Identification of a 150‐kDa membrane component which is modulated by parathyroid hormone

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Abstract
Monoclonal antibodies have been produced against primary bone cells obtained from the collagenase digestion of mouse cranial bone. Antibodies were selected on the basis of their immunoglobulin class and those which were identified as IgG were further screened for their ability to inhibit cAMP accumulation in response to sub-maximal doses of the 1-34 amino-terminal peptide of bovine parathyroid hormone, bPTH(1-34). Nine hybridoma clones were subsequently characterized as inhibitory with respect to parathyroid hormone (PTH) responses in intact mouse cranial bone and which also identified a variety of membrane components from detergent extracts of surface-labeled primary bone cells. Five of these antibodies immunoprecipitated a membrane component with Mr of 80,000 that appeared to be a major component of the extract susceptible to surface-labeling with 125I. All nine monoclonal antibodies were shown to bind to a suspended-cell preparation of primary bone cells with 2-3 orders of magnitude greater binding than that of control antibodies. Using this assay, one clone, designated 3G12 IgG, was observed to exhibit desensitization effects at the binding level with a time course and dose dependency for PTH pre-incubation that was simialr to the establishment of the refractory state in other systems. In addition, the desensitization effect occurred at 37 C but not at 4.degree. C. This antibody was shown to bind saturably to both intact mouse cranial bone and primary bone cells with an apparent affinity constant (Ka) in the range of 109 M. Inhibition of bone cAMP accumulation in response to 2.5 mM bPTH(1-34) was directly correlated to the binding of 3G12 IgG to intact mouse calvariae. A maximum inhibition of approximately 85% was observed. 3G12 IgG immunoprecipitated a single membrane component, Mr 150,000, from NP-40 detergent extracts of 125I-labeled primary mouse bone cells. The molecular mass of this component was also 150,000 daltons when run on polyacrylamide gel slabs under non-reducing conditions. Control and PTH-pre-treated bone cells were surface-labeled, detergent-solubilized and immunoprecipitated with 3G12 IgG in order to investigate further the desensitization effect at the molecular level. Incubation of bone cells with 1 .mu.g/ml bPTH(1-34) for 45 min at 37.degree. C caused an increased susceptibility to surface-labeling with 125I that was approximately three-fold higher in specific activity than that of control cells. Under conditions of quantitative immunoprecipitation, 3G12 IgG precipitated less of the 150-kDa membrane component from the PTH pre-treated bone cell extract, indicating a decreased availability of this membrane component under conditions of desensitization. We propose that the 150-kDa protein from mouse bone cells may be the PTH receptor, a component of the receptor or a membrane protein which is directly involved in the PTH responsiveness of these cells.

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