CHARACTERIZATION OF ALTERNATIVE AMINO-ACID SUBSTITUTIONS AT ARGININE-830 OF THE ANDROGEN RECEPTOR THAT CAUSE COMPLETE ANDROGEN INSENSITIVITY IN 3 FAMILIES

We have studied two different missense mutations at arginine-830 in ex on 7 of the human androgen receptor (hAR) gene that cause complete and rogen insensitivity (CAIS) in three families, These substitutions resu lt from point mutations at nucleotide 2489: a G-->T transversion cause s Arg830Leu and a G-->A transition causes Arg830Gln, Genital skin fibr oblasts of the patients have negligible androgen-binding capacity, The mutations were recreated in an hAR cDNA expression vector that was tr ansiently transfected into COS-1 cells. Both mutant androgen receptors have increased dissociation rate constants and apparent equilibrium r ate constants when measured with 5 alpha-dihydrotestosterone or the sy nthetic, nonmetabolizable androgens, mibolerone or methyltrienolone. T he mutant androgen-binding activities share a distinctive thermal misb ehavior. At 37 degrees C R830Q and R830L are about 40% and 10% of norm al, respectively, At 22 degrees C both mutants gain androgen binding w hile the normal decreases by 20%; for R830Q the augmented value approa ches 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 mut ants decreases by at least 85%. Both mutants have a very reduced abili ty to transactivate a cotransfected androgen-responsive reporter gene, but R830Q is better than R830L. We conclude that arginine-830 is impo rtant for A-R complex stability, and that its replacement by glutamine or leucine yields distinctive functional aberrations. The importance of arginine at residue 830 in the hAR is reflected in the fact that it occupies the homologous position in the hormone-binding domains of th e receptors for progesterone, glucocorticoid, and mineralocorticoid; f urthermore, in the estrogen receptor it is conservatively replaced by lysine.