TRANSCRIPTIONAL ACTIVATION AND DIMERIZATION FUNCTIONS IN THE HUMAN VITAMIN-D-RECEPTOR

The C-terminal domain of the human vitamin D receptor (hVDR) is essent ial for dimerization with retinoid X receptors and for transcriptional activation. To define the dimerization domain of the hVDR, a series o f internal deletion mutants of the receptor were prepared beginning wi thin the E domain and extending through the F domain to the C terminus . These mutant receptors were tested for dimerization and transcriptio nal activities by means of gel shift assay and beta-galactosidase assa y, respectively, in a yeast system. The dimerization domain of the hVD R was localized to two separate but adjacent regions of the receptor m olecule. 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 employ ed using transformed yeast. Two classes of inactive receptors were ide ntified: one in which both transcriptional activation and dimerization were compromised and a second in which only transcriptional activatio n was abolished, Most of the mutations responsible for these phenotype s were single. The studies suggest a separation between dimerization a nd transactivation domains. We reconstituted each of these hVDR mutant s in a mammalian expression vector and evaluated them individually in COS-1 cells. All VDR mutants were transcriptionally active in this cel lular background in response to 1,25-dihydroxyvitamin D-3, although th e 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 a n activation domain in hVDR that is separable from the domain involved in dimerization, Factors that couple hVDR to the general transcriptio n apparatus in yeast through the activation domain in the hVDR, howeve r, appear to be unrelated or dissimilar to those used in COS-1 cells.