Regulation of G-protein signaling in CNS neurons

描述（由申请人提供）：G蛋白偶联受体（GPCR）是大部分临床有用药物和许多滥用药物的靶点。GPCR通常在激动剂的持续存在下脱敏，并且由这些受体产生的生理反应因此减弱。人们认为脱敏是药物耐受性的基础，而不脱敏的受体产生的信号可能导致药物依赖性的适应性变化。因此，重要的是要了解影响GPCR脱敏神经元的因素。由于脱敏可以根据所研究的细胞类型而变化很大，因此在天然神经元中研究这一过程也很重要。使用体外GPCR脱敏模型，我们发现位于突触前末梢的腺苷A[1]受体（A1 Rs）比位于相同神经元突触后树突上的相同受体脱敏慢得多。因此，我们假设神经元GPCR的脱敏可以受到亚细胞位置的影响，并且突触前末梢是GPCR抵抗脱敏的部位。本文提出的工作将确定突触前和/或突触后腺苷A1受体的脱敏是否由保守的（GRK/抑制蛋白）机制介导，该机制负责非神经元细胞中GPCR的脱敏（具体目标1和2），并将确定突触前末梢中的GPCR是否通常缓慢脱敏（具体目标3）。 我们将使用电生理学和放射性配体结合技术研究天然神经元受体，并将使用病毒载体干扰内源性调节途径。我们还将使用非病毒转染和病毒载体在小脑颗粒神经元和A1 R缺陷神经元中表达A1 R和突变A1 R，以试图确定这些受体脱敏的机制。这些实验结果将为神经元GPCR脱敏提供重要的新信息。如果我们的假设是正确的，我们将确定突触前末梢的关键网站的适应性变化负责发展的药物效应，药物耐受性和药物依赖性。

DESCRIPTION (provided by applicant): G-protein-coupled receptors (GPCRs) are the targets of a large fraction of all clinically useful drugs and many drugs of abuse. GPCRs often desensitize in the continued presence of an agonist, and physiological responses produced by these receptors are thus diminished. It is thought that desensitization underlies drug tolerance, whereas signals produced by receptors that do not desensitize may lead to the adaptive changes that underlie drug dependence. It is therefore important to understand the factors that influence GPCR desensitization in neurons. Since desensitization can vary greatly depending on the type of cell studied, it is also essential to study this process in native neurons. Using an in vitro model of GPCR desensitization, we have found that adenosine A[1] receptors (A1Rs) located on presynaptic terminals desensitize much more slowly than the same receptors located on postsynaptic dendrites in the same neurons. Therefore, we hypothesize is that desensitization of neuronal GPCRs can be influenced by subcellular location, and that presynaptic terminals are sites where GPCRs are resistant to desensitization. The work proposed here will determine if desensitization of presynaptic and/or postsynaptic adenosine A1Rs is mediated by the conserved (GRK/arrestin) mechanism responsible for desensitization of GPCRs in non-neuronal cells (Specific Aims 1 and 2), and will determine if GPCRs in presynaptic terminals desensitize slowly as a rule (Specific Aim 3). We will study native neuronal receptors using electrophysiology and radioligand binding techniques, and will use viral vectors to perturb endogenous regulatory pathways. We will also use non-viral transfection and viral vectors to express A1Rs and mutant A1Rs in cerebellar granule neurons and A1R-deficient neurons, in an attempt to determine the mechanism(s) whereby these receptors desensitize. The results of these experiments will provide important new information about GPCR desensitization in neurons. If our hypothesis is correct, we will identify presynaptic terminals as key sites for the adaptive changes responsible for the development of drug effects, drug tolerance and drug dependence.