TRAFFICKING OF ANDROGEN RECEPTOR MUTANTS FUSED TO GREEN FLUORESCENT PROTEIN - A NEW INVESTIGATION OF PARTIAL ANDROGEN INSENSITIVITY SYNDROME

The naturally occurring mutations of the androgen receptor (AR), detec ted in patients with androgen insensitivity syndrome (AIS), are curren tly analyzed by in vitro assays. Unfortunately, these assays do not al ways permit the demonstration of a direct relationship between the in vitro activity of the receptor and the severity of the phenotype (in p articular, for mutations detected in patients with partial AIS). We re cently studied the trafficking of wild-type AR, fused to the green flu orescent protein (GFP) in living cells. In the present study, we appli ed this method for the analysis of AR mutants to find out whether it c ould be a complementary method of investigation of AIS. After construc tion of the GFP-AR mutant fusion proteins, the androgen-binding charac teristics, nuclear transfer capacities, and transcriptional activities were evaluated. The nuclear transfer was quantified in the presence o f various concentrations of dihydrotestosterone (DHT). We studied two mutants associated with partial AIS: G743V and R840C. The androgen-bin ding characteristics of both mutants were affected, in comparison with normal AR. Although the affinities were similar, the dissociation rat e of GFP-AR-G743V was twice that of GFP-AR-R840C. In transcriptional a ssay, both mutants were active only at high concentrations of androgen . The nuclear trafficking of the mutants was;evaluated by two paramete rs: 1) the rate of nuclear transfer; and 2) the maximal amount of rece ptors imported into the nucleus. At 10(-6) mol/L DHT, the GFP-AR mutan ts entered into the nucleus in a fashion similar to that of GFP-AR-wt. At 10(-7) mol/L DHT, the rate and maximal degree of nuclear import we re both reduced, even more, for GFP-AR-G743V. The difference between m utants was more pronounced at 10(-9) mol/L DHT, because GFP-ARG743V en tered into the nucleus with even slower kinetics. Though the androgen- binding affinity and transcriptional activity assays did not reveal ma jor differences between mutants, the dissociation rate and the traffic king capacity measurements permitted the activity of the mutants to be differentiated. We observed that the nuclear transfer capacities of t hese mutants are in correlation with the severity of the phenotype. Th e GFP-AR model provides an opportunity both to observe the dynamics of the hormone/receptor complex in living cells and to study the impact of the ligand-binding domain mutation, as opposed to certain in vitro techniques. Because the nuclear import capacity correlates well with t he degree of androgen insensitivity, the GFP-AR is a useful complement ary tool to understanding the phenotype/genotype relationship of AR fu nction in patients with AIS.