Investigating the Interactions of GRK2 with a G‐protein Signaling‐Biased D₂ Dopamine Receptor

Abstract

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.