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

Recent crystal structures of GPCRs have emphasized the previously unappreciated role of the second extracellular (E2) loop in ligand binding and gating and receptor activation. Here, we have assessed the role of the E2 loop in the activation of the melatonin MT₁ receptor and in the inactivation of the closely related orphan receptor GPR50.

Chimeric MT₁ -GPR50 receptors were generated and functionally analysed in terms of 2-[¹²⁵ I]iodomelatonin binding, G_i /cAMP signalling and β-arrestin2 recruitment. We also used computational molecular dynamics (MD) simulations.

MD simulations of 300 ns revealed (i) the tight hairpin structure of the E2 loop of the MT₁ receptor (ii) the most suitable features for melatonin binding in MT₁ receptors and (iii) major predicted rearrangements upon MT₁ receptor activation, stabilizing interaction networks between Phe179 or Gln181 in the E2 loop and transmembrane helixes 5 and 6. Functional assays confirmed these predictions, because reciprocal replacement of MT₁ and GPR50 residues/domains led to the predicted loss- and gain-of-melatonin action of MT₁ receptors and GPR50 respectively.

Our work demonstrated the crucial role of the E2 loop for MT₁ receptor and GPR50 function by proposing a model in which the E2 loop is important in stabilizing active MT₁ receptor conformations and by showing how evolutionary processes appear to have selected for modifications in the E2 loop in order to make GPR50 unresponsive to melatonin.

This article is part of a themed section on Recent Developments in Research of Melatonin and its Potential Therapeutic Applications. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.16/issuetoc.