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Cloned, stably expressed parathyroid hormone (PTH)/PTH-related peptide receptors activate multiple messenger signals and biological responses in LLC-PK1 kidney cells. 1993

F R Bringhurst, and H Juppner, and J Guo, and P Urena, and J T Potts, and H M Kronenberg, and A B Abou-Samra, and G V Segre
Endocrine Unit, Massachusetts General Hospital, Boston 02114.

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.

UI MeSH Term Description Entries
D007668 Kidney Body organ that filters blood for the secretion of URINE and that regulates ion concentrations. Kidneys
D009893 Opossums New World marsupials of the family Didelphidae. Opossums are omnivorous, largely nocturnal and arboreal MAMMALS, grow to about three feet in length, including the scaly prehensile tail, and have an abdominal pouch in which the young are carried at birth. Didelphidae,Opossum
D010281 Parathyroid Hormone A polypeptide hormone (84 amino acid residues) secreted by the PARATHYROID GLANDS which performs the essential role of maintaining intracellular CALCIUM levels in the body. Parathyroid hormone increases intracellular calcium by promoting the release of CALCIUM from BONE, increases the intestinal absorption of calcium, increases the renal tubular reabsorption of calcium, and increases the renal excretion of phosphates. Natpara,PTH (1-84),PTH(1-34),Parathormone,Parathyrin,Parathyroid Hormone (1-34),Parathyroid Hormone (1-84),Parathyroid Hormone Peptide (1-34),Hormone, Parathyroid
D011506 Proteins Linear POLYPEPTIDES that are synthesized on RIBOSOMES and may be further modified, crosslinked, cleaved, or assembled into complex proteins with several subunits. The specific sequence of AMINO ACIDS determines the shape the polypeptide will take, during PROTEIN FOLDING, and the function of the protein. Gene Products, Protein,Gene Proteins,Protein,Protein Gene Products,Proteins, Gene
D011956 Receptors, Cell Surface Cell surface proteins that bind signalling molecules external to the cell with high affinity and convert this extracellular event into one or more intracellular signals that alter the behavior of the target cell (From Alberts, Molecular Biology of the Cell, 2nd ed, pp693-5). Cell surface receptors, unlike enzymes, do not chemically alter their ligands. Cell Surface Receptor,Cell Surface Receptors,Hormone Receptors, Cell Surface,Receptors, Endogenous Substances,Cell Surface Hormone Receptors,Endogenous Substances Receptors,Receptor, Cell Surface,Surface Receptor, Cell
D011994 Recombinant Proteins Proteins prepared by recombinant DNA technology. Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D002118 Calcium A basic element found in nearly all tissues. It is a member of the alkaline earth family of metals with the atomic symbol Ca, atomic number 20, and atomic weight 40. Calcium is the most abundant mineral in the body and combines with phosphorus to form calcium phosphate in the bones and teeth. It is essential for the normal functioning of nerves and muscles and plays a role in blood coagulation (as factor IV) and in many enzymatic processes. Coagulation Factor IV,Factor IV,Blood Coagulation Factor IV,Calcium-40,Calcium 40,Factor IV, Coagulation
D002455 Cell Division The fission of a CELL. It includes CYTOKINESIS, when the CYTOPLASM of a cell is divided, and CELL NUCLEUS DIVISION. M Phase,Cell Division Phase,Cell Divisions,Division Phase, Cell,Division, Cell,Divisions, Cell,M Phases,Phase, Cell Division,Phase, M,Phases, M
D002460 Cell Line Established cell cultures that have the potential to propagate indefinitely. Cell Lines,Line, Cell,Lines, Cell
D003001 Cloning, Molecular The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells. Molecular Cloning

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