We have characterized proteolytic fragments of the chick intestinal 1,25-dihydroxyvitamin 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. 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.
|Original language||English (US)|
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|State||Published - 1987|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology