Two basic amino acids C-terminal of the proximal box specify functional binding of the vitamin D receptor to its rat osteocalcin deoxyribonucleic acid-responsive element.
Nuclear hormone receptor-responsive element binding specificity has been reported to reside predominantly in the proximal box (P-box), three amino acids located in a DNA-recognition alpha-helix situated on the C-terminal side of the first zinc finger. To further define the residues in the vitamin D receptor (VDR) DNA binding domain (DBD) that mediate its interaction as a retinoid X receptor (RXR) heterodimer with the rat osteocalcin vitamin D-responsive element (VDRE), chimeric receptors were created in which the core DBD of VDR was replaced with that of the homodimerizing glucocorticoid receptor (GR). Systematic alteration of GR DBD amino acids in these chimeras to VDR DBD residues identified arg-49 and lys-53, just C-terminal of the P-box within the base recognition alpha-helix of human VDR (hVDR), as the only two amino acids among 36 differences required to convert the GR core zinc finger domain to that of the VDR. Gel mobility shift and 1,25-dihydroxyvitamin D3-stimulated transcription assays verified that an hVDR-GR DBD chimera is functional on the rat osteocalcin VDRE with only the conservative change of lys-49 to arg, and of the negatively charged glu-53 to a basic amino acid (lys or arg). Thus, for RXR heterodimerizing receptors like VDR, the P-box requires redefinition and expansion to include a DNA specificity element corresponding to arg-49 and lys-53 of hVDR. Examination of DNA specificity element amino acids in other nuclear receptors in terms of conservation and base contact in cocrystal structures supports the conclusion that these residues are crucial for selective DNA recognition.
Hsieh
Jui-Cheng
JC
Department of Biochemistry and Molecular Biophysics, College of Medicine, The University of Arizona, Tucson, Arizona 85724, USA.
Whitfield
G Kerr
GK
Jurutka
Peter W
PW
Haussler
Carol A
CA
Thatcher
Michelle L
ML
Thompson
Paul D
PD
Dang
Hope T L
HT
Galligan
Michael A
MA
Oza
Anish K
AK
Haussler
Mark R
MR
D000595:Amino Acid Sequence / Q000235:genetics; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D003461:Crystallography; D004247:DNA / Q000378:metabolism*; D019281:Dimerization; D006801:Humans; D051379:Mice; D015675:Osteocalcin / Q000235:genetics*; D017434:Protein Structure, Tertiary / Q000502:physiology*; D051381:Rats; D018167:Receptors, Calcitriol / Q000235:genetics* / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics; D011965:Receptors, Glucocorticoid / Q000737:chemistry; D018168:Receptors, Retinoic Acid / Q000737:chemistry / Q000378:metabolism; D011988:Receptors, Thyroid Hormone / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D047488:Retinoid X Receptors; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism; D015533:Transcriptional Activation; D030181:Vitamin D Response Element / Q000235:genetics / Q000502:physiology*
D018167:Receptors, Calcitriol; D018160:Receptors, Cytoplasmic and Nuclear; D011965:Receptors, Glucocorticoid; D018168:Receptors, Retinoic Acid; D011988:Receptors, Thyroid Hormone; D011993:Recombinant Fusion Proteins; D047488:Retinoid X Receptors; D014157:Transcription Factors; D015675:Osteocalcin; D004247:DNA