837.13 - G protein-coupled receptor kinases regulate β-arrestin interactions with the D2 dopamine receptor in an isoform-specific manner and in the absence of direct receptor phosphorylation

Noelia Boldizsar (Molecular Neuropharmacology Section, NINDS, NIH), Marta Sanchez-Soto (Molecular Neuropharmacology Section, NINDS, NIH), Kayla Schardien (Molecular Neuropharmacology Section, NINDS, NIH), Julia Drube (Institute of Molecular Cell Biology, Jena University Hospital), Raphael Haider (Institute of Molecular Cell Biology, Jena University Hospital), R. Free (Molecular Neuropharmacology Section, NINDS, NIH), Carsten Hoffmann (Institute of Molecular Cell Biology, Jena University Hospital), David Sibley (Molecular Neuropharmacology Section, NINDS, NIH)

The D2 dopamine receptor (D2R) regulates numerous CNS functions including movement, cognition, emotion, and reward circuitry. Agonist binding to the D2R promotes the activation of G protein-mediated signaling and the recruitment of β-arrestin to the receptor. Activated D2Rs are also phosphorylated by members of the G protein-coupled receptor kinase (GRK) family. GRK-mediated receptor phosphorylation is canonically thought to promote β-arrestin recruitment to GPCRs, however, previous research has shown that receptor phosphorylation is not required for β-arrestin recruitment to the D2R. Thus, the role of GRKs in D2R-β-arrestin interactions remains unclear. In the current study, we examined the relationship between GRKs and β-arrestin recruitment using GRK overexpression and GRK knockout cells engineered using CRISPR-Cas9 technology. Overexpression of individual GRK isoforms in parental HEK293 cells potentiated D2R-β-arrestin interactions to varying degrees depending on the isoform. Complete knockout of all GRK isoforms in HEK293 cells (GRK-KO) significantly abrogated, but did not eliminate, β-arrestin recruitment to the D2R. Further, knockout of individual GRK isoforms had variable effects on D2R-β-arrestin interactions ranging from no effect to significant inhibition. Expression of individual GRK isoforms in the GRK-KO cells rescued D2R-mediated β-arrestin recruitment to levels seen in the parental HEK293 cells. Overall, in these experiments, GRK2 seemed to exhibit the greatest modulatory effects on D2R-β-arrestin interactions. We next evaluated the role of receptor phosphorylation in GRK-mediated effects using a phosphorylation-deficient (PO4-null) D2R mutant (Namkung et. al., JBC 284:34103, 2009). The WT-D2R and PO4-null-D2R exhibited identical levels of agonist-stimulated β-arrestin interactions as well as receptor internalization. Notably, inhibition of GRK catalytic activity attenuated agonist-stimulated β-arrestin recruitment to both the WT-D2R and PO4-null-D2R, suggesting the existence of an unknown GRK substrate. Upon recruitment to GPCRs, β-arrestins undergo conformational changes that correlate with their downstream signaling effects. Consequently, we sought to investigate if GRKs play a role in β-arrestin conformational changes seen upon D2R activation. To probe these phenomena, we used intramolecular fluorescent arsenical hairpin (FlAsH) nanoBRET β-arrestin biosensors.  We found that GRK2 increased β-arrestin conformational changes induced by the WT-D2R and PO4-null-D2R. Further, activation of the PO4-null-D2R induced greater conformational changes in β-arrestin than with the WT-D2R, suggesting that receptor phosphorylation does not mediate β-arrestin activation by this GPCR. Taken together, these data suggest that GRKs function noncanonically to modulate D2R-β-arrestin interactions and highlight the need to identify additional GRK substrates to better inform our understanding of GPCR signaling.     

Support or Funding Information

Support for this work was provided by NINDS IRP.