Abstract
The specific binding of radiolabeled 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) to intact rat osteosarcoma (ROS 17/2) cells was followed for 24 h. In the presence of 0.5-1.5 nM 1,25(OH)2D3, hormone binding increased over a period of 12 h, from 1.1 X 10(4) to 1.3 X 10(5) receptors/cell. The elevated level of hormone binding persisted through 24 h provided that the initial concentration of hormone was maintained. The concentration dependence of this increase in receptor level was centered between 10 and 30 pM 1,25(OH)2D3, and the binding at 12 h exhibited the metabolite specificity expected for a 1,25(OH)2D3 receptor. The t 1/2 values for the disappearance of unoccupied and occupied receptors were roughly the same, approximately 2.7 h; therefore, the increase in hormone binding was not due to receptor stabilization. In comparison, hormone-receptor complexes appeared to dissociate with a t 1/2 of 1 h. alpha-Amanitin treatment reduced the magnitude of receptor accumulation by 50-60%, indicating that mRNA synthesis was required to achieve the maximal response. Ligand-dependent regulation of cellular receptor levels provides a mechanism for amplifying the primary hormonal signal and is predicted to influence the kinetics, magnitude, and dose dependence of cellular responses.
Highlights
To assess turnover of receptor molecules in the presence and receptors/cell (Fig. 2). This is somewhat lower than the preabsence of 1nM 1,25(OH)2D3F. ig. 6 shows the consequences viously reported value of 1.8 X 10' receptors/cell in cytosol of adding 5 PM cycloheximide in the absence of hormone for assays [9,24];the difference may be due to the fact up to 7 h prior to the determination of receptor levels
The that cells were assayed at a higher density in the present decline in receptor levels gave a linear semilogarithmic plot study
An inverse relationship exists between receptor content as a function of time, indicating that at least 85% of the and cell density inROS 17/2 [13].Alternatively, the difference unoccupied receptors behaved as a single class of noninter- in receptor content may reflect clonal variability between the acting molecules with a tlh= 2.7 f 0.2 h
Summary
The receptor levels measured after 1 h of hormone binding to intact ROS 17/2 cells are, comparable to those previously determined by cytosol assays [9, 24].
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