Molecular basis of G protein-coupled receptor function

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

SUMMARY 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. Generation of G protein- and beta-arrestin-biased mutant M3 muscarinic receptors During the past decades, our lab has extensively analyzed the M3 muscarinic acetylcholine receptor (M3R) as a prototypic class A GPCR (rhodopsin-like GPCR). Following the binding of an extracellular agonist, M3Rs undergo conformational changes, leading to the activation of G proteins of the Gq family. In addition, like most other GPCRs, the activated M3R also recruits beta-arrestin-1 and -2 (barr1 and barr2, respectively) which mediate M3R desensitization but can also act as signaling molecules in their own right. To explore the contribution of barr1/2 signaling to M3R function, we used site-directed mutagenesis to generate two biased mutant M3Rs. One of these mutant M3Rs couples to Gq but does not recruit barr1/2 (G-protein biased M3R). The other mutant M3R does not activate Gq but is still able to interact with barr1/2 (arrestin-biased M3R). Ongoing studies focus on identifying the structures of these receptors and their functional properties both in vitro and in vivo.