AENDO June 41/6

Nemere, Ilka, Rahul Ray, and William McManus. Immunochemical studies on the putative plasmalemmal receptor for 1,25(OH)2D3. I. Chick intestine. Am J Physiol Endocrinol Metab 278: E1104–E1114, 2000.—Antisera were raised against the NH2-terminus of the putative basal lateral membrane (BLM) receptor for 1,25-dihydroxyvitamin D3 [1,25(OH)2D3; BLM-VDR]. In Western analyses of BLM proteins, antibody (Ab) 099 was monospecific for a 64.5-kDa band. A protein of 64.5 kDa was also labeled by the affinity ligand [C]1,25(OH)2D3-bromoacetate; label was diminished in the presence of excess unlabeled secosteroid. The monoclonal antibody against the nuclear VDR (9A7) failed to detect an appropriate band in BLM fractions. Preincubation of isolated intestinal cells with Ab 099, but not 9A7, affected the following two 1,25(OH)2D3-mediated signal transduction events: augmented intracellular calcium and protein kinase C activity. Subcellular distribution of Ab 099 reactivity by Western analyses and fluorescence microscopy revealed the highest concentrations in BLM followed by the endoplasmic reticulum. Exposure of isolated intestinal cells to 1,25(OH)2D3 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 electron microscopy, whereas 24,25-dihydroxyvitamin D3 failed to increase antigenic labeling in nuclei. Densitometric quantitation of Western blots of subcellular fractions prepared from isolated intestinal cells treated with vehicle or 1,25(OH)2D3 confirmed a hormoneinduced increase of putative BLM-VDR in the nucleus. It is concluded that a novel cell surface binding protein for 1,25(OH)2D3 has been identified.