Conformational studies of human vitamin-D receptor by antipeptide antibodies, partial proteolytic digestion and ligand binding.
We have studied conformational changes of human vitamin-D receptor by using antipeptide antibodies, partial proteolytic digestion and binding of the natural ligand calcitriol or its synthetic analogs. Before exposing either [35S]methionine-labelled in vitro translated human vitamin-D receptor or a recombinant human vitamin-D receptor produced either in Escherichia coli or in Sf9 insect cells to limited proteolysis by trypsin or chymotrypsin, the proteins were treated with calcitriol or its synthetic analogs. The digestion products were analyzed by SDS/PAGE, immunoblotting with polyclonal antipeptide antibodies targeted against different domains of the receptor, and Edman N-terminal sequencing. After limited proteolysis with trypsin, two fragments of Mr 21,000 and Mr 34,000 could be localized into N-terminus and C-terminus of the receptor, respectively, by antipeptide antibodies. We found that treatment 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 Mr 34,000 was further analyzed by Edman N-terminal sequencing, the major cleavage site in the receptor between amino acids Arg173 and His174 was revealed.
Väisänen
S
S
Department of Biochemistry and Biotechnology, University of Kuopio, Finland. vaisanen@messi.uku.fi
Juntunen
K
K
Itkonen
A
A
Vihko
P
P
Mäenpää
P H
PH
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001665:Binding Sites / Q000235:genetics; D002117:Calcitriol / Q000031:analogs & derivatives / Q000494:pharmacology; D002460:Cell Line; D010450:Endopeptidases; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D008024:Ligands; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D018167:Receptors, Calcitriol / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera; D014357:Trypsin
D000906:Antibodies; D008024:Ligands; D010455:Peptides; D018167:Receptors, Calcitriol; D011994:Recombinant Proteins; D010450:Endopeptidases; D014357:Trypsin; D002117:Calcitriol