The Experts below are selected from a list of 111 Experts worldwide ranked by ideXlab platform
Roberta L Mckee - One of the best experts on this subject based on the ideXlab platform.
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tumor necrosis factor and interleukin 1 inhibit Parathyroid Hormone responsive adenylate cyclase in clonal osteoblast like cells by down regulating Parathyroid Hormone Receptors
Journal of Cellular Physiology, 1992Co-Authors: Michael S Katz, Gloria Gutierrez, Gregory R Mundy, Tazuko K Hymer, Michael P Caulfield, Roberta L MckeeAbstract:The effects of the monokines tumor necrosis factor α (TNF) and interleukin 1 (IL 1) on Parathyroid Hormone (PTH)-responsive adenylate cyclase were examined in clonal rat osteosarcoma cells (UMR-106) with the osteoblast phenotype. Recombinant TNF and IL 1 incubated with UMR-106 cells for 48 hr each produced concentration-dependent inhibition of PTH-sensitive adeylate cyclase, with maximal inhibition of PTH response (40% for TNF. 24% for IL 1) occuring at 10−8 M of either monikine. Both monokines also decreased adenylate cyclase stimulation by the tumor-derived PTH-related Protein (PTHrP). In contrast, TNF and IL 1 had little or no inhibitory effect on receptor-mediated stimulation of adenylate cyclase by isoproterenol and nonReceptors-mediated enzyme activation by cholera toxin forskolin; both monokines increased prostaglandin E2 stimulation of adenylate cyclase. Binding of the radiodinated agonistt mono-[125I]-[Nle3, 18, Tyr34]bPTH-(1–34)NH2 to UMR-106 cells in the presence of increasing concentrations of unlabeled [Nle8,18, Tyr34]bPTH-(1–34)NH2 revealed a decline in PTH receptor density (Bmax) without change in receptor binding affinity (dissociation constant, Kd) after treatment with TNF or IL 1. Pertusis toxin increased PTH-sensitive adenylate cyclase activity but did not attenuate monokine-induced inhibiton of PTH response. In time course studies, brief (1hr) exposure of cells to TNF or IL 1 during early culture was sufficient to decrease PTH response but only after exposed cells were subsequently allowed to grow for prolonged periods. Inhibition of PTH response by monokines was blocked by cycloheximide. The results and indicate that TNF and IL 1 impair responsiveness to PTH (and PTHrP) by a time protein synthesis-dependent down-regulation of PTH Receptors linked to adenylate cyclase. © 1992 Wiley-Liss, Inc.
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A pharmacological comparison of Parathyroid Hormone Receptors in human bone and kidney.
Endocrinology, 1992Co-Authors: John J. Orloff, Roberta L Mckee, Andrea E. Ribaudo, Michael Rosenblatt, Andrew F. StewartAbstract:While abundant information is available characterizing PTH receptor properties in other species, data on human PTH Receptors is very limited. We have been interested in the possibility that tissue-specific differences among human PTH Receptors (i.e. bone vs. kidney) might exist. We have, therefore, compared pharmacological profiles for a wide array of PTH and PTH-related peptide (PTHrP) analogs in human osteoblast-like cells (SaOS-2) and human renal cortical membranes (RCM) using radioiodinated (Tyr36)hPTHrP(1-36)NH2 as a probe for PTH receptor function. The rank order of receptor affinity for 10 PTH/PTHrP receptor agonists tested was very similar in the bone and kidney assay systems. Binding affinity for these peptides was greater in human (h)RCMs and SaOS-2 membranes than in SaOS-2 intact cells. The relative binding affinities for (Tyr36)hPTHrP(1-36)amide, hPTH(1-34), bovine (b)PTH(1-34), and rat PTH(1-34) were similar in human RCMs, SaOS-2 membranes, and SaOS-2 cells. bPTH(1-84) and hPTHrP(1-74) both m...
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tumor necrosis factor and interleukin 1 inhibit Parathyroid Hormone responsive adenylate cyclase in clonal osteoblast like cells by down regulating Parathyroid Hormone Receptors
Journal of Cellular Physiology, 1992Co-Authors: Michael S Katz, Gloria Gutierrez, Gregory R Mundy, Tazuko K Hymer, Michael P Caulfield, Roberta L MckeeAbstract:The effects of the monokines tumor necrosis factor alpha (TNF) and interleukin 1 (IL 1) on Parathyroid Hormone (PTH)-responsive adenylate cyclase were examined in clonal rat osteosarcoma cells (UMR-106) with the osteoblast phenotype. Recombinant TNF and IL 1 incubated with UMR-106 cells for 48 hr each produced concentration-dependent inhibition of PTH-sensitive adenylate cyclase, with maximal inhibition of PTH response (40% for TNF, 24% for IL 1) occurring at 10(-8) M of either monokine. Both monokines also decreased adenylate cyclase stimulation by the tumor-derived PTH-related protein (PTHrP). In contrast, TNF and IL 1 had little or no inhibitory effect on receptor-mediated stimulation of adenylate cyclase by isoproterenol and nonreceptor-mediated enzyme activation by cholera toxin and forskolin; both monokines increased prostaglandin E2 stimulation of adenylate cyclase. Binding of the radioiodinated agonist mono-[125I]-[Nle8,18, Tyr34]bPTH-(1-34)NH2 to UMR-106 cells in the presence of increasing concentrations of unlabeled [Nle8,18, Tyr34]bPTH-(1-34)NH2 revealed a decline in PTH receptor density (Bmax) without change in receptor binding affinity (dissociation constant, Kd) after treatment with TNF or IL 1. Pertussis toxin increased PTH-sensitive adenylate cyclase activity but did not attenuate monokine-induced inhibition of PTH response. In time course studies, brief (1 hr) exposure of cells to TNF or IL 1 during early culture was sufficient to decrease PTH response but only after exposed cells were subsequently allowed to grow for prolonged periods. Inhibition of PTH response by monokines was blocked by cycloheximide. The results indicate that TNF and IL 1 impair responsiveness to PTH (and PTHrP) by a time- and protein synthesis-dependent down-regulation of PTH Receptors linked to adenylate cyclase.
Jan A. Fischer - One of the best experts on this subject based on the ideXlab platform.
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Apical and basolateral Parathyroid Hormone Receptors in rat renal cortical membranes
Endocrinology, 1994Co-Authors: Margit Kaufmann, Roman Muff, Heini Murer, Bruno Stieger, Juerg Biber, Jan A. FischerAbstract:Brush border (BBM) and basolateral membranes (BLM) of rat renal cortical cells separated by free flow electrophoresis revealed two distinct peaks of BBM-specific leucine aminopeptidase and Na+/K(+)-ATPase for BLM. PTH/PTH-related protein (PTHrP) Receptors were identified in BBM and BLM. Specific binding of 125 pM [125I]chicken [Tyr36]-PTHrP-(1-36)amide [chPTHrP-(1-36)] to individual fractions of membranes separated by free flow electrophoresis overlapped with the leucine aminopeptidase and Na+/K(+)-ATPase profiles. Binding to pooled BBM was 53 +/- 5% (mean +/- SEM) of that to BLM (P < 0.01). In BBM and BLM, half-maximal inhibition of binding was obtained with 0.4-0.9 nM chPTHrP-(1-36) and 0.2-0.6 nM rat PTH-(1-34). Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S; 100 microM) lowered chPTHrP-(1-36) binding to 50% of control levels, and half-maximal inhibition of binding was obtained with 480 and 8 nM GTP gamma S in BBM and BLM, respectively. Cross-linking of the PTH/PTHrP Receptors with [125I]chPTHrP-(1-36) modified with N-hydroxysuccinimidyl-4-azidobenzoate revealed indistinguishable doublets of 83 and 73 kilodaltons in both BBM and BLM. Adenylyl cyclase was stimulated 6- and 10-fold by chPTHrP-(1-36) and GTP gamma S, respectively, in BLM and 1.3- and 1.9-fold in BBM. In conclusion, PTH Receptors were recognized in both the basolateral and brush border membranes. Different receptor coupling to G-proteins and minimal cAMP stimulation in BBM provide evidence for PTH/PTHrP receptor isotypes and/or different postreceptor activation in BBM and BLM.
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Comparison of Parathyroid Hormone Receptors in rat osteosarcoma cells and kidney
Biochimica et biophysica acta, 1993Co-Authors: Margit Kaufmann, Jan A. Fischer, Roman MuffAbstract:Abstract 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 μM 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.
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Parathyroid Hormone Receptors in control of proximal tubule function.
Annual review of physiology, 1992Co-Authors: Roman Muff, Jan A. Fischer, Jiirg Biber, Heini MurerAbstract:Parathyroid Hormone action on renal proximal tubule function involves phospholipase C/protein kinase C as well as adenylate cyclase/protein kinase A mediated regulatory pathways. Tissue culture experiments suggest that low concentrations of PTH affect preferentially the phospholipase C/protein kinase C pathway. In vivo, both regulatory cascades are probably involved in the regulation of proximal tubule function. It is not clear at present whether the two intracellular pathways are linked to one or two PTH Receptors. A polarized distribution of PTH receptor(s) involving different second messengers appears possible in proximal tubule epithelial cells. High-affinity (Kd 10(-11)-10(-12) M) PTH Receptors in the range of circulating PTH concentrations in vivo remain to be identified. Structural and functional characterization of PTH Receptors as well as of the PTH-sensitive intracellular mediators and transport systems form the basis for a better understanding of PTH-dependent regulation of proximal tubule function.
Roman Muff - One of the best experts on this subject based on the ideXlab platform.
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Apical and basolateral Parathyroid Hormone Receptors in rat renal cortical membranes
Endocrinology, 1994Co-Authors: Margit Kaufmann, Roman Muff, Heini Murer, Bruno Stieger, Juerg Biber, Jan A. FischerAbstract:Brush border (BBM) and basolateral membranes (BLM) of rat renal cortical cells separated by free flow electrophoresis revealed two distinct peaks of BBM-specific leucine aminopeptidase and Na+/K(+)-ATPase for BLM. PTH/PTH-related protein (PTHrP) Receptors were identified in BBM and BLM. Specific binding of 125 pM [125I]chicken [Tyr36]-PTHrP-(1-36)amide [chPTHrP-(1-36)] to individual fractions of membranes separated by free flow electrophoresis overlapped with the leucine aminopeptidase and Na+/K(+)-ATPase profiles. Binding to pooled BBM was 53 +/- 5% (mean +/- SEM) of that to BLM (P < 0.01). In BBM and BLM, half-maximal inhibition of binding was obtained with 0.4-0.9 nM chPTHrP-(1-36) and 0.2-0.6 nM rat PTH-(1-34). Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S; 100 microM) lowered chPTHrP-(1-36) binding to 50% of control levels, and half-maximal inhibition of binding was obtained with 480 and 8 nM GTP gamma S in BBM and BLM, respectively. Cross-linking of the PTH/PTHrP Receptors with [125I]chPTHrP-(1-36) modified with N-hydroxysuccinimidyl-4-azidobenzoate revealed indistinguishable doublets of 83 and 73 kilodaltons in both BBM and BLM. Adenylyl cyclase was stimulated 6- and 10-fold by chPTHrP-(1-36) and GTP gamma S, respectively, in BLM and 1.3- and 1.9-fold in BBM. In conclusion, PTH Receptors were recognized in both the basolateral and brush border membranes. Different receptor coupling to G-proteins and minimal cAMP stimulation in BBM provide evidence for PTH/PTHrP receptor isotypes and/or different postreceptor activation in BBM and BLM.
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Comparison of Parathyroid Hormone Receptors in rat osteosarcoma cells and kidney
Biochimica et biophysica acta, 1993Co-Authors: Margit Kaufmann, Jan A. Fischer, Roman MuffAbstract:Abstract 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 μM 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.
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Parathyroid Hormone Receptors in control of proximal tubule function.
Annual review of physiology, 1992Co-Authors: Roman Muff, Jan A. Fischer, Jiirg Biber, Heini MurerAbstract:Parathyroid Hormone action on renal proximal tubule function involves phospholipase C/protein kinase C as well as adenylate cyclase/protein kinase A mediated regulatory pathways. Tissue culture experiments suggest that low concentrations of PTH affect preferentially the phospholipase C/protein kinase C pathway. In vivo, both regulatory cascades are probably involved in the regulation of proximal tubule function. It is not clear at present whether the two intracellular pathways are linked to one or two PTH Receptors. A polarized distribution of PTH receptor(s) involving different second messengers appears possible in proximal tubule epithelial cells. High-affinity (Kd 10(-11)-10(-12) M) PTH Receptors in the range of circulating PTH concentrations in vivo remain to be identified. Structural and functional characterization of PTH Receptors as well as of the PTH-sensitive intracellular mediators and transport systems form the basis for a better understanding of PTH-dependent regulation of proximal tubule function.
Tazuko K Hymer - One of the best experts on this subject based on the ideXlab platform.
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tumor necrosis factor and interleukin 1 inhibit Parathyroid Hormone responsive adenylate cyclase in clonal osteoblast like cells by down regulating Parathyroid Hormone Receptors
Journal of Cellular Physiology, 1992Co-Authors: Michael S Katz, Gloria Gutierrez, Gregory R Mundy, Tazuko K Hymer, Michael P Caulfield, Roberta L MckeeAbstract:The effects of the monokines tumor necrosis factor α (TNF) and interleukin 1 (IL 1) on Parathyroid Hormone (PTH)-responsive adenylate cyclase were examined in clonal rat osteosarcoma cells (UMR-106) with the osteoblast phenotype. Recombinant TNF and IL 1 incubated with UMR-106 cells for 48 hr each produced concentration-dependent inhibition of PTH-sensitive adeylate cyclase, with maximal inhibition of PTH response (40% for TNF. 24% for IL 1) occuring at 10−8 M of either monikine. Both monokines also decreased adenylate cyclase stimulation by the tumor-derived PTH-related Protein (PTHrP). In contrast, TNF and IL 1 had little or no inhibitory effect on receptor-mediated stimulation of adenylate cyclase by isoproterenol and nonReceptors-mediated enzyme activation by cholera toxin forskolin; both monokines increased prostaglandin E2 stimulation of adenylate cyclase. Binding of the radiodinated agonistt mono-[125I]-[Nle3, 18, Tyr34]bPTH-(1–34)NH2 to UMR-106 cells in the presence of increasing concentrations of unlabeled [Nle8,18, Tyr34]bPTH-(1–34)NH2 revealed a decline in PTH receptor density (Bmax) without change in receptor binding affinity (dissociation constant, Kd) after treatment with TNF or IL 1. Pertusis toxin increased PTH-sensitive adenylate cyclase activity but did not attenuate monokine-induced inhibiton of PTH response. In time course studies, brief (1hr) exposure of cells to TNF or IL 1 during early culture was sufficient to decrease PTH response but only after exposed cells were subsequently allowed to grow for prolonged periods. Inhibition of PTH response by monokines was blocked by cycloheximide. The results and indicate that TNF and IL 1 impair responsiveness to PTH (and PTHrP) by a time protein synthesis-dependent down-regulation of PTH Receptors linked to adenylate cyclase. © 1992 Wiley-Liss, Inc.
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tumor necrosis factor and interleukin 1 inhibit Parathyroid Hormone responsive adenylate cyclase in clonal osteoblast like cells by down regulating Parathyroid Hormone Receptors
Journal of Cellular Physiology, 1992Co-Authors: Michael S Katz, Gloria Gutierrez, Gregory R Mundy, Tazuko K Hymer, Michael P Caulfield, Roberta L MckeeAbstract:The effects of the monokines tumor necrosis factor alpha (TNF) and interleukin 1 (IL 1) on Parathyroid Hormone (PTH)-responsive adenylate cyclase were examined in clonal rat osteosarcoma cells (UMR-106) with the osteoblast phenotype. Recombinant TNF and IL 1 incubated with UMR-106 cells for 48 hr each produced concentration-dependent inhibition of PTH-sensitive adenylate cyclase, with maximal inhibition of PTH response (40% for TNF, 24% for IL 1) occurring at 10(-8) M of either monokine. Both monokines also decreased adenylate cyclase stimulation by the tumor-derived PTH-related protein (PTHrP). In contrast, TNF and IL 1 had little or no inhibitory effect on receptor-mediated stimulation of adenylate cyclase by isoproterenol and nonreceptor-mediated enzyme activation by cholera toxin and forskolin; both monokines increased prostaglandin E2 stimulation of adenylate cyclase. Binding of the radioiodinated agonist mono-[125I]-[Nle8,18, Tyr34]bPTH-(1-34)NH2 to UMR-106 cells in the presence of increasing concentrations of unlabeled [Nle8,18, Tyr34]bPTH-(1-34)NH2 revealed a decline in PTH receptor density (Bmax) without change in receptor binding affinity (dissociation constant, Kd) after treatment with TNF or IL 1. Pertussis toxin increased PTH-sensitive adenylate cyclase activity but did not attenuate monokine-induced inhibition of PTH response. In time course studies, brief (1 hr) exposure of cells to TNF or IL 1 during early culture was sufficient to decrease PTH response but only after exposed cells were subsequently allowed to grow for prolonged periods. Inhibition of PTH response by monokines was blocked by cycloheximide. The results indicate that TNF and IL 1 impair responsiveness to PTH (and PTHrP) by a time- and protein synthesis-dependent down-regulation of PTH Receptors linked to adenylate cyclase.
Thomas J Gardella - One of the best experts on this subject based on the ideXlab platform.
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international union of basic and clinical pharmacology xciii the Parathyroid Hormone Receptors family b g protein coupled Receptors
Pharmacological Reviews, 2015Co-Authors: Thomas J Gardella, Jeanpierre VilardagaAbstract:The type-1 Parathyroid Hormone receptor (PTHR1) is a family B G protein–coupled receptor (GPCR) that mediates the actions of two polypeptide ligands; Parathyroid Hormone (PTH), an endocrine Hormone that regulates the levels of calcium and inorganic phosphate in the blood by acting on bone and kidney, and PTH-related protein (PTHrP), a paracrine-factor that regulates cell differentiation and proliferation programs in developing bone and other tissues. The type-2 Parathyroid Hormone receptor (PTHR2) binds a peptide ligand, called tuberoinfundibular peptide-39 (TIP39), and while the biologic role of the PTHR2/TIP39 system is not as defined as that of the PTHR1, it likely plays a role in the central nervous system as well as in spermatogenesis. Mechanisms of action at these Receptors have been explored through a variety of pharmacological and biochemical approaches, and the data obtained support a basic “two-site” mode of ligand binding now thought to be used by each of the family B peptide Hormone GPCRs. Recent crystallographic studies on the family B GPCRs are providing new insights that help to further refine the specifics of the overall receptor architecture and modes of ligand docking. One intriguing pharmacological finding for the PTHR1 is that it can form surprisingly stable complexes with certain PTH/PTHrP ligand analogs and thereby mediate markedly prolonged cell signaling responses that persist even when the bulk of the complexes are found in internalized vesicles. The PTHR1 thus appears to be able to activate the Gαs/cAMP pathway not only from the plasma membrane but also from the endosomal domain. The cumulative findings could have an impact on efforts to develop new drug therapies for the PTH Receptors.
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selective and nonselective inverse agonists for constitutively active type 1 Parathyroid Hormone Receptors evidence for altered receptor conformations
Endocrinology, 2001Co-Authors: Percy H Carter, John T Potts, Brian D Petroni, Robert C Gensure, Ernestina Schipani, Thomas J GardellaAbstract:The spontaneous signaling activity of some G protein-coupled Receptors and the capacity of certain ligands (inverse agonists) to inhibit such constitutive activity are poorly understood phenomena. We investigated these processes for several analogs of PTH-related peptide (PTHrP) and the constitutively active human PTH/PTHrP Receptors (hP1Rcs) hP1Rc-H223R and hP1Rc-T410P. The N-terminally truncated antagonist PTHrP(5-36) functioned as a weak partial/neutral agonist with both mutant Receptors but was converted to an inverse agonist for both Receptors by the combined substitution of Leu(11) and D-Trp(12). The N-terminally intact analog [Bpa(2)]PTHrP(1-36)-a partial agonist with the wild-type hP1Rc-was a selective inverse agonist, in that it depressed basal cAMP signaling by hP1Rc-H223R but enhanced signaling by hP1Rc-T410P. The ability of [Bpa(2)]PTHrP(1-36) to discriminate between the two receptor mutants suggested that H223R and T410P confer constitutive receptor activity by inducing distinct conformational changes. This hypothesis was confirmed by the observations that: 1) the double mutant receptor hP1Rc-H223R/T410P exhibited basal cAMP levels that were 2-fold higher than those of either single mutant; and 2) hP1Rc-H223R and hP1Rc-T410P internalized (125)I-PTHrP(5-36) to markedly different extents. The overall results thus reveal that two different types of inverse agonists are possible for PTHrP ligands (nonselective and selective) and that constitutively active PTH-1 Receptors can access different conformational states.
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autoactivation of type 1 Parathyroid Hormone Receptors containing a tethered ligand
Journal of Biological Chemistry, 2000Co-Authors: Masaru Shimizu, Percy H Carter, Thomas J GardellaAbstract:Interactions between the N-terminal residues of Parathyroid Hormone (PTH) and the region of the PTH receptor containing the extracellular loops and transmembrane domains are thought to be critical for receptor activation. We evaluated this hypothesis by replacing the large N-terminal extracellular domain of the human type 1 PTH receptor (hP1Rc-WT) with residues 1–9 of PTH (AVSEIQLMH) using a tetraglycine linker between His-9 of the ligand and Glu-182 of the receptor near the extracellular terminus of transmembrane domain-1. Expression of this construct, hP1Rc-Tether(1–9), in COS-7 cells resulted in basal cAMP levels that were 10-fold higher than those seen in control cells transfected with hP1Rc-WT. Extending the ligand sequence to include Asn-10 and the activity-enhancing substitution of Leu-11 → Arg yielded hP1Rc-[Arg11]Tether(1–11), for which we observed basal cAMP levels that were 50-fold higher than those seen with P1Rc-WT. An alanine-scan analysis of hP1Rc-[Arg11]Tether(1–11) revealed that Gln-6 and His-9 were not critical for autoactivation, whereas Val-2, Ile-5, and Met-8 were. The data show that tethered PTH/PTH Receptors can autoactivate. Analysis of the structure-activity relationships in these tethered receptor constructs can provide new information concerning how the N-terminal residues of PTH interact with the extracellular loops and transmembrane regions of the PTH-1 receptor, particularly in regard to receptor activation.
