Key phosphorylation sites in GPCR s orchestrate the contribution of β‐Arrestin 1 in ERK 1/2 activation

Abstract

β‐arrestins (βarrs) are key regulators of G protein‐coupled receptor (GPCR) signaling and trafficking, and their knockdown typically leads to a decrease in agonist‐induced ERK1/2 MAP kinase activation. Interestingly, for some GPCRs, knockdown of βarr1 augments agonist‐induced ERK1/2 phosphorylation although a mechanistic basis for this intriguing phenomenon is unclear. Here, we use selected GPCRs to explore a possible correlation between the spatial positioning of receptor phosphorylation sites and the contribution of βarr1 in ERK1/2 activation. We discover that engineering a spatially positioned double‐phosphorylation‐site cluster in the bradykinin receptor (B2R), analogous to that present in the vasopressin receptor (V2R), reverses the contribution of βarr1 in ERK1/2 activation from inhibitory to promotive. An intrabody sensor suggests a conformational mechanism for this role reversal of βarr1, and molecular dynamics simulation reveals a bifurcated salt bridge between this double‐phosphorylation site cluster and Lys294 in the lariat loop of βarr1, which directs the orientation of the lariat loop. Our findings provide novel insights into the opposite roles of βarr1 in ERK1/2 activation for different GPCRs with a direct relevance to biased agonism and novel therapeutics.

Arrestin-Dependent and -Independent Internalization of G Protein–Coupled Receptors: Methods, Mechanisms, and Implications on Cell Signaling

“…Although it is tempting to speculate that some of these biosensors could provide evidence for arrestin activation without arrestin C-tail displacement, such inferences are notoriously difficult to make for biosensors. Recently, the biosensor experiments were corroborated by a study using an intracellularly expressed antibody fragment (intrabody) specific for active arrestin-2 (Baidya et al, 2020). The binding of the intrabody to arrestin-2 was triggered by ligand stimulation of the V 2 vasopressin receptor, but not by the B 2 bradykinin receptor, although both recruited arrestin-2 to a similar extent.…”

The G Protein-Coupled Receptor Kinases (GRKs) in Chemokine Receptor-Mediated Immune Cell Migration: From Molecular Cues to Physiopathology

“…This coordinated action of different GRKs would be translated into distinct biological outcomes upon phosphorylation barcoding [ 42 , 43 , 44 ], which is envisioned as a general signalling hub that would be biased by the conformation of the receptor notably induced by the binding of arrestins and cellular effectors, the nature of the chemokine ligand ( Figure 1 b) and the possible allosteric interactions with other receptors (i.e., oligomerization, Figure 1 c), including atypical chemokine receptors, which are known for their regulatory effect on chemoattractant signals. Functional evidence for this concept of phosphorylation barcoding was provided by recent studies revealing that switching the code pattern of phosphosites between GPCRs reprograms arrestin activation [ 45 , 46 ].…”

Keys to the Kingdom: GPCR phosphorylation patterns direct β‐arrestin

“…Function-based evidence and recent structure-based evidence have suggested that patterns of receptor phosphorylation ("barcodes") may be a critical determinant of b-arrestin action. In this issue of EMBO Reports, Baidya and colleagues (Baidya et al, 2020a) report that specific receptor phosphorylation site clusters ("codes") determine whether b-arrestin 1 acts to promote or inhibit receptor activation of Erk mitogen-activated protein kinases. They provide direct evidence for a functional barcode system by transferring inhibitory and stimulatory codes between receptors, suggesting future work to understand just how code site location in a receptor and its phosphorylation status can lead to very different functions of bound b-arrestin proteins.…”

“…The authors also make use of an intrabody as a sensor to label a b-Arr1 active conformation after activation of their mutant receptors, which demonstrates a shift in b-Arr1 active conformation upon switching phosphocodes (Baidya et al, 2020a). A recent study by the same authors highlights the need for additional such sensors that detect further distinct active conformations (Baidya et al, 2020b).…”

“…Since b-arrestins are general phospho-GPCR binding proteins, the rules governing their recognition of specific phosphocodes and their translation of that into activity of bound signaling partners may be quite complex. Indeed, putative code sites very often are multiplexed/interleaved due to clustering of Ser/Thr residues in GPCRs; for example, a PxxPxxP motif in the V2 receptor is also a PxPxxP motif, due to additional phosphorylation sites within this short stretch of amino acids, and two PxxPxxP motifs in the B2 receptor overlap (i.e., PxxPxxPxxP) (Baidya et al, 2020a). Another complication is that due to ease of purification, b-Arr1 has been used in most structural studies to date, while functional studies often focus on b-Arr2, so how applicable these models are to GPCR recognition by b-Arr2 remains to be determined.…”