Discordant Measures of Androgen-Binding Kinetics in Two Mutant Androgen Receptors Causing Mild or Partial Androgen Insensitivity, Respectively1
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We have characterized two different mutations of the human androgen receptor (hAR) found in two unrelated subjects with androgen insensitivity syndrome (AIS): in one, the external genitalia were ambiguous (partial, PAIS); in the other, they were male, but small (mild, MAIS). Single base substitutions have been found in both individuals: E772A in the PAIS subject, and R871G in the MAIS patient. In COS-1 cells transfected with the E772A and R871G hARs, the apparent equilibrium dissociation constants (Kd) for mibolerone (MB) and methyltrienolone are normal. Nonetheless, the mutant hAR from the PAIS subject (E772A) has elevated nonequilibrium dissociation rate constants (k(diss)) for both androgens. In contrast, the MAIS subject's hAR (R871G) has k(diss) values that are apparently normal for MB and methyltrienolone; in addition, the R871G hAR's ability to bind MB resists thermal stress better than the hAR from the PAIS subject. The E772A and R871G hARs, therefore, confer the same pattern of discordant androgen-binding parameters in transfected COS-1 cells as observed previously in the subjects' genital skin fibroblasts. This proves their pathogenicity and correlates with the relative severity of the clinical phenotype. In COS-1 cells transfected with an androgen-responsive reporter gene, trans-activation was 50% of normal in cells containing either mutant hAR. However, mutant hAR-MB binding is unstable during prolonged incubation with MB, whereas normal hAR-MB binding increases. Thus, normal equilibrium dissociation constants alone, as determined by Scatchard analysis, may not be indicative of normal hAR function. An increased k(diss) despite a normal Kd for a given androgen suggests that it not only has increased egress from a mutant ligand-binding pocket, but also increased access to it. This hypothesis has certain implications in terms of the three-dimensional model of the ligand-binding domain of the nuclear receptor superfamily.
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