A novel mutation in helix 12 of the vitamin D receptor impairs coactivator interaction and causes hereditary 1,25-dihydroxyvitamin D-resistant rickets without alopecia

Hereditary vitamin D-resistant rickets (HVDRR) is a genetic disorder most often caused by mutations in the vitamin D receptor (VDR). The patient in this study exhibited the typical clinical features of HVDRR with early onset rickets, hypocalcemia, secondary hyperparathyroidism, and elevated serum concentrations of alkaline phosphatase and 1,25-dihydroxyvitamin D [1,25-(OH)(2)D(3)]. The patient did not have alopecia. Assays of the VDR showed a normal high affinity low capacity binding site for [(3)H]1,25-(OH)(2)D(3) in extracts from the patient's fibroblasts. However, the cells were resistant to 1,25-dihydroxyvitamin D action as demonstrated by the failure of the patient's cultured fibroblasts to induce the 24-hydroxylase gene when treated with either high doses of 1,25-(OH)(2)D(3) or vitamin D analogs. A novel point mutation was identified in helix H12 in the ligand-binding domain of the VDR that changed a highly conserved glutamic acid at amino acid 420 to lysine (E420K). The patient was homozygous for the mutation. The E420K mutant receptor recreated by site-directed mutagenesis exhibited many normal properties including ligand binding, heterodimerization with the retinoid X receptor, and binding to vitamin D response elements. However, the mutant VDR was unable to elicit 1,25-(OH)(2)D(3)-dependent transactivation. Subsequent studies demonstrated that the mutant VDR had a marked impairment in binding steroid receptor coactivator 1 (SRC-1) and DRIP205, a subunit of the vitamin D receptor-interacting protein (DRIP) coactivator complex. Taken together, our data indicate that the mutation in helix H12 alters the coactivator binding site preventing coactivator binding and transactivation. In conclusion, we have identified the first case of a naturally occurring mutation in the VDR (E420K) that disrupts coactivator binding to the VDR and causes HVDRR.     

Reciprocal recruitment of DRIP/mediator and p160 coactivator complexes in vivo by estrogen receptor

Two functionally distinct classes of coactivators are recruited by liganded estrogen receptor, the DRIP/Mediator complex and p160 proteins, although the relative dynamics of recruitment is unclear. Previously, we have shown a direct, estradiol-dependent interaction between the DRIP205 subunit of the DRIP complex and the estrogen receptor (ER) AF2 domain. Here we demonstrate the in vivo recruitment of other endogenous DRIP subunits to ER in response to estradiol treatment in MCF-7 cells. To explore the relationship between DRIP and p160 coactivators, we examined the kinetics of coactivator recruitment to the ER target promoter, pS2, by chromatin immunoprecipitation. We observed a cyclic association and dissociation of coactivators with the promoter, with recruitment of p160s and DRIPs occurring in opposite phases, suggesting an exchange between these coactivator complexes at the target promoter.     

Coactivators for the orphan nuclear receptor RORalpha.

A mutation in the nuclear orphan receptor RORalpha results in a severe impairment of cerebellar development by unknown mechanisms. We have shown previously that RORalpha contains a strong constitutive activation domain in its C terminus. We therefore searched for mammalian RORalpha coactivators using the minimal activation domain as bait in a two-hybrid screen. Several known and putative coactivators were isolated, including glucocorticoid receptor-interacting protein-1 (GRIP-1) and peroxisome proliferator-activated receptor (PPAR)-binding protein (PBP/TRAP220/DRIP205). These interactions were confirmed in vitro and require the intact activation domain of RORalpha although different requirements for interaction with GRIP-1 and PBP were detected. Even in the absence of exogenous ligand, RORalpha interacts with a complex or complexes of endogenous proteins, similar to those that bind to ligand-occupied thyroid hormone and vitamin D receptors. Both PBP and GRIP-1 were shown to be present in these complexes. Thus we have identified several potential RORalpha coactivators that, in contrast to the interactions with hormone receptors, interact with RORalpha in yeast, in bacterial extracts, and in mammalian cells in vivo and in vitro in the absence of exogenous ligand. GRIP-1 functioned as a coactivator for the RORalpha both in yeast and in mammalian cells. Thus, GRIP-1 is the first proven coactivator for RORalpha.