Molecular Analysis of Human Androgen Receptor Mutations Causing Motor Neuronopathy or Infertility

Abstract

Abstract : hAR mutations cause, or predispose to, androgen insensitivity syndrome (AIS), spinobulbar muscular atrophy (SBMA, a motor neuronodegenerative disease), and prostate cancer. I investigated the pathogenic mechanism of three AR mutations: expansion of the trinucleotide repeat (CAG)n coding for the polymorphic polyglutamine (polyGln) tract in exon 1 of the AR, which causes SBMA, and mutations in exon 5 (Asn727Lys) and exon 8 (Met886Val) of the AR ligand-binding domain (LBD), each of which causes oligospermic infertility. In the transiently transfected motor neuron hybrid cell line (NSC34), the polyGln-expanded AR is ∼40% less transactive than the wild-type receptor despite a higher AR protein content. The polyGln-expanded AR has normal androgen-binding kinetics. A polyGln-expanded AR made from mixed CAG and CAA codons is also hypotransactive, suggesting protein dysfunction. The polyGln-expanded AR is more resistant than normal to short, cell-free trypsinolysis and yields a distinct set of Gln-expanded fragments even after extended exposure with a denaturant, suggesting conformational changes. COS cells transfected with (CAG)- or (CAG/CAA)-expanded AR cDNA generate a unique 75 kDa fragment containing the Gln tract and are twice as likely to die apoptotically. Androgen caused a negative shift in the voltage-dependence of activation, resulting in an increase in the T-type Ca2+-channel window current in two motor neuron cell lines expressing polyGln-expanded AR Accumulation of an aberrant or protease-resistant polyGln-expanded AR firument may be the proximate motor neuronopathic agent in SBMA. This agent may cause Ca2+ overload and motor-neuron apoptosis in SBMA by abnormal activation of voltage-gated Ca2+ channels Neither the Met886Val nor the Asn727Lys mutation affects androgen binding in genital skin fibroblasts or transfected COS cells. However, both reduce transactivation by ~40%, a result paralleled by A-R binding to a steroid response element. To my knowledge, these are the first AR LBD mutations that impair DNA binding. Mesterolone normalized spermatogenesis in Asn727Lys subjects. Interestingly, the Asn727Lys AR is 50% hypertransactive with mesterolone at 37°C but not at 34°C, and trypsinolysis indicates that it is conformationally normal with mesterolone but not with 5α-DHT