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Endocrinology
Pines, M., Department of Medicine, Belli Israel Hospital, Harvard Medical School, Boston, MA, 02215, United States, Institute of Animal Science, Volcani Center, Israel
Adams, A.E., Department of Medicine, Belli Israel Hospital, Harvard Medical School, Boston, MA, 02215, United States
Stueckle, S., Department of Medicine, Belli Israel Hospital, Harvard Medical School, Boston, MA, 02215, United States
Bessalle, R., Department of Medicine, Belli Israel Hospital, Harvard Medical School, Boston, MA, 02215, United States
Rashti-Behar, V., Department of Medicine, Belli Israel Hospital, Harvard Medical School, Boston, MA, 02215, United States
Chorev, M., Department of Medicine, Belli Israel Hospital, Harvard Medical School, Boston, MA, 02215, United States
Rosenblatt, M., Department of Medicine, Belli Israel Hospital, Harvard Medical School, Boston, MA, 02215, United States
Suva, L.J., Department of Medicine, Belli Israel Hospital, Harvard Medical School, Boston, MA, 02215, United States, Div. of Bone and Mineral Metabolism, Beth Israel Hospital, 330 Brookline Av, Boston, MA 02215, United States
Parathyroid hormone (PTH) exerts its biological action by binding to membrane-bound, G-protein coupled receptors expressed predominantly in bone and kidney. In this study, we describe the production and characterization of a panel of cell lines, derived from a human embryonic kidney cell line (HEK- 293), each of which stably express different amounts of the recombinant human PTH/parathyroid hormone-related protein (PTHrP) receptor (Rc). A total of 52 distinct clones displaying different levels of PTH-responsive cAMP production were analyzed; three clones were chosen for more detailed evaluation. These clones (and the receptor-lacking parental cell line) were examined for PTH binding, PTH-stimulated cyclic AMP accumulation and PTH/PTHrP Rc mRNA expression. Receptor-positive clones display a spectrum of PTH-responsiveness that correlates with receptor number/cell and level of receptor mRNA present. The interaction of a C-terminal hPTH-(52-84) peptide with the stably expressed human receptor was examined in cells expressing the highest amount of Rc (>400,000 Rc/cell). There was no direct binding of hPTH-(52-84) or specific competition versus radiolabeled PTH-(1-34). However, competition versus radiolabeled PTH-(1-34) was observed with bPTH-(1-34), hPTH-(1-84) and hPTHrP-(1-34). These data suggest that hPTH-(52-84) does not interact with the only known form of the human PTH/PTHrP Rc. Therefore, the reported effects of PTH-(52-84) in other systems must be via an alternate (as yet unidentified) mechanism(s). The expression of various amounts of the human PTH/PTHrP Rc in a human target cell background should facilitate characterization of the ligand-binding properties and physiological signal transduction mechanism of the Rc.
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Generation and characterization of human kidney cell lines stably expressing recombinant human PTH/PTHrP receptor: Lack of interaction with a C-terminal human PTH peptide
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Pines, M., Department of Medicine, Belli Israel Hospital, Harvard Medical School, Boston, MA, 02215, United States, Institute of Animal Science, Volcani Center, Israel
Adams, A.E., Department of Medicine, Belli Israel Hospital, Harvard Medical School, Boston, MA, 02215, United States
Stueckle, S., Department of Medicine, Belli Israel Hospital, Harvard Medical School, Boston, MA, 02215, United States
Bessalle, R., Department of Medicine, Belli Israel Hospital, Harvard Medical School, Boston, MA, 02215, United States
Rashti-Behar, V., Department of Medicine, Belli Israel Hospital, Harvard Medical School, Boston, MA, 02215, United States
Chorev, M., Department of Medicine, Belli Israel Hospital, Harvard Medical School, Boston, MA, 02215, United States
Rosenblatt, M., Department of Medicine, Belli Israel Hospital, Harvard Medical School, Boston, MA, 02215, United States
Suva, L.J., Department of Medicine, Belli Israel Hospital, Harvard Medical School, Boston, MA, 02215, United States, Div. of Bone and Mineral Metabolism, Beth Israel Hospital, 330 Brookline Av, Boston, MA 02215, United States
Generation and characterization of human kidney cell lines stably expressing recombinant human PTH/PTHrP receptor: Lack of interaction with a C-terminal human PTH peptide
Parathyroid hormone (PTH) exerts its biological action by binding to membrane-bound, G-protein coupled receptors expressed predominantly in bone and kidney. In this study, we describe the production and characterization of a panel of cell lines, derived from a human embryonic kidney cell line (HEK- 293), each of which stably express different amounts of the recombinant human PTH/parathyroid hormone-related protein (PTHrP) receptor (Rc). A total of 52 distinct clones displaying different levels of PTH-responsive cAMP production were analyzed; three clones were chosen for more detailed evaluation. These clones (and the receptor-lacking parental cell line) were examined for PTH binding, PTH-stimulated cyclic AMP accumulation and PTH/PTHrP Rc mRNA expression. Receptor-positive clones display a spectrum of PTH-responsiveness that correlates with receptor number/cell and level of receptor mRNA present. The interaction of a C-terminal hPTH-(52-84) peptide with the stably expressed human receptor was examined in cells expressing the highest amount of Rc (>400,000 Rc/cell). There was no direct binding of hPTH-(52-84) or specific competition versus radiolabeled PTH-(1-34). However, competition versus radiolabeled PTH-(1-34) was observed with bPTH-(1-34), hPTH-(1-84) and hPTHrP-(1-34). These data suggest that hPTH-(52-84) does not interact with the only known form of the human PTH/PTHrP Rc. Therefore, the reported effects of PTH-(52-84) in other systems must be via an alternate (as yet unidentified) mechanism(s). The expression of various amounts of the human PTH/PTHrP Rc in a human target cell background should facilitate characterization of the ligand-binding properties and physiological signal transduction mechanism of the Rc.
Scientific Publication
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