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

Abstract

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.

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

“…2B), demonstrating that VDR⅐Hr interaction does not require the functional AF-2/helix 12 in hVDR that is known to contact coactivators (40,53). Intriguingly, there exists a natural AF-2 mutation (E420K) in hVDR that causes vitamin D-resistant rickets but does not elicit alopecia (18). The phenotype of this mutant implies that the transactivation function (calcium absorption) and repression actions (hair cycling) of VDR are separable, perhaps with the former mediated by AF-2-coactivator association and the latter effected by Hr contact with a novel domain in VDR.…”

“…2 and 4), whereas the AF-2 of ROR is an important determinant of Hr interaction specificity (31). Interestingly, a reported natural mutation in the hVDR AF-2/helix that compromises coactivator binding and results in rickets does not elicit alopecia (18), implying that VDR function in hair cycling does not require transcriptional activation via the AF-2/helix 12 coactivator binding motif. This genetic insight is consistent with the potential relevance of Hr⅐VDR association in controlling the hair cycle, because Hr binds to VDR in an AF-2-independent manner.…”

“…VDR is required primarily for the following: (i) stimulation of calcium and phosphate absorption from the intestine to prevent rickets, (ii) induction of the CYP24 enzyme that initiates the catabolism of 1,25(OH) 2 D 3 , and (iii) progression of the normal hair cycle in mammalian skin. Although VDR gene ablation in mice elicits both rickets and hair loss (13,14), point mutations in human VDR that specifically compromise either 1,25(OH) 2 D 3 ligand (16,17) or coactivator (18) contacts confer rickets without disruption of the hair cycle. Loss of function mutations in human VDR that do result in both rickets and congenital hair loss (alopecia or atrichia) abolish either VDR DNA binding (19 -21) or VDR⅐RXR heterodimerization (17).…”

Transcriptional activation of the wild-type and mutant vitamin D receptors by vitamin D3 analogs

“…Mutation positions are indicated in Fig. 1A: Q259E on helix 4 (Macedo et al 2008), I268T on helix5 (Malloy et al 2004), H305Q on the loop connecting two α-helices (Malloy et al 1997), I314S and G319V on helix 7 (Whitfield et al 1996, Macedo et al 2008, R391C on helix 10 (Whitfield et al 1996), and E420K on helix 12 (Malloy et al 2002). The WT and mutant receptors were subcloned into the pCMX, and the WT receptor was pCMX-VP16 vectors described previously (Umesono et al 1991, Tagami et al 1999 for in vitro transcription/translation and transient expression in transfected cells.…”

The phenotype and function of murine bone marrow-derived dendritic cells is not affected by the absence of VDR or its ability to bind 1α,25-dihydroxyvitamin D3

“…This interaction between VDR and repressor is independent of its ligand 1a,25 (OH) 2 D 3 nor of the AF-2 region of the receptor [22]. Therefore, mutations in the AF-2 domain do not affect the hair cycle, while the expression of other genes is hampered, resulting in rickets [23]. However, as vitamin D 3 deficiency rather than absence of the VDR and possible associated repressors, is able to induce more severe autoimmune disease phenotypes, it is unlikely that the molecular pathways behind these immune observations will be the same.…”