Unexpected role of a conserved domain in the first extracellular loop in G protein-coupled receptor trafficking

Highlights: • Most Class A GPCRs contain a conserved WxFG motif. • Substitution of the W with a non-aromatic amino acid disrupts GPCR functioning. • Disrupted GPCR functioning is the due to aberrant plasma membrane localization. G protein-coupled receptors are the largest superfamily of cell surface receptors in the Metazoa and play critical roles in transducing extracellular signals into intracellular responses. This action is mediated through conformational changes in the receptor following ligand binding. A number of conserved motifs have critical roles in GPCR function, and here we focus on a highly conserved motif (WxFG) in extracellular loop one (EL1). A phylogenetic analysis documents the presence of the WxFG motif in ∼90% of Class A GPCRs and the motif is represented in 17 of the 19 Class A GPCR subfamilies. Using site-directed mutagenesis, we mutagenized the conserved tryptophan residue in eight receptors which are members of disparate class A GPCR subfamilies from different taxa. The modification of the Drosophila leucokinin receptor shows that substitution of any non-aromatic amino acid for the tryptophan leads to a loss of receptor function. Additionally, leucine substitutions at this position caused similar signaling defects in the follicle-stimulating hormone receptor (FSHR), Galanin receptor (GALR1), AKH receptor (AKHR), corazonin receptor (CRZR), and muscarinic acetylcholine receptor (mACHR1). Visualization of modified receptors through the incorporation of a fluorescent tag revealed a severe reduction in plasma membrane expression, indicating aberrant trafficking of these modified receptors. Taken together, these results suggest a novel role for the WxFG motif in GPCR trafficking and receptor function.