adenylyl cyclase.

Canine renal receptors for parathyroid hormone. Down-regulation in vivo by exogenous parathyroid hormone.

Chronic elevation of circulating parathyroid hormone (PTH) is associated with decreased target cell responsiveness to PTH. To study the subcellular mechanism of this phenomenon we evaluated PTH receptors and adenylate cyclase activity in renal cortical membranes prepared before and after infusion of bovine parathyroid gland extract (PTE) into thyroparathyroidectomized dogs. PTE infusion resulted in a 53% decrease in the number of high-affinity receptors (P less than 0.01) associated with a 66% decrease in PTH-stimulated adenylate cyclase (P less than 0.01) relative to paired base-line values. Both the equilibrium constant of dissociation (KD) for PTH binding and the concentration of PTH that caused half-maximal stimulation of adenylate cyclase were in the range of 1 to 4 nM, and were unaffected by the PTE infusion. Responsiveness of the renal adenylate cyclase to sodium fluoride was 88% of base-line values. Infusion of the PTE vehicle alone did not affect PTH receptor number or blunt the adenylate cyclase response to PTH. Pretreatment of the membranes made after PTE infusion with guanosine triphosphate (GTP), which is known to produce dissociation of receptor-bound PTH, failed to restore either receptor number or PTH-stimulated adenylate cyclase. This finding was not due to a lack of efficacy of the GTP pretreatment, because identical GTP pretreatment restored PTH binding to base-line values in membranes partially occupied by incubation with PTH in vitro. Thus, simple residual occupancy of PTH receptors by the infused hormone did not appear to account for the observed receptor loss. The results of this study suggest that target cell resistance to PTH in patients with hyperparathyroidism might occur, at least in part, due to down-regulation of PTH receptors by circulating hormone.

Human renal carcinoma cells produce hypercalcemia in the nude mouse and a novel protein recognized by parathyroid hormone receptors.

When grown in nude mice, cultured renal carcinoma cells from a hypercalcemic patient produced marked hypercalcemia that was reversed by resection of tumor. Conditioned medium from this cell line contained a protein with activity in a renal adenylate cyclase bioassay for parathyroid hormone (PTH) which was blocked by the competitive PTH antagonist [8norleucyl, 18norleucyl, 34tyrosinyl]bPTH (3-34)amide. However, the biologically active protein was eluted from gel filtration columns as a larger molecular size component that PTH and was not recognized by any of four region-specific PTH antisera. The properties of this factor resemble those of the postulated PTH-like substance(s) in humoral hypercalcemia of malignancy.

Structural requirements for parathyroid hormone action in mature bone. Effects on release of cyclic adenosine monophosphate and bone gamma-carboxyglutamic acid-containing protein from perfused rat hindquarters.

To determine the structural requirements for parathyroid hormone (PTH) activity in mature bone, we perfused the surgically isolated hindquarters of adult male rats with either native bovine PTH-(1-84) [bPTH-(1-84)] or the synthetic amino-terminal fragment, bovine PTH-(1-34) [bPTH-(1-34)]. Changes in the release of cyclic AMP (cAMP) and bone Gla protein (BGP) were monitored as evidence of bone-specific response to PTH; tissue specificity of the cAMP response was confirmed through in vitro examination on nonskeletal tissue response to PTH. Biologically active, monoiodinated 125I-bPTH-(1-84) was administered to determine if mature murine bone cleaves native hormone. We found that perfused rat bone continuously releases BGP, and that both bPTH-(1-84) and bPTH-(1-34) acutely suppress this release. In addition, both hormones stimulate cAMP release from perfused rat hindquarters. When examined on a molar basis, the magnitude of the cAMP response was dose-dependent and similar for both hormones, with doses yielding half-maximal cAMP responses. The response for bPTH-(1-34) was 0.5 nmol and for bPTH-(1-84) was 0.7 nmol. Moreover, biologically active 125I-bPTH-(1-84) was not metabolized in our hindquarter perfusion system. These findings indicate that PTH-(1-84) does not require extraskeletal or skeletal cleavage to an amino-terminal fragment in order to stimulate cAMP generation in, or suppress BGP release from, mature rat bone.

Human transforming growth factor-alpha stimulates bone resorption in vitro.

Tumor-derived transforming growth factors (TGF) have been proposed as possible mediators of hypercalcemia in malignancy. We have studied the action of recombinant human TGF-alpha in cultured bone cells and in bone explant cultures. In clonal UMR-106 rat osteosarcoma cells, TGF-alpha and epidermal growth factor (EGF) were equipotent in binding to the EGF receptor. TGF-alpha and EGF both stimulated resorption of neonatal mouse calvaria, and maximal responses were obtained with 10 ng/ml of TGF-alpha after 72 h in culture. The effects of both TGF-alpha and EGF in calvaria, but not those of parathyroid hormone, were inhibited by 5 X 10(-7) M indomethacin. Fetal rat limb bone cultures were less sensitive to TGF-alpha than neonatal mouse calvaria, with a concentration of 30 ng/ml being required to stimulate resorption in this system. The bone-resorbing activity of TGF-alpha in fetal rat bones was inhibited by 10 ng/ml calcitonin but not by 5 X 10(-7) M indomethacin. EGF at concentrations up to 300 ng/ml did not stimulate resorption of the limb bones at time periods up to 66 h. The results indicate that human TGF-alpha is a potent bone-resorbing agent, and support the concept that this growth factor exhibits some effects distinct from those of EGF. TGF-alpha could play an etiologic role in the hypercalcemia of malignancy.

Parathyroid hormonelike protein from human renal carcinoma cells. Structural and functional homology with parathyroid hormone.

A variety of solid tumors secrete proteins that are immunochemically distinct from parathyroid hormone (PTH) but activate PTH-responsive adenylate cyclase. Such PTH-like proteins have been proposed as mediators of the hypercalcemia and hypophosphatemia frequently associated with malignancies. We purified to apparent homogeneity a PTH-like protein with a molecular weight of 6,000, that is produced by human renal carcinoma cells. The amino-terminal sequence of the PTH-like protein and that of human PTH were found to display at least five identities in the first 13 positions. The purified protein bound to PTH receptors, activated adenylate cyclase in renal plasma membranes, and stimulated cAMP formation in rat osteosarcoma cells. The PTH-like protein reproduced two additional effects of PTH, stimulation of bone resorption in fetal rat limb bone cultures and inhibition of phosphate uptake in cultured opossum kidney cells. These properties are consistent with a role for PTH-like proteins as mediators of the syndrome of malignancy-associated hypercalcemia.

Activation of the parathyroid hormone receptor-adenylate cyclase system in osteosarcoma cells by a human renal carcinoma factor.

Human renal carcinoma cell line 786-0 elaborates a protein that is structurally and immunochemically distinct from parathyroid hormone (PTH) and that activates renal cortical adenylate cyclase via an interaction with the PTH receptor. Because of the high frequency of excessive bone resorption and resultant hypercalcemia in patients with malignant disease we evaluated the ability of this 786-0 cell factor to reproduce PTH action in bone-derived cells. The 786-0 factor as well as bovine PTH (BPTH) (1-34) and prostaglandin E1 produced marked increases in cyclic adenosine 3':5'-monophosphate (cAMP) accumulation in the clonal rat osteosarcoma cell line UMR-106. A competitive antagonist of PTH action, [norleucine8, norleucine18, tyrosine34] BPTH(3-34)amide, blocked the cAMP stimulation produced by 786-0 factor and BPTH(1-34) but not that produced by prostaglandin E1. In the presence of forskolin (0.1 microM) UMR-106 cells were extremely sensitive to 786-0 factor, showing significant increases in cAMP production at a concentration 10-fold less than that required to activate adenylate cyclase in renal membranes. In contrast UMR-106 cells were less sensitive to BPTH(1-34) than were renal membranes. This preferential increase in sensitivity to 786-0 factor was not seen in membranes prepared from UMR-106 cells suggesting the importance of cytosolic components. Six additional human genitourinary carcinoma cell lines were found to produce factors that increased cAMP levels in UMR-106 cells. We conclude that 786-0 factor is a potent activator of the PTH receptor-adenylate cyclase system in these bone-derived cells. These findings are consistent with the view that cancer-associated hypercalcemia may frequently be attributable to tumor secretion of proteins (such as 786-0 factor) that are distinct from PTH but are capable of activating skeletal PTH receptors.

Oxytocin receptors coupled to uterine contraction in estrogen-dominated rabbits.

The present study investigated whether specific [3H]oxytocin binding sites previously demonstrated in estrogen-dominated rabbit uterus have properties expected of physiologic receptors coupled to uterine contraction. Microsomal membranes from estrogen-dominated rabbit uterus were found to contain high-affinity specific oxytocin binding sites with Kd = 2-3 nM. These sites were predominantly myometrial in locus. Specific oxytocin binding exhibited a pH optimum between 7.5 and 8.0. Mg2+ or Mn2+ was necessary for maximal specific [3H]oxytocin binding; in contrast, Ca2+ at submillimolar concentrations inhibited specific binding. Oxytocin binding sites were not detectable in microsomal membranes isolated from progesterone-dominated rabbit uterus. Relative binding and uterotonic activities of 10 synthetic neurohypophyseal hormone analogues were determined in estrogen-dominated rabbit uterus. A qualitative correlation was observed between binding and uterotonic responses. Angiotensin II and insulin did not compete with [3H]oxytocin for uterine binding sites. It is concluded that the specific high affinity [3H]oxytocin binding sites demonstrated in estrogen-dominated rabbit uterus have the selectivity for neurohypophyseal hormone analogues expected for physiologic receptors coupled to uterine contraction.

Mutations of neighboring polar residues on the second transmembrane helix disrupt signaling by the parathyroid hormone receptor.

Site-directed mutagenesis was used to assess the role of transmembrane (TM)-charged amino acids in the expression and function of the G protein-coupled receptor for PTH and PTH-related protein (PTHrP). Charged residues that are conserved in the TM regions of most or all members of the PTH/secretin receptor subfamily were targeted. Four mutants (E296A, R337A, H414A, and E459K) displayed properties similar to the wild type PTH/PTHrP receptor with respect to agonist binding and stimulation of adenylyl cyclase when expressed in COS-7 cells. Several mutations, all in TM II, produced receptors that signaled extremely poorly. Mutation of three residues (227S, 230R, and 233S), predicted to be aligned on one helical face of TM II, displayed a similar phenotype: markedly blunted adenylyl cyclase activity in response to PTH (20-30% of the wild type response) and a lower binding affinity for agonist, with no reduction in cell surface receptor expression. These results suggest that TM II contains a polar face that is involved in TM signaling by the PTH/PTHrP receptor. Two of these mutations were made at the corresponding sites in the secretin receptor, and a similar reduction in secretin-stimulated adenylyl cyclase activity was observed. Thus this region of TM II may participate in a mechanism of TM signal transduction that is shared by the PTH/secretin sub-family of G protein-coupled receptors.

Analysis of parathyroid hormone (PTH)/secretin receptor chimeras differentiates the role of functional domains in the pth/ pth-related peptide (PTHrP) receptor on hormone binding and receptor activation.

The type 1 parathyroid hormore receptor (PTH1r) belongs to the class II family of G protein-coupled receptors. To delineate the sites in the PTH1r's N-terminal region, and the carboxy-core domain (transmembrane segments + extracellular loops) involved in PTH binding, we have evaluated the functional properties of 27 PTH1-secretin chimeras receptors stably expressed in HEK-293 cells. The wild type and chimeric receptors were analyzed for cell surface expression, binding for PTH and secretin, and functional responsiveness (cAMP induction) toward secretin and PTH. The expression levels of the chimeric receptors were comparable to that of the PTH1r (60-100%). The N-terminal region of PTH1r was divided into three segments that were replaced either singly or in various combinations with the homologous region of the secretin receptor (SECr). Substitution of the carboxy-terminal half (residues 105-186) of the N-terminal region of PTH1r for a SECr homologous segment did not reduced affinity for PTH but abolished signaling in response to PTH. This data indicate that receptor activation is dissociable from high affinity hormone binding in the PTH1r, and that the N-terminal region might play a critical role in the activation process. Further segment replacements in the N-termini focus on residues 105-186 and particularly residues 146-186 of PTH1r as providing critical segments for receptor activation. The data obtained suggest the existence of two distinct PTH binding sites in the PTH1r's N-terminal region: one site in the amino-terminal half (residues 1-62) (site 1) that participates in high-affinity PTH binding; and a second site of lower affinity constituted by amino acid residues scattered throughout the carboxy-terminal half (residues 105-186) (site 2). In the absence of PTH binding to site 1, higher concentrations of hormone are required to promote receptor activation. In addition, elimination of the interaction of PTH with site 2 results in a loss of signal transduction without loss of high-affinity PTH binding. Divers substitutions of the extracellular loops of the PTH1r highlight the differential role of the first- and third extracellular loop in the process of PTH1r activation after hormone binding. A chimera containing the entire extracellular domains of the PTH1r and the transmembrane + cytoplasmic domains of SECr had very low PTH binding affinity and did not signal in response to PTH. Further substitution of helix 5 of PTH1r in this chimera increased affinity for PTH that is close to the PTH affinity for the wild-type PTH1r but surprisingly, did not mediate signaling response. Additional substitutions of PTH1r's helices in various combinations emphasize the fundamental role of helix 3 and helix 6 on the activation process of the PTH1r. Overall, our studies demonstrated that several PTH1r domains contribute differentially to PTH binding affinity and signal transduction mechanism and highlight the role of the N-terminal domain and helix 3 and helix 6 on receptor activation.

Apoptosis mediated by activation of the G protein-coupled receptor for parathyroid hormone (PTH)/PTH-related protein (PTHrP).

The present studies were carried out to evaluate the mechanisms by which PTH/PTHrP receptor (PTHR) activation influences cell viability. In 293 cells expressing recombinant PTHRs, PTH treatment markedly reduced the number of viable cells. This effect was associated with a marked apoptotic response including DNA fragmentation and the appearance of apoptotic nuclei. Similar effects were evidenced in response to serum withdrawal or to the addition of tumor necrosis factor (TNFalpha). Addition of caspase inhibitors or overexpression of bcl-2 partially abrogated apoptosis induced by serum withdrawal. Caspase inhibitors also protected cells from PTH-induced apoptosis, but overexpression of bcl-2 did not. The effects of PTH on cell number and apoptosis were neither mimicked by activators of the cAMP pathway (forskolin, isoproterenol) nor blocked by an inhibitor (H-89). However, elevation of Ca(i)2+ by addition of thapsigargin induced rapid apoptosis, and suppression of Ca(i)2+ by overexpression of the calcium- binding protein, calbindin D28k, inhibited PTH-induced apoptosis. The protein kinase C inhibitor GF 109203X partially inhibited PTH-induced apoptosis. Regulator of G protein signaling 4 (RGS4) (an inhibitor of the activity of the alpha-subunit of Gq) suppressed apoptotic signaling by the PTHR, whereas the C-terminal fragment of GRK2 (an inhibitor of the activity of the beta(gamma)-subunits of G proteins) was without effect. Chemical mutagenesis allowed selection of a series of 293 cell lines resistant to the apoptotic actions of PTH; a subset of these were also resistant to TNFalpha. These results suggest that 1) apoptosis produced by PTHR and TNF receptor signaling involve converging pathways; and 2) Gq-mediated phospholipase C/Ca2+ signaling, rather than Gs-mediated cAMP signaling, is required for the apoptotic effects of PTHR activation.

Identification of phosphorylation sites in the G protein-coupled receptor for parathyroid hormone. Receptor phosphorylation is not required for agonist-induced internalization.

In some G protein-coupled receptors (GPCRs), agonist-dependent phosphorylation by specific GPCR kinases (GRKs) is an important mediator of receptor desensitization and endocytosis. Phosphorylation and the subsequent events that it triggers, such as arrestin binding, have been suggested to be regulatory mechanisms for a wide variety of GPCRs. In the present study, we investigated whether agonist-induced phosphorylation of the PTH receptor, a class II GPCR, also regulates receptor internalization. Upon agonist stimulation, the PTH receptor was exclusively phosphorylated on serine residues. Phosphoamino acid analysis of a number of receptor mutants in which individual serine residues had been replaced by threonine identified serine residues in positions 485, 486, and 489 of the cytoplasmic tail as sites of phosphorylation after agonist treatment. When serine residues at positions 483, 485, 486, 489, 495, and 498 were simultaneously replaced by alanine residues, the PTH receptor was no longer phosphorylated either basally or in response to PTH. The substitution of these serine residues by alanine affected neither the number of receptors expressed on the cell surface nor the ability of the receptor to signal via Gs. Overexpression of GRK2, but not GRK3, enhanced PTH-stimulated receptor phosphorylation, and this phosphorylation was abolished by alanine mutagenesis of residues 483, 485, 486, 489, 495, and 498. Thus, phosphorylation of the PTH receptor by the endogenous kinase in HEK-293 cells occurs on the same residues targeted by overexpressed GRK2. Strikingly, the rate and extent of PTH-stimulated internalization of mutated PTH receptors lacking phosphorylation sites were identical to that observed for the wild-type PTH receptor. Moreover, overexpressed GRK2, while enhancing the phosphorylation of the wild-type PTH receptor, had no affect on the rate or extent of receptor internalization in response to PTH. Thus, the agonist-occupied PTH receptor is phosphorylated by a kinase similar or identical to GRK2 in HEK-293 cells, but this phosphorylation is not requisite for efficient receptor endocytosis.

Constitutive activation of the cyclic adenosine 3',5'-monophosphate signaling pathway by parathyroid hormone (PTH)/PTH-related peptide receptors mutated at the two loci for Jansen's metaphyseal chondrodysplasia.

Two different activating PTH/PTH-related peptide (PTHrP) receptor mutations, H223R and T410P, were recently identified as the most likely cause of Jansen's metaphyseal chondrodysplasia. To assess the functional importance of either amino acid position in the human PTH/PTHrP receptor, H223 and T410 were individually replaced by all other amino acids. At position 223, only arginine and lysine led to agonist-independent cAMP accumulation; all other amino acid substitutions resulted in receptor mutants that lacked constitutive activity or were uninformative due to poor cell surface expression. In contrast, most amino acid substitutions at position 410 conferred constitutive cAMP accumulation and affected PTH/PTHrP receptor expression not at all or only mildly. Mutations corresponding to the H223R or T410P exchange in the human PTH/PTHrP receptor also led to constitutive activity when introduced into the opossum receptor homolog, but showed little or no change in basal cAMP accumulation when introduced into the rat PTH/PTHrP receptor. The PTH/PTHrP receptor residues mutated in Jansen's disease are conserved in all mammalian members of this family of G protein-coupled receptors. However, when the equivalent of either the H223R or the T410P mutation was introduced into several other related receptors, including the PTH2 receptor and the receptors for calcitonin, secretin, GH-releasing hormone, glucagon-like peptide I, and CRH, the resulting mutants failed to induce constitutive activity. These studies suggest that two residues in the human PTH/PTHrP receptor, 223 and 410, have critical roles in signal transduction, but with different sequence constrains.

Expression of a parathyroid hormone-like protein and its receptor during differentiation of embryonal carcinoma cells.

Differentiation of mouse embryonal carcinoma cells to the parietal endoderm phenotype is associated with expression of PTH-responsive adenylate cyclase. A PTH-like protein (PLP), which binds to PTH receptors and activates adenylate cyclase in classical PTH target cells was recently isolated and cloned. We assessed whether the parietal endoderm phenotype is associated with the expression of PLP or its receptor. A 1.4-kilobase PLP transcript was detected in the mouse parietal endoderm cell line PYS-2. No hybridizing transcripts were evident in undifferentiated mouse embryonal carcinoma cells PSA-1 or F9. However, differentiation of these cells to parietal endoderm, either spontaneously (PSA-1) or by treatment with retinoic acid and dibutyryl cAMP (F9), resulted in expression of the 1.4-kilobase PLP message. Undifferentiated F9 cells displayed negligible specific binding of [125I]PLP-(1-34)amide. When F9 cells were induced to differentiate to parietal endoderm, specific binding sites for [125I]PLP-(1-34)amide were expressed in parallel with PLP-responsive adenylate cyclase. These receptors, like those in classical PTH target tissues, displayed identical affinity (Kd = 5.2 nM) for bPTH-(1-34) and hPLP-(1-34)amide; with binding capacity (Bmax) of 6.6 x 10(4) sites/cell. In the presence of retinoic acid, exogenous PLP substituted for dibutyryl cAMP in a concentration-dependent fashion in promoting the differentiation of F9 cells to parietal endoderm. Thus, both PLP mRNA and PLP receptors coupled to adenylate cyclase are expressed during the differentiation of mouse embryonal carcinoma cells. Increased cAMP levels produced by autocrine stimulation of PLP receptors by PLP may contribute to differentiation of embryonal carcinoma cells into parietal endoderm.

Mutational analysis of the cytoplasmic tail of the G protein-coupled receptor for parathyroid hormone (PTH) and PTH-related protein: effects on receptor expression and signaling.

The present studies were undertaken to examine the role of the cytoplasmic tail of the G protein-coupled receptor for PTH and PTH-related protein (PTHrP) on receptor signaling and expression. The wild type (WT) receptor (585 amino acids) and five truncated receptors whose cytoplasmic tails terminated at residues 507, 494, 474, 466, and 458 were expressed in COS-7 cells. Based on [125I]PTHrP binding, mutants T507, T494, and T466 displayed progressively decreased levels of expression, compared with WT. The tailless mutant T458 was not expressed in a functional form, whereas T474 was expressed at a level similar to WT. Comparable results were obtained when expression levels of WT and mutated PTH/PTHrP receptors were evaluated by Western blotting. Binding affinities were similar for all mutated receptors (IC50 = 1-2 nM). Immunocytochemistry showed that WT and mutated receptors were diffusely distributed, presumably at the cell surface, except for the tailless mutant T458, which displayed striking perinuclear localization. T458 did not display an adenylyl cyclase response to PTH, while the other mutants were similar to WT both with respect to their maximal adenylyl cyclase responses to PTH and to their EC50 values. Cai2+ signaling properties of these mutants were assessed as PTH-stimulated 45Ca efflux from Xenopus oocytes that had been injected with in vitro transcribed PTH/PTHrP receptor cRNAs. The WT and mutated receptors (except for T458) responded to PTH with significant (6- to 27-fold) increases in 45Ca efflux.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of alternatively spliced isoforms of the parathyroid hormone (PTH)/PTH-related peptide receptor messenger RNA in human kidney and bone cells.

Using a PCR-based strategy, two variants of the PTH/PTH-related peptide (PTH-rp) receptor mRNA were identified in human kidney, SaOS-2 human osteoblast cells, and rat bone that are produced by alternative splicing of exons coding for the N-terminal portion of the receptor. In the S-N3-E2 isoform, the exon coding the signal peptide (S) is spliced to an alternative 3'-acceptor site, producing a product respecting the reading frame, but in which the E1 exon is replaced by 12 amino acids derived from the N3 intron. In the S-E2 isoform, in which the E1 exon is deleted by cassette exclusion, the reading frame is changed, but a truncated receptor may be produced by reinitiation of translation at an overlapping stop/start codon. After transfection of COS and Chinese hamster ovary cells with the originally described S-E1-E2 isoform and the two splice variants, active transcription of PTH/PTH-rp receptor mRNA was detected by RT-PCR in all cases. Cell lines transfected with the S-E1-E2 and S-N3-E2 isoforms displayed a 15- to 25-fold and 2- to 3-fold increase, respectively, in cAMP content after stimulation with 2.4 x 10(-7) M human PTH(1-34), whereas cells transfected with the S-E2 isoform did not respond. PTH elicited an increase in intracellular calcium only in cells transfected with the S-E1-E2 isoform. Studies evaluating the surface expression of receptors using anti-human PTH/PTH-rp receptor antibodies and the ability of transfected cells to bind [125I]PTH-rp indicated that the low or absent responses to PTH stimulation resulted, at least in part, from low surface expression of the S-N3-E2 and S-E2 isoforms. These studies support the conclusion that exon E1 is extremely important in promoting surface expression of the PTH/PTH-rp receptor but indicate that isoforms lacking this exon can retain the ability to recognize PTH. The possible intracellular expression of these splice variants, which account for 15-20% of total PTH/PTH-rp receptor mRNA, needs to be evaluated.

G protein-dependent activation of a phosphoinositide-specific phospholipase C in UMR-106 osteosarcoma cell membranes.

Recent evidence suggests that guanyl nucleotide binding (G) proteins are involved in receptor-mediated bone resorption and in osteoblastic function, but the nature of the G protein coupled to effectors that are involved in these skeletal effects is unknown. The purposes of this study were to determine (1) whether a G protein mediates activation of phosphoinositide-specific phospholipase C in UMR-106 rat osteosarcoma cells, and (2) whether parathyroid hormone (PTH) and a PTH-like protein (PLP) associated with humoral hypercalcemia of malignancy promote GTP-dependent PIP2 hydrolysis. Addition of GTP (10(-4) M) or guanosine 5'-0-(3-thiotriphosphate, GTP gamma S, 10(-5) M) to membranes prepared from UMR-106 cells labeled with [3H]myo-inositol increased both [3H]inositol trisphosphate (IP3) and [3H]inositol bisphosphate (IP2) formation. The increases in [3H]IP2 and [3H]IP3 produced by GTP were 8.6- and 4.3-fold, respectively. GTP gamma S produced a 17.6- and 11.9-fold increase in [3H]IP2 and [3H]IP3, respectively. The stimulatory effects of GTP and GTP gamma S were dose dependent (GTP ED50 = 3.9 x 10(-6) M; GTP gamma S ED50 = 2.5 x 10(-7) M) and progressive over 10 minutes and required the presence of Mg2+.GTP (10(-4) M) and GTP gamma S (10(-5) M) decreased membrane [3H]phosphoinositides concomitantly with increased [3H]IP2 and [3H]IP3. The GDP analog guanosine 5'-O-(2-thiodiphosphate, GDP beta S) alone did not alter [3H]IP2 or [3H]IP3 production but at 10(-4) M blocks the stimulatory effects of GTP and GTP gamma S. NaF (3 x 10(-2)M) produced a 2.8- and 2.0-fold stimulation of [3H]IP2 and [3H]IP3, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Parathyroid hormone receptors in human dermal fibroblasts: structural and functional characterization.

Previous studies have established the presence of parathyroid hormone (PTH)-sensitive adenylate cyclase activity in cultured human skin fibroblasts. The present study was undertaken to identify and quantitate PTH receptors directly in such cells. Human dermal fibroblast cell line CRL 1564 was found to possess specific binding sites for [125I]PTH(1-34). These sites bound PTH selectively; bovine and human PTH(1-34) and PTH(1-84) competed for [125I]PTH(1-34) binding sites, whereas the unrelated peptides calcitonin, insulin, AVP, angiotensin II, and ACTH(1-24) were inactive even at micromolar concentrations. Competitive binding experiments demonstrated the presence of binding site heterogeneity. These data fit a "two-site" model (p less than 0.001) in which one binding component has high affinity (Kd = 2.5 ng/ml = 0.6 nM) and low capacity (10(4) sites/cell) while the other has low affinity (Kd = 5.9 micrograms/ml = 1.5 microM) and high capacity (greater than 10(7) sites/cell). Similar high- and low-affinity [125I]bPTH(1-34) binding sites were seen also in CRL 1564 membranes containing a PTH-responsive adenylate cyclase. The Kd of the high-affinity sites was identical to the concentration of unlabeled bPTH(1-34) (4.2 ng/ml = 1.0 nM) required to half-maximally elevated cyclic AMP in CRL 1564 cells. Affinity labeling of specific PTH binding sites revealed the presence of multiple components with Mrs of 85, 70, 40, 33, and 23 kD on SDS-PAGE. Competition experiments did not disclose structurally discrete high- and low-affinity sites. Thus, structurally homologous PTH receptors in human skin fibroblasts apparently can assume two affinity states: (i) a high-affinity state coupled to adenylate cyclase and (ii) a low-affinity state that may represent uncoupled receptors.