Molecular basis of G protein-coupled receptor function

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

SUMMARY Agonist-activated GPCRs are phosphorylated by a class of protein kinases known as GPCR kinases (GRKs). GRK-phosphorylated receptors can recruit members of the arrestin protein family (beta-arrestin-1 and -2), interfering with receptor/G protein coupling and promoting GPCR internalization. Besides these canonical functions of beta-arrestins, beta-arrestins can also represent signaling molecules in their own right (non-canonical actions of beta-arrestins). 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 Since the early 90s, our lab has extensively analyzed the M3 muscarinic acetylcholine receptor (M3R), a prototypic class A GPCR (rhodopsin-like GPCR). When activated by extracellular ligands, M3Rs undergo conformational changes, leading to the activation of G proteins of the Gq family. Moreover, like most other GPCRs, the activated M3R also recruits beta-arrestin-1 and -2 which mediate M3R desensitization but can also act as signaling molecules in their own right. To explore the contribution of beta-arrestin-1/2 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 beta-arrestins (G-protein biased M3R). The other mutant M3R does not activate Gq but is still able to interact with beta-arrestin-1/2 (arrestin-biased M3R). We currently carry out studies to elucidate the structures of these receptors and their functional properties both in vitro and in vivo.