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

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.