Studies on the Mode of Action of Calciferol. XXIX.Biochemical Characterization of 1,25-Dihydroxyvitamin D3Receptors in Chick Pancreas and Kidney Cytosol*

Abstract

Macromolecular cytoplasmic components which specifically bind the steroid hormone 1α,25-dihydroxyvitamin D₃ [lα,25-(OH)₂D₃] were identified in rachitic chick pancreas and kidney. These receptors were very similar to the analogous 1α,25-(OH)₂D₃ receptors present in chick intestinal cytosol. Similar to the intestinal receptors, the cytoplasmic hormone macromolecular complex formed by the incubation of chick kidney or pancreas cytosol with tritiated hormone exhibited a sedimentation coefficient of 3.6S in 0.3-M KC1 sucrose gradients. The interaction of 1,25-(OH)₂-[³H]D₃ occurred at low concentrations of hormone (-8 M) and with high affinity [Kd = 4.0± 1 × 10_-10 M at 4 C for the binding of 1,25-(OH)₂D₃ to pancreas cytosol receptor and 6.6 ± 2.7 × 10^-10 M for kidney cytosol receptor]. The time course of association of lα,25-(OH)₂D₃ to kidney and pancreatic cytosol at 4 and 24 C was found to be similar to the time course of association of 1α,25-(OH)₂D₃ to intestinal cytosol. The concentration of the kidney receptor was less than one third the concentration of the receptor in the intestine. Thermolability studies indicated that for all three tissues, the presence of ligand as well as reduced temperature act to stabilize the receptor. The t_½ for the aporeceptors at 4 C was 15 ± 1 h for kidney cytosol, 11 ± 0.5 h for pancreas cytosol, and ± 3 h for intestinal cytosol. The t_½ of the aporeceptors at 24 C was 3.6 ± 0.4 h for kidney and 51 ± 6 min and 64 ± 7 min for pancreas and intestine, respectively. All of the receptors have similar molecular weights and a similar time course of unfilled receptor concentration after injection with 1α,25-(OH)₂D₃. The participation of protein in the steroid-binding site was suggested by the destruction of binding activity by pronase and trypsin but not by ribonuclease and deoxyribonuclease. In addition, kidney and pancreas receptors possessed reduced cysteine residues at or near the 1α,25-(OH)₂D₃-binding site which was critical for binding activity. Finally [³H]25-hydroxyvitamin D₃ administration studies indicated that pancreas and kidney were capable of accumulating 4.3- and 9.2-fold more endogenously produced 1,25-(OH)₂D₃, respectively, than a nontarget tissue, skeletal muscle. The similarities to the chick intestinal receptor suggest that pancreas and kidney, like the intestine, may be target tissues of 1,25-(OH)₂D₃ action.