Importance of the second extracellular loop for melatonin MT1 receptor function and absence of melatonin binding in GPR50

Background and Purpose Recent crystal structures of G protein-coupled receptors (GPCRs) highlight the previously unappreciated role of the 2nd extracellular (E2) loop in ligand binding and gating and receptor activation. Here we studied the role of the E2 loop in the activation of the melatonin MT1 receptor (MT1) and in the inactivation of the closely related orphan GPR50 receptor. Experimental Approach Chimeric MT1-GPR50 receptors were generated and analyzed for 2-[125I]iodomelatonin binding, Gi/cAMP signaling and β-arrestin 2 recruitment assisted by computational molecular dynamics (MD) simulations. Key Results MD simulations of 300 ns revealed (i) the tight hairpin structure of the E2 loop of MT1, (ii) the most suitable features for melatonin binding in MT1 and (iii) major predicted rearrangements upon MT1 activation stabilizing interaction networks between Phe179 or Gln181 in the E2 loop and transmembrane helixes 5 and 6. Functional assays confirmed these predictions as reciprocal replacement of MT1 and GPR50 residues/domains led to the predicted loss- and gain-of-melatonin action of MT1 and GPR50, respectively. Conclusion and Implications Our work demonstrates the crucial role of the E2 loop for MT1 and GPR50 function by proposing a model in which the E2 loop is important in stabilizing active MT1 conformations and by showing how evolutionary processes selected E2 loop modifications to inactive GPR50.