Analysis of hormone- and polynucleotide/histone-binding sites of the chicken intestinal 1,25-dihydroxyvitamin D3 receptor by means of proteolysis.
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, alpha-chymotrypsin, and papain. The holoreceptor complex was found to be asymmetric and have a Stoke's radius of 37 A. Depending on the concentration of protease, several sterol-binding fragments of 33 and 27 A were produced which tended to be more globular in shape. The 27 A 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 A complex eluted at 1.1 M KCl, and the 33 A form eluted at 0.6 M KCl, whereas the 27 A complex was not retained by the column and eluted in the 0.15 M KCl wash. The specificity of the 37 A complex for histone binding was assessed. The order of binding preference was: histone f3 greater than histone f2a approximately equal to histone f2b much greater than histone f1. 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.
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D000818:Animals; D001665:Binding Sites; D002645:Chickens; D002918:Chymotrypsin / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D006657:Histones / Q000378:metabolism*; D006728:Hormones / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000378:metabolism*; D008297:Male; D009711:Nucleotides / Q000378:metabolism*; D010206:Papain / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid / Q000378:metabolism*; D012685:Sepharose / Q000031:analogs & derivatives / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D006657:Histones; D006728:Hormones; D009711:Nucleotides; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid; C017842:cibacron blue-sepharose; D004247:DNA; D012685:Sepharose; D010447:Peptide Hydrolases; D002918:Chymotrypsin; D014357:Trypsin; D010206:Papain