A mutant androgen receptor from patients with Reifenstein syndrome: identification of the function of a conserved alanine residue in the D box of steroid receptors.

Abstract

Reifenstein syndrome is an eponymic term that describes partial androgen-insensitive disorders. Androgen receptor isolated from five patients with this syndrome contains a specific mutation in the DNA binding domain of the receptor. This mutation converts an alanine to a threonine at position 596 next to the zinc catenation site at the second finger. The threonine 596 mutant receptor mediated normal androgen response at promoters with closely positioned multiple regulatory elements for the androgen receptor and other transcription factors.Promoters with single isolated androgen response elements were not transactivated by the mutant receptor. In in vitro receptor-DNA binding studies, interaction with DNA by the mutant receptor was achieved only in the presence of an anti-androgen receptor antibody. Exchanging alanine 596 in the wild-type androgen receptor with serine or valine produced mutants with properties indistinguishable from those of the naturally occurring threonine 596 mutant receptor. These results indicate that an alanine residue at position 596 contributes important structural and functional activities to the androgen receptor. In the androgen receptor from the patients with Reifenstein syndrome, in which this alanine is converted to a threonine, wild-type receptor properties can be restored by exchanging an additional threonine at position 602 to an alanine. An alanine residue at position 596 or 602 in the DNA binding domain of the androgen receptor is therefore important for the full function of this receptor. In all steroid receptors that bind the core sequence AGAACANNNTGTTCT, an alanine residue is also present at a position equivalent to alanine 596 in the androgen receptor.The androgen receptor (AR) belongs to a large family of ligand-binding transcription factors which include receptors for steroid/thyroid hormones (1, 13), retinoic acids (13), peroxisomal activators (22), vitamin D (13), certain regulators of early development in Drosophila melanogaster (28,29), and other proteins of unknown function (13, 34). These receptors consist of carboxy-terminal ligand binding domains, N-terminal modulator domains, and conserved central portions that bind DNA (1). The DNA binding domains contain two zinc atoms tetrahedrally coordinated by conserved cysteine residues required for proper folding and sequence-specific DNA binding (17).Like other steroid receptors, the AR is thought to induce gene expression by binding to DNA (4,7,31). High-affinity DNA binding is enhanced by the ability of the receptors to dimerize, a function encoded by specific sequences in both the DNA and hormone binding domains. In the DNA binding domain, this function is mediated by sequences in the stem of the second zinc finger in a region designated D box (9, 13, 26). In the hormone binding domain, the relevant sequences are included in a heptamer repeat of hydrophobic residues conserved in all members of the steroid hormone receptor superfamily (14,16). How these two dimerization regions function is not known, and whether one o...

Determinants of Subnuclear Organization of Mineralocorticoid Receptor Characterized through Analysis of Wild Type and Mutant Receptors

“…1B. The ZF2 dimer interface stabilizes receptor DNA binding to some but not all palindromic sites (7,18), whereas an ␣-helix within ZF1 intercalates into the major groove of the HRE and is essential for DNA binding (34,35). The above observations suggested that the receptor DBD and associated NLS are sufficient to mediate nuclear localization but not cluster formation.…”

“…Moreover, the dimer interface mutations do not interfere with receptor-mediated repression, and the replacement of the wild type GR gene with a GR dimer mutant is non-lethal in mice in contrast to GR deletion, which is uniformly lethal (36,37). Hence, the disruption of the DBD dimer interface interaction surface imparts a complex phenotype, and the dimer mutants do not reflect the simple abrogation of DNA binding (7,18).…”

“…Much has been learned about the mechanisms of transcriptional regulation by the steroid receptors through the functional analysis of both naturally occurring (18,19) and artificially constructed (7,20) mutant receptors at these various types of HREs. In particular, some receptor functions can be reconstituted by isolated structural motifs that are transferable from the receptor to a heterologous context (21,22).…”

Androgen and Glucocorticoid Receptor Heterodimer Formation

“…It is interesting to note that a mutation within the DBD dimer interface of AR has been implicated in the human disease, Reifenstein's syndrome (24). In transfection experiments, Kaspar et al found that the mutant AR was surprisingly unaffected by the mutation at paired HREs while its activity was decreased at a simple HRE.…”

The molecular basis of male sexual differentiation

“…Mutations within the ligand binding domain may alter androgen binding but may, in addition, influence dimerisation due to disruption of N-C-terminal structural interactions (82)(83)(84). Mutations within the DNA binding domain have been demonstrated to affect receptor binding to target DNA (85). Mutations may also impair AR mRNA stability, leading to additional dysfunction of androgen action (86).…”