Cleavage of the rat intestinal 1,25-dihydroxyvitamin D3 receptor by an endogenous protease to a form with defective DNA binding.
In this report we describe a form of the 1,25(OH)2D3 receptor which no longer binds to DNA. The defective form of the receptor was produced by the action of an endogenous protease. Rat intestinal receptors, obtained by a two-step procedure of a low salt homogenization followed by extraction of the chromatin pellet with high salt, fail to bind to DNA-cellulose. Inclusion of various serine protease inhibitors during the preparation protects against the loss of DNA binding. Sedimentation analysis in sucrose gradients indicates that the defective receptor is measurably smaller than the native receptor and is unable to aggregate normally under low salt conditions. The size difference, as determined by gel chromatography, is approximately 9,000 Da (56,000 for the protected receptor, 47,000 for the cleaved form). The elution from DEAE-cellulose indicates that the overall charge of both intact and cleaved receptor forms is very similar. Cell fractionation and mixing experiments suggest the enzyme may be located in the lysosomal compartment, organelles which are susceptible to breakage during the extraction procedure. The results demonstrate that an endogenous enzyme preferentially cleaves the 1,25(OH)2D3 DNA binding site resulting in a receptor with altered characteristics. Such an enzymatic activity has not been previously described for the 1,25(OH)2D3 receptor from other tissues or species. Since rat intestine is a classically studied target organ, these findings have additional relevance in receptor purification or other studies to characterize the receptor.
D000818:Animals; D002478:Cells, Cultured; D002482:Cellulose / Q000031:analogs & derivatives; D002499:Centrifugation, Density Gradient; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004247:DNA / Q000031:analogs & derivatives / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000378:metabolism; D008297:Male; D010447:Peptide Hydrolases / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D011919:Rats, Inbred Strains; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid / Q000378:metabolism*
C033190:DNA-cellulose; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid; D002482:Cellulose; D004247:DNA; D010447:Peptide Hydrolases