Molecular defect in activation of a mutant Stat5b

最近的一份报告确定了一名表现为生长激素不敏感的临床特征的患者，该患者在Stat5b基因中携带纯合错义突变，导致在靠近SH2结构域的氨基酸630处丙氨酸被脯氨酸取代。由突变的Stat5b蛋白引起的确切分子缺陷尚未被阐明。初步实验表明，在培养的哺乳动物细胞中瞬时转染可以检测到突变型Stat5b蛋白，但其水平始终低于野生型Stat5b蛋白的50%。在重组实验中，突变体Stat5b在生长激素刺激下未能增强报告基因活性。本研究计划试图确定突变体Stat5b在失活周期中的特定分子缺陷，突出两个假设的功能障碍区域。第一个特定目标是研究突变体Stat5b的蛋白质稳定性和亚细胞定位。第二个特定目标关注的是附近SH2结构域的功能是否被破坏，从而突变的Stat5b不能与激活的生长激素受体/Jak2复合物或在二聚化过程中与其他Stat5b分子相互作用。

A recent report identified a patient presenting with clinical features of growth hormone insensitivity, who harbored a homozygous mis-sense mutation in the Stat5b gene that resulted in an alanine to proline substitution at amino acid 630, adjacent to the SH2 domain. The precise molecular defect caused by the mutant Stat5b protein has not been elucidated. Preliminary experiments have demonstrated that the mutant Stat5b protein is detected upon transient transfection in cultured mammalian cells, however at consistently <50% the level of wild-type Stat5b protein. In reconstitution experiments, mutant Stat5b failed to enhance reporter gene activity upon growth hormone stimulation. This research proposal attempts to identify the specific molecular defect of the mutant Stat5b in its inactivation cycle, highlighting two hypothesized areas of dysfunction. The first specific aim addresses protein stability and subcellular location of mutant Stat5b. The second specific aim focuses on whether the function of the nearby SH2 domain is disrupted, such that the mutant Stat5b can not interact with the activated growth hormone receptor/Jak2 complex or with other Stat5b molecules in the dimerization process.