PMID- 11948698
OWN - NLM
STAT- MEDLINE
DCOM- 20020722
LR  - 20191025
IS  - 0730-2312 (Print)
IS  - 0730-2312 (Linking)
VI  - 85
IP  - 2
DP  - 2002
TI  - Isolation of baculovirus-expressed human vitamin D receptor: DNA responsive 
      element interactions and phosphorylation of the purified receptor.
PG  - 435-57
AB  - Two controversial aspects in the mechanism of human vitamin D receptor (hVDR) 
      action are the possible significance of VDR homodimers and the functional role of 
      receptor phosphorylation. To address these issues, milligram quantities of 
      baculovirus-expressed hVDR were purified to 97% homogeneity, and then tested for 
      binding to the rat osteocalcin vitamin D responsive element (VDRE) via 
      electrophoretic mobility shift and half-site competition assays in the presence 
      or absence of a CV-1 nuclear extract containing retinoid X receptor (RXR). 
      Methylation interference analysis revealed that both the hVDR homodimer and the 
      VDR-RXR heterodimer display similar patterns of VDRE G-base protection. However, 
      in competition studies, the relative dissociation of the homodimeric hVDR complex 
      from the VDRE was extremely rapid (t1/2 < 30 s) compared to the dissociation of 
      the heteromeric complex (t1/2 > 5 min), thus illustrating the relative 
      instability and low affinity of homodimeric VDR binding to DNA. These results 
      indicate that VDR-RXR heterodimers are the preferred VDRE binding species. 
      Further, two dimensional gel electrophoresis of hVDR demonstrated several 
      isoelectric forms of the receptor, suggesting that it is subject to multiple 
      phosphorylation events. In vitro kinase assays confirmed that purified hVDR is an 
      efficient substrate for protein kinases A and Cbeta, as well as casein kinase II. 
      In vivo studies of the expressed receptor in intact cells, namely baculovirus 
      vector infected Sf9 insect cells and transfected mammalian COS-7 cells, 
      demonstrated that hVDR was phosphorylated in a hormone-enhanced fashion. 
      Functional consequences of hVDR phosphorylation were suggested by the 
      observations that: (i) potato acid phosphatase (PAP)-treated hVDR no longer 
      interacted with the VDRE as either a homodimer or a heteromeric complex with RXR, 
      and (ii) treatment of transfected COS-7 cells with a phosphatase inhibitor 
      (okadaic acid) along with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) resulted in a 
      synergistic enhancement of both hVDR phosphorylation and transactivation of a 
      VDRE-linked reporter gene, compared to the effect of treatment with either agent 
      alone. These studies point to a significant role for phosphorylation of VDR in 
      regulating high-affinity VDR-RXR interactions with VDREs, and also in modulating 
      1,25(OH)2D3-elicited transcriptional activation in target cells.
CI  - Copyright 2002 Wiley-Liss, Inc.
FAU - Jurutka, Peter W
AU  - Jurutka PW
AD  - Department of Biochemistry and Molecular Biophysics, College of Medicine, 
      University of Arizona, Tucson, Arizona 85724, USA.
FAU - MacDonald, Paul N
AU  - MacDonald PN
FAU - Nakajima, Shigeo
AU  - Nakajima S
FAU - Hsieh, Jui-Cheng
AU  - Hsieh JC
FAU - Thompson, Paul D
AU  - Thompson PD
FAU - Whitfield, G Kerr
AU  - Whitfield GK
FAU - Galligan, Michael A
AU  - Galligan MA
FAU - Haussler, Carol A
AU  - Haussler CA
FAU - Haussler, Mark R
AU  - Haussler MR
LA  - eng
PT  - Journal Article
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - J Cell Biochem
JT  - Journal of cellular biochemistry
JID - 8205768
RN  - 0 (DNA Primers)
RN  - 0 (Receptors, Calcitriol)
RN  - 0 (Receptors, Retinoic Acid)
RN  - 0 (Recombinant Proteins)
RN  - 0 (Retinoid X Receptors)
RN  - 0 (Transcription Factors)
RN  - 104982-03-8 (Osteocalcin)
RN  - 1406-16-2 (Vitamin D)
RN  - 1W21G5Q4N2 (Okadaic Acid)
RN  - 5688UTC01R (Tretinoin)
RN  - 9007-49-2 (DNA)
RN  - EC 3.1.3.2 (Acid Phosphatase)
SB  - IM
MH  - Acid Phosphatase/metabolism
MH  - Animals
MH  - Baculoviridae/*genetics
MH  - Binding Sites
MH  - Blotting, Western
MH  - COS Cells
MH  - DNA/*metabolism
MH  - DNA Primers/chemistry
MH  - Dimerization
MH  - Electrophoretic Mobility Shift Assay
MH  - Genes, Regulator/*genetics
MH  - Genetic Vectors
MH  - Humans
MH  - Okadaic Acid/pharmacology
MH  - Osteocalcin/genetics/metabolism
MH  - Phosphorylation
MH  - Promoter Regions, Genetic/*genetics
MH  - Rats
MH  - Receptors, Calcitriol/*genetics/isolation & purification/metabolism
MH  - Receptors, Retinoic Acid/genetics
MH  - Recombinant Proteins/genetics/metabolism
MH  - Response Elements/*genetics
MH  - Retinoid X Receptors
MH  - Transcription Factors/genetics
MH  - Tretinoin/*physiology
MH  - Vitamin D/metabolism
EDAT- 2002/04/12 10:00
MHDA- 2002/07/23 10:01
CRDT- 2002/04/12 10:00
PHST- 2002/04/12 10:00 [pubmed]
PHST- 2002/07/23 10:01 [medline]
PHST- 2002/04/12 10:00 [entrez]
AID - 10.1002/jcb.10134 [pii]
AID - 10.1002/jcb.10134 [doi]
PST - ppublish
SO  - J Cell Biochem. 2002;85(2):435-57. doi: 10.1002/jcb.10134.