title: Immunochemical detection of unique proteolytic fragments of the chick 1,25-dihydroxyvitamin D3 receptor. Distinct 20-kDa DNA-binding and 45-kDa hormone-binding species.
abstract: We have characterized proteolytic fragments of the chick intestinal 1,25-dihyroxyvitamin D3 (1,25-(OH)2D3) receptor, produced through either exogenous or endogenous protease action, utilizing a variety of physical and functional assays coupled to immunoblot detection methodology. Treatment of intestinal cytosol with increasing concentrations of trypsin resulted in a progressive diminishment of the 60-kDa receptor concomitant with the appearance of a 20-kDa fragment reactive by Western blot analysis to an anti-1,25-(OH)2D3 receptor monoclonal antibody. Cleveland analysis supported the receptor-origin of this 20-kDa fragment: a common immunoreactive species of 12 kDa could be generated by Staphylococcus aureus V8 protease treatment of the intact 60-kDa receptor as well as the 20-kDa proteolytic product. The 20-kDa fragment did not bind hormone but was capable of interacting with DNA-cellulose in a fashion identical to that of the 60-kDa receptor and, therefore, may contain the functional DNA-binding domain of the chick 1,25-(OH)2D3 receptor. Thus, this fragment likely represents the complement of a larger hormone-bound fragment that we have previously described (Allegretto, E. A., and Pike, J.W. (1985) J. Biol. Chem. 260, 10139-10145). In contrast to the exogenous effect of trypsin, incubation of cytosol resulted in the time-dependent formation of an endogenous protease-derived fragment of 45 kDa. Cleveland analysis was consistent with the 60-kDa receptor derivation of the 45-kDa fragment. This species retained the hormone-binding site and the antibody determinant but was devoid of DNA-binding activity. Moreover, it generated neither the trypsin-dependent 20-kDa fragment nor the V8 protease-dependent 12-kDa species and, therefore, was derived from the opposite end of the receptor molecule. These data have facilitated the construction of a schematic model of the chick receptor in which the immunoreactive epitope is located between the functional domains for hormone binding and DNA binding.
mesh_terms: D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D002117:Calcitriol / Q000378:metabolism*; D002645:Chickens / Q000378:metabolism*; D002846:Chromatography, Affinity; D003600:Cytosol / Q000032:analysis; D004247:DNA / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D006728:Hormones / Q000378:metabolism; D007159:Immunologic Tests; D007413:Intestinal Mucosa / Q000032:analysis*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism*; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid / Q000276:immunology / Q000378:metabolism*; D012697:Serine Endopeptidases; D014357:Trypsin / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D006728:Hormones; D010446:Peptide Fragments; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid; D002731:4-Chloromercuribenzenesulfonate; D004247:DNA; D010450:Endopeptidases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin; D002117:Calcitriol