Mutations in the 1,25-dihydroxyvitamin D3 receptor identifying C-terminal amino acids required for transcriptional activation that are functionally dissociated from hormone binding, heterodimeric DNA binding, and interaction with basal transcription factor IIB, in vitro.
To investigate a potential ligand-dependent transcriptional activation domain (AF-2) in the C-terminal region of the human vitamin D receptor (hVDR), two conserved residues, Leu-417 and Glu-420, were replaced with alanines by site-directed mutagenesis (L417A and E420A). Transcriptional activation in response to 1, 25-dihydroxyvitamin D3 (1,25-(OH)2D3) was virtually eliminated when either point mutant was transfected into several mammalian cell lines. Furthermore, both mutants exhibited a dominant negative phenotype when expressed in COS-7 cells. Scatchard analysis at 4 degrees C and a ligand-dependent DNA binding assay at 25 degrees C revealed essentially normal 1,25-(OH)2D3 binding for the mutant hVDRs, which were also equivalent to native receptor in associating with the rat osteocalcin vitamin D responsive element as a presumed heterodimer with retinoid X receptor. Glutathione S-transferase-human transcription factor IIB (TFIIB) fusion protein linked to Sepharose equally coprecipitated the wild-type hVDR and the AF-2 mutants. These data implicate amino acids Leu-417 and Glu-420, residing in a putative alpha-helical region at the extreme C terminus of hVDR, as critical in the mechanism of 1, 25-(OH)2D3-stimulated transcription, likely mediating an interaction with a coactivator(s) or a component of the basal transcriptional machinery distinct from TFIIB.
Jurutka
P W
PW
Department of Biochemistry, College of Medicine, The University of Arizona, Tucson, Arizona 85724, USA.
Hsieh
J C
JC
Remus
L S
LS
Whitfield
G K
GK
Thompson
P D
PD
Haussler
C A
CA
Blanco
J C
JC
Ozato
K
K
Haussler
M R
MR
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002117:Calcitriol / Q000494:pharmacology; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism*; D019281:Dimerization; D018698:Glutamic Acid; D006801:Humans; D007700:Kinetics; D007930:Leucine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015675:Osteocalcin / Q000096:biosynthesis; D010641:Phenotype; D017354:Point Mutation; D017433:Protein Structure, Secondary; D051381:Rats; D018167:Receptors, Calcitriol / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D018168:Receptors, Retinoic Acid / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D047488:Retinoid X Receptors; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D035581:Transcription Factor TFIIB; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection
D018167:Receptors, Calcitriol; D018168:Receptors, Retinoic Acid; D011994:Recombinant Proteins; D047488:Retinoid X Receptors; D035581:Transcription Factor TFIIB; D014157:Transcription Factors; D015675:Osteocalcin; D018698:Glutamic Acid; D004247:DNA; D002117:Calcitriol; D007930:Leucine