Functional Role of Odorant Receptor Phosphorylation

描述（由申请人提供）：嗅觉神经元使用大量G蛋白偶联的气味受体来检测数千种气味。配体刺激的受体磷酸化是许多G蛋白偶联受体（GPCR）的主要受体功能调节机制，并且对于终止GPCR信号转导至关重要。与其他G蛋白偶联信号一样，嗅觉传导可能使用受体磷酸化机制来调节对气味刺激的反应。然而，仍然缺乏直接和明确的证据，气味刺激气味受体磷酸化。气味刺激的气味受体磷酸化如何以及有多少对嗅觉信号转导有贡献仍然是未知的。这个项目的总体目标是了解嗅觉信号转导是如何在气味受体水平上调节的。该项目提出的两个具体目标包括：1）证明气味刺激的气味受体磷酸化; 2）揭示气味刺激的气味受体磷酸化在嗅觉信号转导中的作用。第一个目标将通过基因靶向方法在小鼠的所有嗅觉神经元中表达具有已知配体特异性的表位标记的气味受体来实现。气味刺激的气味受体磷酸化将通过使用针对表位标签的明确定义的抗体检测磷酸化的受体蛋白来显示。第二个目标将通过在小鼠的所有嗅觉神经元中表达突变的气味受体来实现，其中潜在的磷酸化残基发生突变。这些小鼠中的气味诱发反应将通过电生理记录来测量，并与携带野生型受体的普遍表达的小鼠中的反应进行比较。这项研究将加深我们对嗅觉神经元如何控制其对气味刺激的敏感性和反应动力学的理解。

DESCRIPTION (provided by applicant): Olfactory neurons use a large repertoire of G protein-coupled odorant receptors to detect thousands of odors. Ligand-stimulated receptor phosphorylation is a major regulatory mechanism to receptor function for many G-protein coupled receptors (GPCRs) and is essential to the termination of GPCR signal transduction. As with other G protein-coupled signaling, olfactory transduction may use the receptor phosphorylation mechanism to regulate the response to odor stimuli. Yet, there is still lack of a direct and unambiguous evidence for odorant-stimulated odorant receptor phosphorylation. How and how much odorant-stimulated odorant receptor phosphorylation contributes to olfactory signal transduction remains largely unknown. The overall goal of this project is to understand how olfactory signal transduction is regulated at the level of the odorant receptor. Two specific aims proposed in this project include: 1) to demonstrate odorant-stimulated odorant receptor phosphorylation; and 2) to reveal the role of odorant-stimulated odorant receptor phosphorylation in olfactory signal transduction. The first aim will be achieved by expressing an epitope tagged odorant receptor with known ligand specificity in all olfactory neurons in mice through a gene targeting approach. The odorant-stimulated odorant receptor phosphorylation will be displayed by detecting the phosphorylated receptor protein using well-defined antibodies against the epitope tag. The second aim will be achieved by expressing a mutated odorant receptor, in which the potential phosphorylation residues are mutated, in all olfactory neurons in mice. The odor-evoked responses in these mice will be measured by electrophysiological recordings and be compared to responses in the mouse carrying ubiquitous expression of the wild-type receptor. The proposed research will enhance our understanding of how olfactory neurons control their sensitivity and response dynamics to odor stimuli.