We recently identified a signaling‐biased agonist of the D2 dopamine receptor (D2R), MLS1547, that stimulates G protein‐mediated signaling, but is ineffective in recruiting β‐arrestin. MLS1547 was found to uniquely interact with a hydrophobic binding pocket formed by residues I184, F189, and V190 at the interface between the fifth transmembrane segment (TM5) and the second extracellular loop (EL2) of the D2R. Detailed investigations on the role of these specific residues lead to the identification of F189 (residue 5.38 using Ballesteros‐Weinstein numbering) as a micro‐switch for regulating D2R interactions with β‐arrestin. As position 5.38 is relatively conserved (frequently Phe or Tyr) in class A GPCRs, we constructed alanine mutations (Phe/Tyr to Ala) at this position within all D2‐like receptors (D2R, D3R, and D4R), the β2‐adrenergic receptor (β2R) and the V2 vasopressin receptor (V2R). Strikingly, we found that the alanine 5.38 mutations negated the receptors' ability to recruit β‐arrestin in response to agonists, while G protein‐signaling efficacy was maintained. These data suggest that the presence of a Phe or Tyr residue at position 5.38 in these GPCRs is critical for stabilizing an activate state for recruiting β‐arrestin. To investigate how alterations at this position produce conformational rearrangements resulting in signaling bias, we used the β2R, for which active state crystal structures are available, to build both β2R‐WT and β2R‐Y199A models in complex with the full agonist BI‐167107, and performed extensive molecular dynamics simulations. Using this approach, we identified residues that differentially interact with BI‐167107 in the β2R‐WT vs. β2R‐Y199A leading to conformational rearrangements that propagate through the TM3‐TM4‐TM5 interface to the intracellular side of the receptor. These coordinated changes result in a different tilt of TM4, face shift of TM4 and TM5 on their extracellular sides, and an altered orientation of IL2 in the β2R‐Y199A compared to the β2R‐WT. Strikingly, such coordinated changes and altered IL2 conformations are reminiscent of the differences between the recently solved cryo‐EM structure of the rhodopsin‐Gi complex and the crystal structure of the rhodopsin‐β‐arrestin complex. These results describe a structural basis for how ligand binding site alterations can modulate GPCR coupling to different transducers resulting in biased signaling.Support or Funding InformationNINDS Intramural Research ProgramThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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 modeling led to a structural model for biased signaling that entails a hydrophobic binding pocket formed by residues I184, F189, and V190 at the interface between the fifth transmembrane segment (TM5) and the second extracellular loop of the D2R. In the current study, we used mutagenesis to investigate the role of these residues in regulating signaling by the D2‐like receptors (D2R, D3R, and D4R), and the β2‐adrenergic receptor (B2R). We constructed point mutations at I184, V190 and F189 of the D2R, and at the aligned residues for F189 (i.e., position 5.38) within the D3R, D4R, and B2R, and studied their effects on G protein‐mediated signaling and β‐arrestin recruitment using BRET‐based biosensors and in vitro signaling assays. The D2R point mutations I184A and V190A produced a small change in the potency of DA for stimulating β‐arrestin recruitment or G protein activation. Strikingly, the F189A mutation ablated the ability of DA and other D2R agonists to recruit β‐arrestin while G protein‐signaling efficacy was maintained. Further, the D2R F189A mutant was unable to directly interact with GRK2 and failed to internalize from the cell surface following DA stimulation. In addition, we found that mutating the residues in the D3R, D4R, and B2R at position 5.38 to Ala resulted in parallel findings (i.e., loss of agonist‐stimulated β‐arrestin recruitment, but minimal to no effects on G protein‐mediated signaling). These data demonstrate that the D2R F189A mutant, and similarly mutated D3R, D4R, and B2R, are highly biased towards G protein‐mediated signaling and suggest that the presence of a Phe or Tyr residue at this position (5.38) is important for stabilizing an activated state for recruiting β‐arrestin. Conformational changes propagated through TM5 might thus act as a molecular switch for receptor signaling via β‐arrestin recruitment. These results may have implications for the design of novel signaling‐biased compounds for the treatment of GPCR‐related disorders.Support or Funding InformationNINDS IRPThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

D2 dopamine receptors (D2Rs) are a part of the G protein‐coupled receptor (GPCR) superfamily, and have been linked to the etiology and/or therapy of numerous neuropsychiatric disorders, such as Parkinson's disease and schizophrenia. While the D2R is a highly validated drug target, the receptor signals through multiple pathways, some of which may result in adverse side effects in patients. Signaling‐biased agents that selectivity stimulate, or block, specific D2R‐mediated pathways could represent improved therapeutics. We recently identified a G‐protein signaling‐biased D2R agonist (MLS1547) that has minimal ability to recruit β‐arrestin. Molecular modeling of MLS1547‐D2R interactions revealed a key residue, F189, at the interface between the fifth transmembrane segment (TM5) and the second extracellular loop that is involved in the biased signaling properties of MLS1547. We mutated this residue in the D2R (F189A) and found, using a variety of signaling assays, that this mutation prevents the recruitment of β‐arrestin with minimal effects on G protein signaling, i.e., the F189A D2R is a G protein signaling‐biased mutant. To investigate the mechanisms of this biased signaling we explored the interactions of the wild‐type D2R and F189 mutant with G‐protein coupled receptor kinase 2 (GRK2), which has been shown to phosphorylate the D2R. Initially, we found that over‐expression of GRK2 can enhance the ability of dopamine to recruit β‐arrestin to the WT receptor. Similarly, using a GRK2‐D2R BRET assay to directly assess D2R‐GRK2 interactions, we found that dopamine stimulation increases GRK2‐D2R interactions. Surprisingly, we found that the F189A D2R mutant is unable to recruit GRK2 upon agonist activation suggesting that GRK2‐D2R interactions may be linked to recruiting β‐arrestin. In another series of experiments, we sought to determine if these effects of GRK2 are mediated by receptor phosphorylation. We examined agonist‐induced β‐arrestin and GRK2 recruitment using previously described phosphorylation‐defective D2R mutants (Namkung et. al., JBC 284:34103, 2009). Interestingly, we found that the D2R phosphorylation‐null mutants were not impaired in their ability to recruit either GRK2 or β‐arrestin – in fact, β‐arrestin recruitment and receptor internalization were actually enhanced. These results suggest that GRK2‐D2R interactions may play a critical role in regulating the biased signaling properties of the D2R, but not through phosphorylation of the receptor. Further, GRK2 may enhance β‐arrestin recruitment by mechanisms other than D2R phosphorylation.Support or Funding InformationNINDS IRPThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.