A Critical Role for Transmembrane Five in Regulating Biased Signaling of the D2 Dopamine Receptor
Authors: Gonye Elizabeth C. , Moore Annah M. , Chun Lani S. , Moritz Amy E. , Shi Lei , Sibley David R. , Free R. Benjamin 
Published: November 2023

D2 dopamine receptors (D2Rs) signal through a variety of second messenger pathways which makes it challenging to discern which are linked to specific desired or undesirable effects of D2R‐targeted drugs; however, this complexity also provides a unique opportunity to develop pathway‐selective therapeutics. Our laboratory has previously described MLS1547 as a D2R G‐protein‐biased agonist using a variety of in vitro functional assays.1 MLS1547 robustly activates G‐protein signaling at the receptor with minimal recruitment of β‐arrestin. A molecular model of biased signaling was developed using structure‐activity analyses of a series of MLS1547 analogs with varying bias. The receptor docking site for the biased analogs as defined by the model includes a hydrophobic pocket formed by residues I184, F189, and V190, all located within the fifth transmembrane region (TM5) of the D2R. Here we present an investigation of this receptor region, as well as the role of TM5 in regulating signaling bias at the D2R. We constructed five independent point mutations (I184A, V190A, F189A, F189L, and F189Y) in the D2R. These mutants, along with the wild‐type receptor, were assessed for both G protein‐mediated signaling and β‐arrestin recruitment using BRET and enzyme complementation‐based technologies. Mutation of I184 and V190 decreased MLS1547's efficacy for stimulating G protein‐mediated signaling without affecting DA's efficacy at activation of G‐protein‐mediated signaling or β‐arrestin recruitment. The F189A mutant caused the complete loss of MLS1547's ability to stimulate G protein‐mediated signaling, even though the compound still bound to the receptor and acted as an antagonist; however, the efficacy of DA on G‐protein signaling was unaffected by this mutation. The efficacy of MLS1547 for stimulating G protein‐mediated signaling was also lost in the F189L mutant, but partially maintained in the more conservatively substituted F189Y receptor. In contrast, G protein‐mediated signaling by DA was unaffected by either mutation. Surprisingly, the ability of DA and other orthosteric agonists to recruit β‐arrestin to the F189A mutant was lost while G protein‐signaling efficacy was maintained. DA‐stimulated β‐arrestin recruitment was also lost in F189L mutant, but partially maintained in F189Y receptor. These data suggest that the F189A and F189L D2R mutants are highly biased towards G protein‐mediated signaling. Interestingly, mutation of an analogous TM5 residue in the D3R (F188), resulted in parallel findings (i.e., loss of β‐arrestin recruitment). Our results extend our TM5 model underlying the G protein‐mediated signaling bias of MLS1547, and more importantly show that a single phenylalanine residue in TM5 of either D2R or D3R can affect their recruitment of β‐arrestin. Taken together, these results suggest that conformational changes in TM5 can act as a molecular switch for receptor signaling via β‐arrestin recruitment.

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NINDS Intramural Research Program