Figure 3.

Disruption of key MT₂ activation switches by genetic variants characterized by a global signaling impairment. (a) Side view of the crystal structure of the agonist-bound human MT₂ in the inactive state showing the location of conserved residues of the water-mediated polar network—depicted by red dots. S123^3.35 (green) is in the vicinity of D86^2.50, while I223^5.61 and F250^6.34 (orange) are packed against each other below Y220^5.58 and nearby the key functional residues R138^3.50 of the DRY motif and Y308^7.49 of the NPxxY motif. (b) Melatonin dose–response curves of Gα_z activation by WT MT₂ (black curve), MT₂-S123R^3.35 (dotted red curve), MT₂-S123E^3.35 (dotted blue curve), or in the absence of transfected receptor (Mock; dotted gray curve). (c) Side view of the crystal structure of MT₂ showing R222^5.60 (orange) and R316^8.51 (green) facing the nearby DRY and NPxxY motifs and ICL4. (d) Melatonin dose–response curves of Gα_z activation by WT MT₂ (black curve), MT₂-R222H^5.60 or MT₂-R316H^8.51 (red dotted curve), and MT₂-R222E^5.60 or MT₂-R316E^8.51 (blue dotted curve). Each point represents the mean ± SEM of five independent experiments performed in quadruplicate (distinct samples). The statistical differences between melatonin-induced maximal responses (E_max) were assessed by comparing the best-fit values of top (E_max) (*p = 0.0109, **p = 0.0038, and ****p < 0.0001).