Real-time imaging of μ-opioid receptor signaling, phosphorylation and desensitization (C05)

G蛋白受体激酶（GRKs）和β-抑制蛋白（β-arrestins）是μ-阿片受体（莫尔）信号转导和转运的关键调节因子。在之前的资助期间，我们发现GRK 2/3比β-arrestins更快地被招募。β-Arrestin的募集需要GRK 2活性和莫尔磷酸化，但GRK的募集也依赖于C-末端尾部的磷酸化位点。与此相一致，磷酸化缺陷MORs介导强烈增强的体内急性镇痛作用，并且在长期给药后，对芬太尼的镇痛耐受被消除。在这个提议中，我们将（I）描述PKC在非经典莫尔脱敏和耐受中的作用，（II）评估体内和体外非常规β-arrestin信号传导的生理作用，和（III）利用先进的成像技术监测活神经元中内源性莫尔受体组织的激动剂依赖性变化。

G protein receptor kinases (GRKs) and β-arrestins are key regulators of μ-opioid receptor (MOR) signaling and trafficking. In the previous funding period, we showed that GRK2/3 is recruited more quickly than β-arrestins. β-Arrestin recruitment required GRK2 activity and MOR phosphorylation, but GRK recruitment also depended on phosphorylation sites in the C-terminal tail. Consistent with this, phosphorylation-deficient MORs mediate strongly enhanced acute analgesia in vivo and upon chronic administration analgesic tolerance to fentanyl is abrogated. In this proposal, we will (I) delineate the role of PKC in non-canonical MOR desensitization and tolerance, (II) evaluate the physiological role of unconventional β-arrestin signaling in vivo and in vitro, and (III) utilize advanced imaging techniques to monitor agonist-dependent changes in endogenous MOR receptor organization in living neurons.