Changing serine-485 to alanine in the opossum parathyroid hormone (PTH)/PTH-related peptide receptor enhances PTH stimulation of phospholipase C in a stably transfected human kidney cell line: a useful model for PTH-analog screening?
Using site-directed mutagenesis, we have introduced a serine-485-to-alanine mutation in the opossum parathyroid hormone (PTH) receptor. This amino acid is considered to be phosphorylated by protein kinase A upon ligand binding. Both wild-type (WT) and mutant receptor were stably expressed in 293-EBNA HEK cells. The mutant receptor showed comparable binding characteristics and only a slight increase in cAMP production compared with WT. However, the PTH dose-dependent increase in inositol phosphate production was 24-fold for the mutant receptor vs. 6-fold for the WT receptor. This mutant might prove useful in the sensitive detection of phospholipase C activation through various ligands, as the PTH receptor becomes a target of therapeutic intervention in osteoporosis.
John
M R
MR
Department of Internal Medicine I-Endocrinology and Metabolism, University of Heidelberg, Heidelberg, Germany.
Bösel
J
J
Breit
S
S
Wickert
H
H
Ziegler
R
R
Blind
E
E
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000502:physiology; D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007457:Iodine Radioisotopes; D007668:Kidney / Q000166:cytology; D016297:Mutagenesis, Site-Directed / Q000502:physiology; D009893:Opossums; D010281:Parathyroid Hormone / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011869:Radioligand Assay; D018016:Receptors, Parathyroid Hormone / Q000737:chemistry / Q000235:genetics*; D012694:Serine / Q000235:genetics; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism*
D007457:Iodine Radioisotopes; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D018016:Receptors, Parathyroid Hormone; D012694:Serine; D015544:Inositol 1,4,5-Trisphosphate; D000242:Cyclic AMP; D010738:Type C Phospholipases; D000409:Alanine