Program

PO1-1-119

Identification of residues in the fifth transmembrane-spanning domain of the D2-like dopamine receptors that engender signaling bias

[Speaker] Marta Sanchez-Soto:1
[Co-author] Blair K A Willette:1, Elizabeth C Gonye:1, Annah M Moore:1, Lani S Chun:1, Amy E Moritz:1, Hideaki Yano:2, Lei Shi:2, R Benjamin Free:1, David R Sibley:1
1:Molecular Neuropharmacology Section, NINDS, NIH, USA, 2:Computational Chemistry and Molecular Biophysics Unit, NIDA, NIH, USA

BACKGROUND
The D2 dopamine (DA) receptor (D2R) signals through a variety of second messenger pathways making it difficult to discern which of these are linked to specific effects of D2R-targeted drugs; however, this complexity provides a unique opportunity to develop pathway-selective therapeutics. Structure-activity analyses using analogs derived from our previously described D2R G protein-biased agonist, MLS1547, coupled with molecular dynamics led to a molecular model for biased signaling which entailed a hydrophobic binding pocket formed by residues I184, F189, and V190 within the fifth transmembrane region (TM5) and second extracellular loop of the D2R. In the current study, we used mutagenesis to investigate the role of these in regulating signaling bias by the D2-like receptors (D2R, D3R, and D4R), and the B2-adrenergic receptor (B2R).
METHODS
We constructed point mutations at I184, V190 and F189 of the D2R, and at the analogous residues for F189 (5.38) within the D3R, D4R, and B2R, and studied their effects on G protein-mediated signaling and B-arrestin recruitment using BRET-based biosensors and in vitro signaling assays.
RESULTS
Mutating D2R residues I184A and V190A produced a small change in the potency of DA for stimulating B-arrestin recruitment or G protein activation. Strikingly, the F189A mutation completely ablated the ability of D2R agonists to recruit B-arrestin while G protein-signaling efficacy was maintained. Further, the D2R F189A mutant is unable to directly interact with GRK2 and failed to internalize from the cell surface following DA stimulation. We extended our study to the D3R, D4R, and B2R and found that mutating the analogous F189 residue (5.38), resulted in parallel findings (i.e., loss of agonist-stimulated B-arrestin recruitment, but minimal to no effect on G protein-mediated signaling).
CONCLUSIONS
These data demonstrate that the F189A mutant D2R, and similarly mutated D3R, D4R, and B2R, are highly biased towards G protein-mediated signaling and suggest that this residue is important for stabilizing an activation state for recruiting B-arrestin. Therefore, conformational changes in TM5 likely act as a molecular switch for receptor signaling via B-arrestin recruitment. These results may have implications for the design of novel signaling-biased compounds for the treatment of GPCR-related disorders.


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