Discordant measures of androgen-binding kinetics in two mutant androgen receptors causing mild or partial androgen insensitivity, respectively

We have characterized two different mutations of the human androgen recepto r (hAR) found in two unrelated subjects with androgen insensitivity syndrom e (AIS): in one, the external genitalia were ambiguous (partial, PAIS); in the other, they were male, but small (mild, MAIS). Single base substitution s 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 h ARs, the apparent equilibrium dissociation constants (K-d) for mibolerone ( MB) and methyltrienolone are normal. Nonetheless, the mutant hAR from the P AIS subject (E772A) has elevated nonequilibrium dissociation rate constants (k(diss)) for both androgens. In contrast, the MAIS subject's hAR (R871G) has kdiss 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, c onfer the same pattern of discordant androgen-binding parameters in transfe cted COS-I cells as observed previously in the subjects' genital skin fibro blasts. This proves their pathogenicity and correlates with the relative se verity of the clinical phenotype. In COS-I cells transfected with an androg en-responsive reporter gene, trans-activation was 50% of normal in cells co ntaining either mutant hAR. However, mutant hAR-MB binding is unstable duri ng prolonged incubation with MB, whereas normal hAR-MB binding increases. T hus, normal equilibrium dissociation constants alone, as determined by Scat chard analysis, may not be indicative of normal hAR function. An increased k(diss) despite a normal K-d for a given androgen suggests tha t it not only has increased egress from a mutant ligand-binding pocket, but also increased access to it. This hypothesis has certain implications in t erms of the three-dimensional model of the ligand-binding domain of the nuc lear receptor superfamily.