Identification of a monoclonal antibody which interacts with the parathyroid hormone receptor-adenylate cyclase system in murine bone.
We have produced monoclonal antibodies which bind specifically to mouse bone cells and then selected these monoclonal antibodies for their ability to inhibit parathyroid hormone (PTH) responses in mouse cranial bone treated with the (1-34) amino terminal peptide of bovine PTH [bPTH(1-34)]. One clone, designated 3-6, characterized as an IgM(kappa), significantly inhibited the accumulation of cAMP in response to bPTH(1-34) at concentrations of hormone between 10(-9) and 10(-7) M. This antibody was subsequently isolated by gel filtration and shown to bind to intact mouse calvariae, with saturation binding occurring at 3 micrograms/ml IgM. A maximal inhibition of approximately 70% of the cAMP accumulation produced in response to 2.5 X 10(-9) M (100 ng/ml) bPTH(1-34) was obtained with 7 micrograms/ml of the purified 3-6 IgM. At this concentration of 3-6 IgM, the half-maximal dose of PTH for activation of cAMP accumulation was increased from 5 X 10(-9) M to 2 X 10(-8) M with no reduction in maximal levels of cAMP production. The utility of this antibody as an inhibitor was further tested by its ability to block the binding of an iodinated PTH analogue, 125I-[Nle8, Nle18, Tyr34]-bPTH(1-34) to mouse cranial bone. The 3-6 IgM at a concentration of 5 X 10(-8) M inhibited 70% of the specific binding of the 125I-labeled analogue. In the absence of parathyroid hormone, 2 X 10(-8) M 3-6 IgM produced a 4-fold increase in cAMP above basal levels, as compared to 40-fold maximal increases observed with PTH, indicating a partial PTH agonist activity of this antibody. When tested for effects on other hormones, 3-6 IgM did not inhibit cAMP accumulation produced in response to salmon calcitonin, epinephrine, prostaglandin E2 or cholera toxin. We propose that the 3-6 monoclonal IgM is specific for the PTH receptor or a component of the PTH receptor-adenylate cyclase system and that this or similar antibodies will serve as useful reagents for future molecular characterization of this receptor.
Weinshank
R L
RL
Cain
C D
CD
Vasquez
N P
NP
Luben
R A
RA
D000262:Adenylyl Cyclases / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001667:Binding, Competitive; D001842:Bone and Bones / Q000276:immunology* / Q000502:physiology; D002460:Cell Line; D002772:Cholera Toxin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D015232:Dinoprostone; D004837:Epinephrine / Q000494:pharmacology; D051379:Mice; D010281:Parathyroid Hormone / Q000276:immunology* / Q000378:metabolism / Q000494:pharmacology; D011458:Prostaglandins E / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000276:immunology*; D018016:Receptors, Parathyroid Hormone
D000911:Antibodies, Monoclonal; D010281:Parathyroid Hormone; D011458:Prostaglandins E; D011956:Receptors, Cell Surface; D018016:Receptors, Parathyroid Hormone; D002772:Cholera Toxin; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D015232:Dinoprostone; D004837:Epinephrine