Characterization of the "Gs-like" activity of Xlas

Xlas“类 Gs”活性的表征

• 批准号: 6766947
• 负责人: MURAT BASTEPE
• 金额: $ 12.99万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6766947
• 关键词: G protein; alleles; biological signal transduction; disease /disorder model; family genetics; gene expression; gene mutation; genetically modified animals; in situ hybridization; kidney; laboratory mouse; molecular pathology; pathologic ossification; protein structure; protein structure function; pseudohypoparathyroidism; receptor binding; renal tubule; sex linked trait

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.

描述(由申请人提供)： 刺激性G蛋白的α亚基(Gsalpha)及其大的变体XLalpas是通过使用替代启动子和前mRNA剪接从GNAS1获得的。与Gsalpha不同的是，XLalpas在组织中的分布有限，包括在肾脏中的表达，并且在所有被研究的组织中都有父系表达。尽管有不同的氨基末端结构域，但这两种蛋白质在一段很长的羧基末端氨基酸上是相同的，几乎包括了Gsalpha特有的所有结构特征。因此，XLalpas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，XLalpas的“Gs样”活动也可能在人体中发挥重要作用。GNASI的Gsalpha编码外显子内的杂合性失活突变与多种表现出父母特有遗传的表型相关，包括假性甲状旁腺功能减退症(PHP)、进行性骨质疏松症(POH)和奥尔布赖特遗传性骨营养不良(Aho)。在父系遗传后，这些突变中的大多数被预测会破坏Gsalpha和XLalpHas，这表明其中一些疾病的发病机制，如POH，可能不仅涉及Gsalpha的缺陷，而且还涉及XLalpHas的缺陷。此外，在母系遗传后发展的疾病中，如PHP-Ia，从完整的父系等位基因表达的XLalpHas的活性可能在某些细胞中提供补偿性的“Gs样”信号，从而在某些类型的PHP中有助于激素抵抗的选择性。因此，我的主要目标是进一步探讨XLalpas的“Gs-like”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进行更详细的体外鉴定，并将其功能特性与Gsalpha的功能特性进行比较，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。我还将确定对其信令活动非常重要的独特的XLalpas特定特性。此外，我将研究XLAS在肾脏中的空间和时间表达，从而为XLAS可能参与甲状旁腺激素在该组织中的作用提供进一步的见解。最后，我将通过建立一个在肾脏近端小管中靶向表达XLalpas的动物模型，来确定XLalpas是否在体内具有“Gs样”的活性。这些研究将有助于阐明XLalpas的生物学作用，并可能进一步提高目前对GNASl相关疾病的理解。