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Analysis of Hormone- and Polynucleotide/Histone-Binding Sites of the Chicken Intestinal 1,25-Dihydroxyvitamin D₃ Receptor by Means of Proteolysis^*

University of Wisconsin School of Pharmacy Madison, Wisconsin 53706

Address requests for reprints to: Dr. William S. Mellon, University of Wisconsin-Madison School of Pharmacy, 425 North Charter Street, Madison, Wisconsin 53706.

Abstract

Several discrete forms of the 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃] receptor from chicken intestinal cytosol were characterized by ultracentrifugation; gel filtration; DNA-, histone-, dye-ligand-binding affinity; and several other chromatographic media. Formation of altered receptor complexes was carried out through partial proteolysis using trypsin, -chymotrypsin, and papain. The holoreceptor complex was found to be asymmetric and have a Stake’s radius of 37 Å. Depending on the concentration of protease, several sterolbinding fragments of 33 and 27 Å were produced which tended to be more globular in shape. The 27 Å complex was incapable of binding DNA, histones, or phosphocellulose, but still retained affinity for DEAE-cellulose. Resolution of three forms of 1,25-(OH)₂D₃ receptors was demonstrated by cibacron blue F3GA-agarose chromatography. The 37 Å complex eluted at 1.1 M KC1, and the 33 Å form eluted at 0.6 M KC1, whereas the 27 Å complex was not retained by the column and eluted in the 0.15 M KC1 wash.

The specificity of the 37 Å complex for histone binding was assessed. The order of binding preference was: histone f₃ > histone f_2a histone f_2b >> histone fi. Results also indicated that DNA could inhibit receptor binding to histone and that this was competitive with respect to histone-agarose binding, suggesting that the interaction of histones and DNA is at a domain common to polynucleotides. It is concluded the 1,25-(OH)₂D₃ receptor has separate and distinct binding sites for hormone and polynucleotides/histones. These in vitro findings of histone binding suggest that the polynucleotide domain of this receptor is capable of recognizing several nuclear derived components that may be important for the alteration of gene expression. (Endocrinology 116: 1408–1417, 1985)

Footnotes

^* This work was supported in part by NIH Grant AM-27234 and the Graduate School of the University of Wisconsin-Madison.

Received May 29, 1984.