Characterization of the "Gs-like" activity of Xlas

描述（由申请人提供）： 刺激性 G 蛋白 (Gsalpha) 的 α 亚基及其大变体 XLalphas 通过使用替代启动子和前 mRNA 剪接衍生自 GNAS1。 XLalphas 与 Gsalpha 不同，显示出有限的组织分布，包括在肾脏中的表达，并且在所有研究的组织中均呈父系表达。尽管具有不同的氨基末端结构域，但这两种蛋白质在很长一段羧基末端氨基酸上是相同的，包括几乎所有 Gsalpha 的结构特征。因此，XLalphas 和 Gsalpha 在细胞中可能具有相似的功能特性，我们最近发表的结果与这一假设一致。鉴于 Gsalpha 在许多不同的生物反应中的重要性，XLalpha 的“类 Gs”活性也可能在体内发挥重要作用。 GNASI 的 Gsalpha 编码外显子内的杂合失活突变与显示亲本特异性遗传的多种表型相关，包括假性甲状旁腺功能减退症 (PHP)、进行性骨异型增生 (POH) 和奥尔布赖特遗传性骨营养不良 (AHO)。在父系遗传后，预计大多数突变都会破坏 Gsalpha 和 XLalphas，这表明其中一些疾病（例如 POH）的发病机制可能不仅涉及 Gsalpha 的缺乏，还涉及 XLalphas 的缺乏。此外，在母系遗传后发生的疾病中，例如 PHP-Ia，完整父系等位基因表达的 XLalpha 活性可能在某些细胞中提供补偿性“Gs 样”信号传导，从而有助于某些类型 PHP 中激素抵抗的选择性。因此，我的主要目标是进一步探索 XLalpha 的“Gs 样”活性在这些疾病的分子和遗传机制中的作用。我的第一个具体目标是对 XLas 进行更详细的体外表征，并将其功能特性与 Gsalpha 进行比较，特别是关于可能的受体选择性和自然发生的 GNASI 突变的差异效应。我还将确定 XLalphas 特有的独特功能，这些功能对其信号活动非常重要。此外，我将检查 XLalphas 在肾脏中的空间和时间表达，从而进一步了解 XLas 可能参与该组织中甲状旁腺激素介导的作用。最后，我将通过生成在肾近曲小管中靶向表达 XLalphas 的动物模型来确定 XLalphas 是否可以在体内具有“Gs 样”活性。这些研究将有助于阐明XLalphas的生物学作用，并可能进一步提高目前对GNAS1相关疾病的理解。

DESCRIPTION (provided by applicant): The alpha subunit of the stimulatory G protein (Gsalpha), and its large variant XLalphas, are derived from GNAS1 through the use of alternative promoters and pre-mRNA splicing. XLalphas, unlike Gsalpha, shows limited tissue distribution, including expression in kidney, and is expressed paternally in all investigated tissues. Despite having distinct amino-terminal domains, the two proteins are identical over a long stretch of carboxyl-terminal amino acids comprising almost all the structural features characterized for Gsalpha. Thus, XLalphas and Gsalpha may have similar functional properties in the cell, and our recently published results are consistent with this hypothesis. Given the importance of Gsalpha in numerous different biological responses, the "Gs-like" activity of XLalphas may also have significant roles in the body. Heterozygous inactivating mutations within Gsalpha-encoding exons of GNASI are associated with multiple phenotypes that show parent-specific inheritance, including pseudohypoparathyroidism (PHP), progressive osseous heteroplasia (POH), and Albright's hereditary osteodystrophy (AHO). After paternal transmission, most of these mutations are predicted to disrupt both Gsalpha and XLalphas, suggesting that pathogenesis of some of these disorders, such as POH, may involve not only the deficiency of Gsalpha but also of XLalphas. Moreover, in disorders that develop after maternal inheritance, such as PHP-Ia, the activity of XLalphas expressed from the intact paternal allele may provide compensatory "Gs-like" signaling in certain cells, thereby contributing to the selectivity of hormone resistance in certain types of PHP. My main objective is thus to further explore the role of the "Gs-like" activity of XLalphas in the molecular and genetic mechanisms underlying these diseases. My first specific aim involves a more detailed in vitro characterization of XLas, and to compare its functional properties with those of Gsalpha, particularly regarding possible receptor selectivity and differential effects of naturally-occurring GNASI mutations. I will also identify the unique XLalphas-specific features that are important for its signaling activity. In addition, I will examine spatial and temporal expression of XLalphas in the kidney to therefore provide further insights into possible involvement of XLas in actions mediated by parathyroid hormone in this tissue. Finally, I will determine whether XLalphas can have "Gs-like" activity in vivo, by generating an animal model with targeted expression of XLalphas in the renal proximal tubule. These studies will be helpful in clarifying the biological roles of XLalphas, and may furthermore improve the current understanding of the GNASl-related disorders.