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The neuronotoxicity of genes with expanded CAG repeats is most likely mediated by their respective polyglutamine (Gln)-expanded gene products. Gln- expanded portions of these products may be sufficient, or necessary, for pathogenesis. We tested whether a Gln-expanded human androgen receptor (AR) is structurally altered, so that it allows for the proteolytic generation of a potentially pathogenic portion that may be resistant to further degradation. We found, in vitro , that a Gln-expanded AR is more proteolytically resistant than normal, and that it yields a distinct set of Gln-expanded fragments even after extended proteolysis in the presence of 2 M urea. Furthermore, COS cells transfected with CAG-expanded AR cDNA generate an aberrant, nuclear-associated 75 kDa derivative containing the Gln-expanded tract. They are also twice as likely to die by 24 h apoptotically than those transfected with normal AR cDNA. Our data support the notion that an unconventional derivative of the Gln- expanded AR is a component of the proximate motor neuronopathic agent in spinobulbar muscular atrophy. They also focus attention on two ways in which neuronotoxic derivatives may originate from various Gln-expanded proteins: (i) generation of an unusual derivative that is pathogenic de novo ; and (ii) the toxic accumulation of a normal derivative because of an inability to dispose of it.

We have studied two different missense mutations at arginine-830 in exon 7 of the human androgen receptor (hAR) gene that cause complete androgen insensitivity (CAIS) in three families. These substitutions result from point mutations at nucleotide 2489: a G-->T transversion causes Arg830Leu and a G-->A transition causes Arg830Gln. Genital skin fibroblasts of the patients have negligible androgen-binding capacity. The mutations were recreated in an hAR cDNA expression vector that was transiently transfected into COS-1 cells. Both mutant androgen receptors have increased dissociation rate constants and apparent equilibrium rate constants when measured with 5 alpha-dihydrotestosterone or the synthetic, nonmetabolizable androgens, mibolerone or methyltrienolone. The mutant androgen-binding activities share a distinctive thermal misbehavior. At 37 degrees C R830Q and R830L are about 40% and 10% of normal, respectively. At 22 degrees C both mutants gain androgen binding while the normal decreases by 20%; for R830Q the augmented value approaches 60% of the normal. During prolonged 18 h incubation at 37 degrees C, androgen binding of the normal AR is stable while that of both mutants decreases by at least 85%. Both mutants have a very reduced ability to transactivate a cotransfected androgen-responsive reporter gene, but R830Q is better than R830L. We conclude that arginine-830 is important for A-R complex stability, and that its replacement by glutamine or leucine yields distinctive functional aberrations.(ABSTRACT TRUNCATED AT 250 WORDS)

We have reported that polyglutamine (polyGln)-expanded human androgen receptors (hAR) have reduced transactivational competence in transfected cells. We presumed that maximal hAR transactivation requires a normal-size polyGln tract. Here we report, however, that hAR transactivity and polyGln-tract length are related inversely: n = 0 > 12 > 20 > 40 > 50. Thus, a normal-size polyGln tract represses the transactivational competence of a polyGln-free hAR, and polyGln expansion increases that negative effect. This observation has pathogenetic implications for X-linked spinobular muscular atrophy (Kennedy syndrome), and possibly for the autosomal dominant central neuronopathies associated with (CAG)n expansion in the translated portion of four different genes.

We describe different single-amino acid aberrations in the DNA-binding domain (DBD) of the human androgen receptor (hAR) in three families with complete androgen insensitivity. No additional alteration was found in the translated portion of each mutant gene. In one family, an in-frame 3 nt deletion removes codon 581-(or 582) and, thereby, one of two phenylalanines that invariably occupy adjacent positions in the N-terminal alpha-helical region of the DBD in the steroid/thyroid/vitamin D receptor superfamily. In the second, an in-frame 3 nt loss deletes Arg614, an invariant residue in the C-terminal alpha-helix of the DBD. In the third, a G-->A transition causes Arg614His. Following transient transfection of COS cells with each mutant AR plasmid, there is a normal concentration of specific androgen-binding activity that has a reduced ability to bind two types of androgen response element (ARE), and to transregulate an androgen-responsive human growth hormone reporter gene. In genital skin fibroblasts with delta Phe581 or Arg614His, androgen-binding, AR protein and AR mRNA are markedly reduced; in gonadal fibroblasts with delta Arg614, AR mRNA may be reduced. Our data substantiate the primary contributions of Phe581 and Arg614 to normal hAR-ARE binding, and expose important secondary effects of the mutations affecting each residue.