Regulation of G-protein signaling in CNS neurons

描述(由申请人提供)：G蛋白偶联受体(GPCRs)是大部分临床有用药物和许多滥用药物的靶标。GPCRs经常在激动剂的持续存在下脱敏，因此这些受体产生的生理反应减弱。脱敏被认为是药物耐受的基础，而不脱敏的受体产生的信号可能导致药物依赖的适应性变化。因此，了解影响神经元GPCR脱敏的因素是很重要的。由于脱敏作用可能因所研究的细胞类型而有很大不同，因此在天然神经元中研究这一过程也是必要的。利用GPCR脱敏的体外模型，我们发现位于突触前终末的腺苷A[1]受体(A1Rs)的脱敏速度比位于突触后树突上的相同受体慢得多。因此，我们假设神经元GPCRs的脱敏可以受到亚细胞位置的影响，而突触前终末是GPCRs抵抗脱敏的部位。这项工作将确定突触前和/或突触后腺苷A1Rs的脱敏是否由负责非神经元细胞GPCRs脱敏的保守机制(GRK/arrestin)介导(特异性目标1和2)，并将确定突触前终末的GPCRs是否通常脱敏缓慢(特异性目标3)。我们将使用电生理学和放射配基结合技术来研究天然神经元受体，并将使用病毒载体来扰乱内源性调节途径。我们还将使用非病毒转染法和病毒载体在小脑颗粒神经元和A1R缺陷神经元中表达A1Rs和突变型A1Rs，试图确定这些受体脱敏的机制(S)。这些实验的结果将为神经元中GPCR脱敏提供重要的新信息。如果我们的假设是正确的，我们将确认突触前终末是导致药物效应、药物耐受和药物依赖发展的适应性变化的关键部位。

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.