Identification of Non-Nuclear Receptors for 1,25-dihydroxyvitamin D3 in Chick Kidney and Brain
1,25 dihydroxyvitamin D3 (1,25(OH)2D3) has been shown to mediate the rapid, non-nuclear stimulation of calcium and phosphate transport in chick intestine through binding to a receptor localized in the basal lateral membrane. By using an antibody to the N-terminus of the membrane receptor, studies were undertaken to determine whether a comparable protein exists in kidney and brain, and whether it is present in a particular subcellular fraction.
The first step was to establish fractionation protocols to separate subcellular organelles as judged by marker enzyme analyses. Differential centrifugation and Percoll gradient fractions were prepared from chick kidney and brain whole homogenates by two methods (method 1 and method 2). Protein and marker enzymes were analyzed in each fraction to determine the distribution of organelles. By method 1, the organelles were not adequately separated. By method 2, chick kidney and brain were found to have the same order of organelle distribution: In the post-nuclear pellet (P2), fraction 1 was found to be enriched for the lysosomal marker acid phosphatase; fractions 2-5 were found to be enriched for the mitochondrial marker succinate dehydrogenase; fraction 8 was found to be enriched for the Golgi marker α-D-mannosidase; and fraction 9 was found to be enriched for the plasma membrane marker Na+,K+ ATPase. In Percoll gradients of microsomal membranes prepared from the 100,000xg pellet (P3), fractions 1-3 contained the endoplasmic reticulum marker enzyme activity glucose-6-phosphatase.
Subsequently, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blots were performed using the antibody to 1,25(OH)2D3 receptors in chick intestinal basal lateral membrane. The areas of the bands were scanned by computer, and analyzed quantitatively. After establishing a suitable protein concentration for Western analysis, differential centrifugation and Percoll gradient fractions were analyzed. Finally, the specific binding of [3H]1,25(OH)2D3 was determined in Percoll gradient fractions to assess whether the receptor is functional. Plasma membrane 1,25(OH)2D3 receptors were found in both chick kidney and brain cells. Golgi membranes also were found to have receptor activity, perhaps since this organelle packages proteins for delivery to other membranes. In kidney, fraction P2 7 demonstrated a very high receptor activity, and [3H[1,25(OH)2D3 specific binding assays showed these membrane receptors are functional. Although this fraction lacks traditional marker enzyme activity, it may contain endocytic vesicles. The physiological function and the mechanism of action of plasma membrane receptors in these two tissues remain to be determined.