CONFORMATIONAL STUDIES OF HUMAN VITAMIN-D-RECEPTOR BY ANTIPEPTIDE ANTIBODIES, PARTIAL PROTEOLYTIC DIGESTION AND LIGAND-BINDING

Citation
S. Vaisanen et al., CONFORMATIONAL STUDIES OF HUMAN VITAMIN-D-RECEPTOR BY ANTIPEPTIDE ANTIBODIES, PARTIAL PROTEOLYTIC DIGESTION AND LIGAND-BINDING, European journal of biochemistry, 248(1), 1997, pp. 156-162
Citations number
32
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biology
ISSN journal
0014-2956
Volume
248
Issue
1
Year of publication
1997
Pages
156 - 162
Database
ISI
SICI code
0014-2956(1997)248:1<156:CSOHVB>2.0.ZU;2-F
Abstract
We have studied conformational changes of human vitamin-D receptor by using antipeptide antibodies, partial proteolytic digestion and bindin g of the natural ligand calcitriol or its synthetic analogs. Before ex posing either [S-35]methionine-labelled in vitro translated human vita min-D receptor or a recombinant human vitamin-D receptor produced eith er in Escherichia coli or in Sf9 insect cells to limited proteolysis b y trypsin or chymotrypsin, the proteins were treated with calcitriol o r its synthetic analogs. The digestion products were analyzed by SDS/P AGE, immunoblotting with polyclonal antipeptide antibodies targeted ag ainst different domains of the receptor, and Edman N-terminal sequenci ng. After limited proteolysis with trypsin, two fragments of M-r 21000 and M-r 34000 could be localized into N-terminus and C-terminus of th e receptor, respectively, by antipeptide antibodies. We found that tre atment with calcitriol or its synthetic analogs leads to differential resistance of the ligand-binding domain of the recombinant receptor to partial proteolysis in vitro. We suggest that this is due to distinct conformational changes in the domain induced by the different ligands . The short N-terminal region and the Zn-finger domain form, however, a protease-resistant structure which is independent on the presence or absence of the ligand. When the C-terminal fragment of M-r 34000 was further analyzed by Edman N-terminal sequencing, the major cleavage si te in the receptor between amino acids Arg173 and His174 was revealed.