Immunochemical studies on the putative plasmalemmal receptor for 1,25(OH)(2)D-3. I. Chick intestine

Antisera were raised against the NH2-terminus of the putative basal lateral membrane (BLM) receptor for 1,25-dihydroxyvitamin D-3 [1,25(OH)(2)D-3; BLM -VDR]. In Western analyses of BLM proteins, antibody (Ab) 099 was monospeci fic for a 64.5-kDa band. A protein of 64.5 kDa was also labeled by the affi nity ligand [C-14]1,25(OH)(2)D-3-bromoacetate; label was diminished in the presence of excess unlabeled secosteroid. The monoclonal antibody against t he nuclear VDR (9A7) failed to detect an appropriate band in BLM fractions. Preincubation of isolated intestinal cells with Ab 099, but not 9A7, affec ted the following two 1,25(OH)(2)D-3-mediated signal transduction events: a ugmented intracellular calcium and protein kinase C activity. Subcellular d istribution of Ab 099 reactivity by Western analyses and fluorescence micro scopy revealed the highest concentrations in BLM followed by the endoplasmi c reticulum. Exposure of isolated intestinal cells to 1,25(OH)(2)D-3 for 10 s or vascular perfusion of duodena for 5 min resulted in a time-dependent increase in nuclear localization of the BLM-VDR antigen, as judged by elect ron microscopy, whereas 24,25-dihydroxyvitamin D-3 failed to increase antig enic labeling in nuclei. Densitometric quantitation of Western blots of sub cellular fractions prepared from isolated intestinal cells treated with veh icle or 1,25(OH)(2)D-3 confirmed a hormone-induced increase of putative BLM -VDR in the nucleus. It is concluded that a novel cell surface binding prot ein for 1,25(OH)(2)D-3 has been identified.