Association of 1α,25-dihydroxyvitamin D3-occupied vitamin D receptors with cellular membrane acceptance sites

Yee S. Kim, Paul N. MacDonald, Shoukat Dedhar, Keith A. Hruska

Research output: Contribution to journalArticle

51 Scopus citations

Abstract

We previously reported nongenomic activation of ROS 17/2.8 cells by vitamin D metabolites (1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], 25- hydroxyvitamin D3, 22-oxa-calcitriol, etc.). The hormone 1α,25-(OH)2D3, or calcitriol, mediated rapid transient changes in intracellular free calcium levels and concomitant stimulation of inositol polyphosphate and diacylglycerol production. These effects resemble the mechanism of cell activation induced by ligands with plasma membrane (PM) receptors. As preliminary studies indicated that PM isolated from ROS 17/2.8 cells lacked specific binding sites for calcitriol alone, we studied the association between calcitriol-occupied vitamin D receptors (VDR) and ROS 17/2.8 cellular membranes. Saturable binding to the PM and the endoplasmic reticulum (ER) of calcitriol-occupied VDR was demonstrated. Binding of the VDR-[3H]calcitriol complex was displaceable by nonradioactive VDR/calcitriol, but not by the unoccupied VDR or by calcitriol alone. ER binding, but not PM binding, was competitively inhibited by a peptide from the VDR sequence recognized by an ER protein, calreticulin, and by an anticalreticulin antibody. The monoclonal antibody (9A7) against the VDR inhibited PM and ER binding of the hormone- occupied VDR. These results were substantiated by studies using baculovirus- expressed human VDR for binding studies with the PM and ER and for immunoblot analysis. We conclude that specific PM and ER sites of association for calcitriol-occupied VDR exist and suggest that these associations could participate in the nongenomic rapid actions of 1α,25-(OH)2D3.

Original languageEnglish
Pages (from-to)3649-3658
Number of pages10
JournalEndocrinology
Volume137
Issue number9
DOIs
StatePublished - Jan 1 1996

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