G protein‐coupled receptor kinases can enhance beta‐arrestin recruitment to the D2 dopamine receptor in the absence of receptor phosphorylation
Authors: Schardien Kayla A. , Soto Marta Sanchez , Willette Blair K.A. , Gonye Elizabeth C. , Moritz Amy E. , Free R. Benjamin , Sibley David R. 
Published: June 2021

The D2 dopamine receptor (D2R) is a highly validated drug target that signals through two major pathways, namely activation of G proteins or the recruitment of β‐arrestin, a multivalent scaffolding protein that coordinates downstream signaling events. Agonist binding to GPCRs is believed to stabilize an active state of the receptor that activates G proteins, but also one that facilitates receptor phosphorylation by one or more members of the G protein‐coupled receptor kinase (GRK) family. GRK‐mediated phosphorylation is widely believed to enhance receptor‐β‐arrestin interactions. We recently identified F189 (residue 5.38 using Ballesteros‐Weinstein numbering) within the ligand binding site as a micro‐switch for regulating D2R interactions with β‐arrestin (see Sanchez‐Soto et al., this meeting). When this residue (F189) is mutated to an alanine, the mutant D2R is still capable of activating G proteins, but is unable to recruit β‐arrestin, i.e., the F189A D2R is a G protein biased mutant. To investigate the mechanisms underlying the biased signaling of the F189A D2R, its interactions with GRKs were explored using a variety of bioluminescence resonance energy transfer (BRET) assays and compared to the wild‐type D2R (D2R‐WT). Both GRK2 and GRK5 have been shown to phosphorylate the D2R‐WT in response to agonist stimulation, and overexpression of either can enhance the ability of dopamine to promote β‐arrestin recruitment to the receptor. Further, we found that, whereas dopamine stimulates direct GRK2 interactions with the D2R‐WT, GRK5 and D2R appear to be constitutively associated. Importantly, we found that the G protein‐biased F189A D2R mutant is incapable of recruiting and interacting with GRK2 upon agonist stimulation suggesting that the absence of these interactions be the underlying basis for the mutant receptor's inability to recruit β‐arrestin. To further test this hypothesis, dopamine‐induced β‐arrestin and GRK2 recruitment were examined using phosphorylation‐defective D2R mutants (Namkung et. al., JBC 284:34103, 2009), catalytically inactive GRK mutants (GRK2‐K220R and GRK5‐K215R), or the GRK2/3 inhibitor compound 101. Strikingly, the phosphorylation‐null D2R mutants were not impaired at all in their ability to recruit either GRK2 or β‐arrestin; in fact, receptor internalization was actually enhanced with these mutants. In addition, GRK2‐K220R or GRK5‐K215R overexpression, or treatment with compound 101, all resulted in decreased β‐arrestin recruitment to the D2R, suggesting that the enzymatic activities of these GRKs are required enhancement of β‐arrestin recruitment. These results suggest that while GRKs play a role in regulating β‐arrestin recruitment to the D2R, and thus can mediate receptor signaling bias, receptor phosphorylation itself does not appear to be the underlying mechanism for this activity.

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This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.