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J Biol Chem, Vol. 274, Issue 23, 16198-16205, June 4, 1999

Differential Regulation of Direct Repeat 3 Vitamin D₃ and Direct Repeat 4 Thyroid Hormone Signaling Pathways by the Human TR4 Orphan Receptor

From the George Whipple Laboratory for Cancer Research, Department of Pathology, Urology, Radiation Oncology, and The Cancer Center, University of Rochester Medical Center, Rochester, New York 14642

In situ hybridization analysis demonstrated that abundant testicular orphan receptor (TR4) transcripts were detected in kidney, intestine, and bone, which are vitamin D₃ target organs. Cell transfection studies also demonstrated that the expression of the vitamin D₃ target gene, 25-hydroxyvitamin D₃ 24-hydroxylase, can be repressed by TR4 through high affinity binding (K_d = 1.32 nM) to the direct repeat 3 vitamin D₃ receptor response element (DR3VDRE). This TR4-mediated repression of DR3VDRE is in contrast to our earlier report that TR4 could induce thyroid hormone target genes containing a direct repeat 4 thyroid hormone response element (DR4T₃RE). Electrophoretic mobility shift assay using several TR4 monoclonal antibodies when combined with either TR4-DR3VDRE or TR4-DR4T₃RE showed a distinct supershifted pattern, and proteolytic analysis further demonstrated distinct digested peptides with either TR4-DR3VDRE or TR4-DR4T₃RE. These results may therefore suggest that TR4 can adapt to different conformations once bound to DR3VDRE or DR4T₃RE. The consequence of these different conformations of TR4-DR3VDRE and TR4-DR4T₃RE may allow each of them to recruit different coregulators. The differential repression of TR4-mediated DR3VDRE and DR4T₃RE transactivation by the receptor interacting protein 140, a TR4 coregulator, further strengthens our hypothesis that the specificity of gene regulation by TR4 can be modulated by protein-DNA and protein-protein interactions.

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