Molecular basis of G protein-coupled receptor function

激动剂与GPCRs结合可通过GPCRK使激活的受体快速磷酸化。这个过程促进arrestin蛋白家族的成员(β-arrestin-1和-2)与激活的受体结合，破坏受体/G蛋白的偶联，并通过将受体靶向笼状蛋白包裹的凹坑来促进GPCR内化。然而，在过去的二十年里，许多研究表明，β-抑制素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。 尽管最近在了解β-arrestin功能的分子机制方面取得了进展，但异源三聚体G蛋白与β-arrestin介导的过程的潜在相互作用仍有待研究。 具有不同功能特性的突变型M3受体的研制(偏向受体) M3受体(M3R)是A类视紫红质样受体的原型。激动剂占据M3R导致GQ家族G蛋白的激活。然而，像绝大多数GPCRs一样，激活的M3R也招募介导M3R脱敏的β-arrestin-1和-2(分别为barr1和barr2)。此外，barr1/2与激活的M3R结合导致构象变化，使barr1和barr2作为各种细胞内信号级联的支架蛋白。为了深入了解Barr1/2信号对M3R功能的贡献，我们开发了两个有偏见的突变体M3R。一个突变的M3R与GQ结合，但不招募β-阻滞剂。另一个突变体M3R不激活GQ，但保留与barr1/2相互作用的能力。目前，我们正在对这两个突变体M3R进行结构和功能研究，以阐明β-arrestins在体外和体内M3R信号转导中的作用。

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 two decades, 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. Development of mutant M3 muscarinic receptors with distinct functional properties (biased receptors) The M3 muscarinic receptor (M3R) is a prototypic class A GPCR (rhodopsin-like GPCR). Agonist occupation of the M3R leads to the activation of G proteins of the Gq family. However, like the vast majority of GPCRs, the activated M3R 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 developed two biased mutant M3Rs. One mutant M3R couples to Gq but does not recruit beta-arrestins. The other mutant M3R does not activate Gq but retains the ability to interact with barr1/2. Currently, we are carrying out structural and functional studies with these two mutant M3Rs to elucidate the roles of beta-arrestins in M3R signaling in vitro and in vivo.