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.
Pines
M
M
Thorndike and Dana Laboratories, Department of Medicine, Beth Israel Hospital, Boston, MA 02215.
Adams
A E
AE
Stueckle
S
S
Bessalle
R
R
Rashti-Behar
V
V
Chorev
M
M
Rosenblatt
M
M
Suva
L J
LJ
D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D004247:DNA / Q000032:analysis / Q000235:genetics; D006801:Humans; D007668:Kidney / Q000737:chemistry* / Q000166:cytology* / Q000378:metabolism; D010281:Parathyroid Hormone / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D019379:Teriparatide
D010281:Parathyroid Hormone; D010446:Peptide Fragments; D012333:RNA, Messenger; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone; D011994:Recombinant Proteins; C035453:parathyroid hormone (53-84); D019379:Teriparatide; D004247:DNA; D000242:Cyclic AMP