人自然突变的雄激素受体结构与功能的研究

Androgens play important roles mediated by androgen receptor (AR) in male sexual differentiation and prostate growth in the adult. Genetic alterations in AR gene may result in androgen insensitivity syndromes (AIS), and are also believed to involved in formation and progression of prostate cancer (PC). We have identified Seven kinds of novel mutations in AR from patients with AIS (T2919 deletion、G2882deletion) or advanced PC (G142V, D221H, S296R, E872Q, and M886I) by PCR-SSCP analysis. In this study, we constructed mutant AR cDNA expression plasmids by site directed mutagenesis and investigated how mutations of AR affect their functions as well as their pathological significance. .Androgen specific binding capacity of the wilde type and mutant AR were determined by whole cell ligand-binding assay in transient expression of wilde typ and mutant AR in COS-7 cell. The androgen binding capacity were lost completely in AR mutants (T2919 deletion、G2882 deletion) with a single base deletion that caused frameshift. Furthermore, the transcriptional activities of these two mutants were also lost completely by assaying in CV-1 cells cotranfected with an androgen responsive Luciferase reporter vector (MMTV-Luc). These functional alterations are consistent with the completely AIS phenotype in affected males. .Equilibrium dissociation constants (Kd) and maximal specific binding capacity (Bmax) of the AR missense mutants (G142V, D221H, S296R,E872Q, and M886I) found in PC were no obviously alterations compared with wild type AR. However, they showed increased transactivational activitiy responsion to different ligands. for example, G142V and D221H variants induced 79% and 69% more transcriptional activities, respectively in the presence of DHT at 2′10-9mol/L than wild type AR (P<0.01). Induced transcriptional activities by E872Q variant in response to 1mmol/L estradiol, progesterone, and the androgen antagonist CPA were about two-fold as wild type AR (P<0.01), indicating that there is alteration of the ligand specificity in E872Q variant. Moreover, in the presence of the AR coactivators TIF-2 or CBP, E872Q and M886I variants showed abnormally increased transcriptional activity in response to DHT (p<0.01). Increased trascription activity of these mutant AR in different conditions may associate with progression of PC and failure of AR-targeted therapies..In this studies we demonstrated that AR mutations we have identified may alterate or lose their functions and provided insight into the molecular mechanisms involved in the role of AR gene mutation in the formation, development and progression of diseases (AIS and prostate cancer)..

以从国人雄激素抵抗征患者和前列腺癌（ＰＣ）组织中发现的种新的雄激素受体（ＡＲ）突变型为模型，研究突变ＡＲ的结合特征，它们与靶基因中雄激素反应元件的结合及转录活性，确定突变ＡＲ对其介导的信号转导通路和转录激活作用的影响环节，以进一步阐明ＡＲ结构与功能的关系，受体对靶基因的转录调节机制，并探讨突变ＡＲ的病理意义及与临床ＰＣ治疗的关系。