Isolation of baculovirus-expressed human vitamin D receptor: DNA responsive element interactions and phosphorylation of the purified receptor.
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.
Jurutka
Peter W
PW
Department of Biochemistry and Molecular Biophysics, College of Medicine, University of Arizona, Tucson, Arizona 85724, USA.
MacDonald
Paul N
PN
Nakajima
Shigeo
S
Hsieh
Jui-Cheng
JC
Thompson
Paul D
PD
Whitfield
G Kerr
GK
Galligan
Michael A
MA
Haussler
Carol A
CA
Haussler
Mark R
MR
D000135:Acid Phosphatase / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001665:Binding Sites; D015153:Blotting, Western; D019556:COS Cells; D004247:DNA / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D019281:Dimerization; D024202:Electrophoretic Mobility Shift Assay; D005809:Genes, Regulator / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D019319:Okadaic Acid / Q000494:pharmacology; D015675:Osteocalcin / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D018167:Receptors, Calcitriol / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D018168:Receptors, Retinoic Acid / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000235:genetics*; D047488:Retinoid X Receptors; D014157:Transcription Factors / Q000235:genetics; D014212:Tretinoin / Q000502:physiology*; D014807:Vitamin D / Q000378:metabolism
D017931:DNA Primers; D018167:Receptors, Calcitriol; D018168:Receptors, Retinoic Acid; D011994:Recombinant Proteins; D047488:Retinoid X Receptors; D014157:Transcription Factors; D015675:Osteocalcin; D014807:Vitamin D; D019319:Okadaic Acid; D014212:Tretinoin; D004247:DNA; D000135:Acid Phosphatase