Molecular basis of G protein-coupled receptor function

与GPCR结合的激动剂引起GPCR激酶对激活受体的快速磷酸化。这一过程促进了捕虏蛋白家族成员（β -捕虏蛋白-1和-2）向激活受体的招募，破坏了受体/G蛋白的偶联，并通过将受体靶向到网格蛋白包被的凹坑上，促进了GPCR的内化。然而，在过去的10-15年里，许多研究已经证明β -阻滞蛋白本身也可以作为信号分子。这一观察结果不仅具有理论意义，而且具有潜在的临床意义。

Agonist binding to GPCRs causes rapid phosphorylation of the activated receptors by GPCR kinases. This process promotes the recruitment of members of the arrestin protein family (beta-arrestin-1 and -2) to the activated receptors, disrupting receptor/G protein coupling and promoting GPCR internalization by targeting the receptors to clathrin-coated pits. However, during the past 10-15 years, many studies have demonstrated that beta-arrestins can also act as signaling molecules in their own right. This observation is not only of theoretical interest but also of potential clinical relevance. Despite recent advances in understanding the molecular mechanisms of beta-arrestin function, the potential interplay of heterotrimeric G proteins with beta-arrestin-mediated processes remains to be investigated. Generation of biased M3 muscarinic receptors Activation of the M3 muscarinic receptor (M3R) leads to the activation of G proteins of the Gq family. The activated receptor also recruits beta-arrestin-1 and -2 (barr1 and barr2, respectively) which mediate M3R desensitization. In addition, binding of barr1/2 to the activated M3R leads to conformational changes that allow barr1 and barr2 to act as scaffolding proteins for various intracellular signaling cascades. To gain insight into the contribution of barr1/2 signaling to M3R function, we generated two biased mutant M3Rs. One of the mutant receptors couples to Gq but is unable to recruit beta-arrestins. The other mutant receptor is no longer capable of activating Gq but retains the ability to interact with barr1/2. We are planning to carry out structural and functional studies with these two mutant M3Rs to gain insight into the role of beta-arrestins in M3R signaling in vitro and in vivo.