Endocytic mechanisms controlling functional selectivity of the CB1R

描述(申请人提供)：G蛋白偶联受体(GPCRs)是最大的跨膜受体家族之一，也是当前治疗药物的主要靶点。在信号水平上，作用于同一受体的配体可以激活多个信号级联，有时甚至是相反的信号级联；这一过程被定义为功能选择性或偏向激动化。功能选择性的主要效应物之一是β-拦阻蛋白，这是一种招募到激活受体的多功能蛋白质。然而，受体激活如何转化为β-arrestin信号并没有明确的定义。我们的初步工作结合了最先进的活细胞成像与分子和生化技术，确定了配体特定的停留时间，即受体在内吞之前聚集到单个内吞凹坑中的时间，作为受体控制β-arrestin介导的信号的机制。我们的假设认为，配体在受体水平上诱导特定的磷酸化，引发特定的内吞停留时间，在此期间，β-抑制素仍然被招募并参与信号传递。我们建议定义一种机制，即大麻素1受体(CB1R)是中枢神经系统中含量最丰富的受体之一，也是大麻的靶标，通过它在胞内停留时间控制β-arrestin信号。我们的目标是：1)表征CB1R的配体特异性驻留时间，以检验我们的假设，即配体可以引起与其内吞效率无关的特定驻留时间。2)确定CB1R配体特异性驻留时间的机制。我们将检验这一假说，即驻留时间由受体的配体特异的磷酸化特征(“条形码”)控制。还将调查替代机制。3)确定β-arrestin信号是否是异源系统和天然组织中配体特异性停留时间的生理靶点。最后，我们将测试通过改变CB1R驻留时间来控制arrestin信号的不同操作。

DESCRIPTION (provided by applicant): G protein-coupled receptors (GPCRs) are one of the largest families of transmembrane receptors and a major target of current therapeutic drugs. At the signaling level, it has become clear that ligands acting on the same receptor can activate multiple and sometime opposing signaling cascades; a process defined as functional selectivity or biased agonism. One of the main effectors of functional selectivity are beta-arrestins, multifunction proteins recruited to activated receptors. However, how receptor activation translates into beta-arrestin signaling is not clearly defined. Our preliminary work combining state-of-the-art live cell imaging with molecular and biochemical techniques identifies ligand-specific dwell times, the time receptors are clustered into individual endocytic pits before endocytosis, as a mechanism by which receptors can control beta-arrestin mediated signaling. Our hypothesis suggests that ligands induce specific phosphorylations at the receptor level, eliciting specific endocytic dwell times during which beta-arrestins remain recruited and engaged in signaling. We propose to define a mechanism by which the Cannabinoid 1 Receptor (CB1R), one of the most abundant receptors in the CNS and target of cannabis, controls beta-arrestin signaling during endocytic dwell times. Our aims are: 1) Characterize ligand-specific dwell times of the CB1R to test our hypothesis that ligands can elicit specific dwell times that ar independent of their endocytic efficacy. 2) Define the mechanisms underlying ligand-specific dwell times of the CB1R. We will test the hypothesis that dwell times are controlled by ligand-specific phosphorylation profiles ("bar-codes") of the receptor. Alternative mechanisms will be also investigated. 3) Determine if beta-arrestin signaling is the physiological target of ligand specific dwell times in heterologous systems and native tissue. Finally, we will test different manipulations to control arrestin signaling by altering CB1R dwell times.