Transcriptional activation and dimerization functions in the human vitamin D receptor.

Abstract

The C-terminal domain of the human vitamin D receptor (hVDR) is essential for dimerization with retinoid X receptors and for transcriptional activation. To define the dimerization domain of the hVDR, a series of internal deletion mutants of the receptor were prepared beginning within the E domain and extending through the F domain to the C terminus. These mutant receptors were tested for dimerization and transcriptional activities by means of gel shift assay and beta-galactosidase assay, respectively, in a yeast system. The dimerization domain of the hVDR was localized to two separate but adjacent regions of the receptor molecule. In these experiments, the activation domain colocalized with dimerization. To more precisely delineate a relationship between these domains, region-specific random mutagenesis was carried out to detect mutants using error-prone PCR and a functional screen strategy employed using transformed yeast. Two classes of inactive receptors were identified: one in which both transcriptional activation and dimerization were compromised and a second in which only transcriptional activation was abolished. Most of the mutations responsible for these phenotypes were single. The studies suggest a separation between dimerization and transactivation domains. We reconstituted each of these hVDR mutants in a mammalian expression vector and evaluated them individually in COS-1 cells. All VDR mutants were transcriptionally active in this cellular background in response to 1,25-dihydroxyvitamin D3 although the potency of the hormone was reduced. The latter observation coincided with the observation that each mutant was compromised to some extent in binding affinity. These data clearly demonstrate the existence of an activation domain in hVDR that is separable from the domain involved in dimerization. Factors that couple hVDR to the general transcription apparatus in yeast through the activation domain in the hVDR, however, appear to be unrelated or dissimilar to those used in COS-1 cells.

Physical and Functional Interaction between the Vitamin D Receptor and Hairless Corepressor, Two Proteins Required for Hair Cycling

“…The CYP24 gene is normally induced by 1,25(OH) 2 D 3 in keratinocytes (56); therefore we used a reporter gene with the human CYP24 promoter linked to a luciferase gene (45). The results (Fig.…”

“…p24-OHaseLuc was constructed by subcloning 5.5 kb of the promoter region (45) of the human CYP24 gene (kindly provided by Drs. S. Christakos and J. W. Pike, New Jersey Medical School and University of Wisconsin, respectively) into a firefly luciferase plasmid, lucp1 (45).…”

Hormone-dependent Translocation of Vitamin D Receptors Is Linked to Transactivation

“…Recent studies showed that steroid receptor activation involves conformational changes (2,3), phosphorylation/dephosphorylation (4), interactions with coregulator proteins (2,5,6), and dimerization (7). The activation process of the glucocorticoid receptor (GR), androgen receptor, and progesterone receptor also includes a translocation of these receptors from the cytoplasm to the nucleus.…”

2-Methylene-19-nor-(20S)-1,25-dihydroxyvitamin D3 Potently Stimulates Gene-specific DNA Binding of the Vitamin D Receptor in Osteoblasts

“…A 2MD-mediated increase in VDR DNA affinity may result from enhanced VDR interaction with RXR and with coactivators such as SRC-1 and DRIP205, both of which could enhance the stability of the VDR on DNA. Plasmids-The human osteocalcin (OC) promoter reporter plasmid phOC-3900 luc, the human Cyp24 reporter plasmid phCyp24-luc (ph24OHase-luc) and pGal4(5ϫ)-luc were previously reported (20,21). pcDNA-hVDR, phVDR-VP16, and pCH110-␤gal have been also previously described (20,22).…”

“…Plasmids-The human osteocalcin (OC) promoter reporter plasmid phOC-3900 luc, the human Cyp24 reporter plasmid phCyp24-luc (ph24OHase-luc) and pGal4(5ϫ)-luc were previously reported (20,21). pcDNA-hVDR, phVDR-VP16, and pCH110-␤gal have been also previously described (20,22). pcDNA-hVDR(R417A/R420A) was prepared from pcDNA-hVDR using the QuikChange site-directed mutagenesis kit from Stratagene (La Jolla, CA).…”