Extracellular Surface Residues of theα1B-Adrenoceptor Critical for G Protein–Coupled Receptor Function

Lotten Ragnarsson, Åsa Andersson, Walter G. Thomas, Richard J. Lewis

Ligand binding and conformational changes that accompany signaling from G protein-coupled receptors (GPCRs) have mostly focused on the role of transmembrane helices (TMHs) and intracellular loop regions. However, recent studies, including several GPCRs co-crystallized with bound ligands, clearly show that the extracellular surface (ECS) of GPCRs plays an important role in ligand recognition, selectivity and binding, as well as potentially contributing to receptor activation and signaling. This study applied alanine-scanning mutagenesis to investigate the role of the complete extracellular surface (ECS) of the α1B-AR on norepinephrine (NE) potency, affinity and efficacy. Half (24 of 48) of the ECS mutations significantly decreased NE potency in an IP-one assay. Most mutations reduced NE affinity (17) determined from 3H-prazosin displacement studies, while four mutations at the entrance to the NE binding pocket enhanced NE affinity. Removing the influence of NE affinity and receptor expression levels on NE potency gave a measure of NE efficacy, which was significantly decreased for 11 of 48 ECS mutants. These different effects tended to cluster to different regions of the ECS, consistent with different regions of the ECS playing discrete functional roles. Exposed ECS residues at the entrance to the NE binding pocket mostly affected NE affinity, while buried or structurally significant residues mostly affected NE efficacy. The broad potential for ECS mutations to affect GPCR function has relevance for the increasing number non-synonymous single nucleotide polymorphisms now being identified in GPCRs.