The second extracellular loop of GPCRs determines subtype-selectivity and controls efficacy as evidenced by loop exchange study at A2 adenosine receptors.

The second extracellular loop (EL2) of G protein-coupled receptors (GPCRs), which represent important drug targets, may be involved in ligand recognition and receptor activation. We studied the closely related adenosine receptor (AR) subtypes A2A and A2B by exchanging the complete EL2 of the human A2BAR for the EL2 of the A2AAR. Furthermore, single amino acid residues (Asp148(45.27), Ser149(45.28), Thr151(45.30), Glu164(45.43), Ser165(45.44), and Val169(45.48)) in the EL2 of the A2BAR were exchanged for alanine. The single mutations did not lead to any major effects, except for the T151A mutant, at which NECA showed considerably increased efficacy. The loop exchange entailed significant effects: The A2A-selective agonist CGS21680, while being completely inactive at A2BARs, showed high affinity for the mutant A2B(EL2-A2A)AR, and was able to fully activate the receptor. Most strikingly, all agonists investigated (adenosine, NECA, BAY60-6583, CGS21680) showed strongly increased efficacies at the mutant A2B(EL2-A2A) as compared to the wt AR. Thus, the EL2 of the A2BAR appears to have multiple functions: besides its involvement in ligand binding and subtype selectivity it modulates agonist-bound receptor conformations thereby controlling signalling efficacy. This role of the EL2 is likely to extend to other members of the GPCR family, and the EL2 of GPCRs appears to be an attractive target structure for drugs.