ABSTRACT
The human vitamin D receptor (hVDR), which is a substrate for several protein kinases, mediates the actions of its 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) ligand to regulate gene expression. To determine the site, and functional impact, of cAMP-dependent protein kinase (PKA)-catalyzed phosphorylation of hVDR, we generated a series of C-terminally truncated and point mutant receptors. Incubation of mutant hVDRs with PKA and [gamma-32P]ATP, in vitro, or overexpressing them in COS-7 kidney cells labeled with [32P]orthophosphate, revealed that serine-182 is the predominant residue in hVDR phosphorylated by PKA. An aspartate substituted mutant (S182D), incorporating a negative charge to mimic phosphorylation, displayed only 50% of the transactivation capacity in response to 1,25(OH)2D3 of either wild-type or an S182A-altered hVDR. When the catalytic subunit of PKA was overexpressed, a similar reduction in wild-type but not S182D hVDR transactivity was observed. In a mammalian two-hybrid system, S182D bound less avidly than wild-type or S182A hVDR to the retinoid X receptor (RXR) heterodimeric partner that co-mediates vitamin D responsive element recognition and transactivation. These data suggest that hVDR serine-182 is a primary site for PKA phosphorylation, an event that leads to an attenuation of both RXR heterodimerization and resultant transactivation of 1,25(OH)2D3 target genes.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/chemistry , Receptors, Calcitriol/chemistry , Serine/chemistry , Animals , Binding Sites , COS Cells , Calcium/metabolism , Catalysis , Cyclic AMP-Dependent Protein Kinases/metabolism , DNA, Complementary/metabolism , Electrophoresis, Polyacrylamide Gel , Humans , Immunoblotting , Immunoprecipitation , Ligands , Mutagenesis, Site-Directed , Mutation , Phosphorylation , Plasmids/metabolism , Retinoid X Receptors/metabolism , Transcriptional Activation , Transfection , Two-Hybrid System TechniquesABSTRACT
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.
Subject(s)
DNA/metabolism , Osteocalcin/genetics , Protein Structure, Tertiary/physiology , Receptors, Calcitriol/genetics , Receptors, Calcitriol/metabolism , Vitamin D Response Element/physiology , Amino Acid Sequence/genetics , Amino Acid Substitution , Animals , COS Cells , Crystallography , Dimerization , Humans , Mice , Rats , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Glucocorticoid/chemistry , Receptors, Retinoic Acid/chemistry , Receptors, Retinoic Acid/metabolism , Receptors, Thyroid Hormone/genetics , Recombinant Fusion Proteins/metabolism , Retinoid X Receptors , Transcription Factors/chemistry , Transcription Factors/metabolism , Transcriptional Activation , Vitamin D Response Element/geneticsABSTRACT
The nuclear vitamin D receptor (VDR) mediates the actions of its 1,25-dihydroxyvitamin D(3) ligand to control gene expression in terrestrial vertebrates. Prominent functions of VDR-regulated genes are to promote intestinal absorption of calcium and phosphate for bone mineralization and to potentiate the hair cycle in mammals. We report the cloning of VDR from Petromyzon marinus, an unexpected finding because lampreys lack mineralized tissues and hair. Lamprey VDR (lampVDR) clones were obtained via RT-PCR from larval protospleen tissue and skin and mouth of juveniles. LampVDR expressed in transfected mammalian COS-7 cells bound 1,25-dihydroxyvitamin D(3) with high affinity, and transactivated a reporter gene linked to a vitamin D-responsive element from the human CYP3A4 gene, which encodes a P450 enzyme involved in xenobiotic detoxification. In tests with other vitamin D responsive elements, such as that from the rat osteocalcin gene, lampVDR showed little or no activity. Phylogenetic comparisons with nuclear receptors from other vertebrates revealed that lampVDR is a basal member of the VDR grouping, also closely related to the pregnane X receptors and constitutive androstane receptors. We propose that, in this evolutionarily ancient vertebrate, VDR may function in part, like pregnane X receptors and constitutive androstane receptors, to induce P450 enzymes for xenobiotic detoxification.
