Purified human vitamin D receptor overexpressed in E. coli and baculovirus systems does not bind 1,25-dihydroxyvitamin D3 hormone efficiently unless supplemented with a rat liver nuclear extract.
We report here that highly purified human vitamin D receptor (hVDR) derived from E. coli or baculovirus expression systems does not exhibit saturable, high affinity 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) ligand binding when these preparations alone are analyzed. Inclusion of rat liver nuclear extract, which does not itself contain detectable 1,25(OH)2D3 binding activity, is required to endow hVDR isolated from bacterial or insect cells with the property of high affinity hormone binding (Kd = 0.13-0.22 nM). This observation should facilitate the valid assay of 1,25(OH)2D3 binding activity and kinetics in samples of overexpressed hVDR. Moreover, since rat liver nuclear extract contains retinoid X receptors and possibly other auxiliary factors capable of forming heterodimers with hVDR that in turn associate with vitamin D responsive elements, we hypothesize that like DNA binding, 1,25(OH)2D3 binding to hVDR requires the cooperation of a co-receptor or some uncharacterized receptor activating/stabilizing factor.
Nakajima
S
S
Department of Biochemistry, College of Medicine, University of Arizona, Tucson 85724.
Hsieh
J C
JC
MacDonald
P N
PN
Haussler
C A
CA
Galligan
M A
MA
Jurutka
P W
PW
Haussler
M R
MR
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002117:Calcitriol / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D009036:Moths; D015675:Osteocalcin / Q000235:genetics; D051381:Rats; D018167:Receptors, Calcitriol / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D014020:Tissue Extracts / Q000378:metabolism*; D014162:Transfection
D004268:DNA-Binding Proteins; D018167:Receptors, Calcitriol; D011994:Recombinant Proteins; D014020:Tissue Extracts; D015675:Osteocalcin; D002117:Calcitriol