Rapid turnover of the 1,25-dihydroxyvitamin d3 receptor in human target cells

Abstract

The early biochemical events following exposure of human target cells to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] have been difficult to study due to the lack of suitable in vitro systems. We have studied the T47 D human breast cancer cell line, which possesses specific high affinity receptors for 1,25-(OH)2D3 and responds to the hormone with changes in cellular replication, as a functional model of 1,25-(OH)2D3- responsive human cells. Specific uptake and binding of [3H]1,25- (OH)2D3 by T47 D cells follows a time course of relatively slow rise and decline. The time of peak binding is delayed relative to that of other steroid hormones in the same system. We have studied the kinetics of 1,25-(OH)2D3 specific uptake and binding in intact cells and Scatchard analysis in broken cell preparations of three T47 D sublines: the wild-type and the 10 and Ml sublines. Binding parameters varied among the three sublines. Thus, peak binding capacity was highest in the wild-type cell and lowest in the Ml subline; ranging from 10–65 fmol/106 cells in intact cell studies and from 23–148 fmol/mg protein (-2.8- 12.7 fmol/106 cells) in broken cell preparations. Receptor affinity in the three sublines ranged from 1.1-2.8 × 10-11 M. The halflife of occupied receptor was estimated in intact cells of all three sublines by incubation in the presence of the protein synthesis inhibitor puromycin. Similar analyses were made of apparent half-life of unoccupied receptor in broken cell preparations of the wild-type and T47-10 cells. The half-life of occupied 1,25-(OH)2D3 receptor was estimated to be 2.8 ± 0.4 h in intact wildtype T47 D cells compared with 3.1 ± 0.7 and 1.4 ± 0.2 hours in intact T47 D-10 and T47 D-Ml cells, respectively. The half-life of unoccupied 1,25-(OH)2D3 receptor was estimated in broken cell preparations to be 4.1 ± 0.7 h in wild-type T47 D and 4.2 ± 0.6 h in the T47 D-10 cells. Receptor half-lives in the wild-type and T47 D-10 cells were not different in either intact or broken cell preparations; however, receptor half-life was 40% longer (P < 0.05) in broken cell preparations (4.2 ± 0.4 h) than in the intact cells (3.0 ± 0.4 h) of the two cell sublines. The differences in time to peak binding in intact cells, which was shortest in the wild-type cells (2.7 h) and longest in the Ml subline (6.7 h), were not explained by the differences in receptor half-life. These data show that the 1,25-(OH)2D3 receptor is turned over rapidly in the intact cell, yet specific uptake and binding are relatively prolonged. Furthermore, binding of hormone to receptor appears to shorten its half-life. We have previously reported rapid entry of 1,25-(OH)2D3 into intact cells, with equilibration within 10–30 sec and maximum formation of hormone-receptor complexes in broken cell preparations within 15 min at 37 C. We, therefore, conclude that there are rate-limiting processes between entry of 1,25-(OH)2D3 into cells and attainment of maximum specific hormone-receptor binding. Possible mechanisms include alterations in receptor levels and changes in components that modify transformation of hormone-receptor complexes. Hormones and factors that modify the cellular response to 1,25-(OH)2D3, e.g. glucocorticoids in the intestine, could do so by altering these processes or receptor turnover. © 1988 by The Endocrine Society.