Hormone-dependent phosphorylation of the 1,25-dihydroxyvitamin D 3 receptor in mouse fibroblasts

Abstract

Experimental results, employing several immunologic techniques, suggest that the mouse receptor for 1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3) undergoes hormone-dependent phosphorylation in intact cells. Treatment of monolayer cultures of mouse 3T6 fibroblasts with 1,25(OH) 2D 3 reveals that the occupied 1,25(OH) 2D 3 receptor displays a minor reduction in electrophoretic mobility as compared to its unoccupied 54,500 dalton counterpart, a change consistent with covalent modification. Similar results were obtained by immunoprecipitation of metabolically-labeled receptors after incubation of 3T6 cells with [ 35S]methionine. This technique also provided greater insight into the precursor-product relationship between the two receptor forms. [ 32P]Orthophosphate-labeling of 3T6 cells, followed by immunoprecipitation indicated that only the form exhibiting covalent modification was phosphorylated. The temporal correspondence between the binding of 1,25(OH) 2D 3 to its cellular receptor and its phosphorylation suggests that the biochemical role of 1,25(OH) 2D 3 may be to induce a conformational change susceptible to phosphorylation and possibly functional activation.