A key GPCR phosphorylation motif discovered in arrestin2•CCR5 phosphopeptide complexes

Abstract

The two non-visual arrestin isoforms, arrestin2 and arrestin3 recognize and bind hundreds of G protein-coupled receptors (GPCRs) with different phosphorylation patterns leading to distinct functional outcomes. The impact of phosphorylation on arrestin interactions has been well studied only for very few GPCRs. Here we have characterized the interactions between the phosphorylated CC chemokine receptor 5 (CCR5) and arrestin2. We detected several new CCR5 phosphorylation sites, which are necessary for stable complex formation with arrestin2. Crystal structures of arrestin2 in apo form and in complexes with CCR5 C-terminal phosphopeptides together with NMR spectroscopy, biochemical and functional assays revealed three phosphoresidues in a pXpp motif that are essential for the arrestin2 interactions and activation. The same phosphoresidue cluster is present in other receptors, which form stable complexes with arrestin2. We propose that the identified pXpp motif is responsible for robust arrestin2 recruitment in many GPCRs. An analysis of available sequences, structural and functional information on other GPCR-arrestin interactions suggests that a particular arrangement of phosphoresidues within the GPCR intracellular loop 3 and C-terminal tail determines arrestin2 and 3 isoform specificity. Taken together, our findings demonstrate how multi-site phosphorylation controls GPCR-arrestin interactions and provide a framework to probe the intricate details of arrestin activation and signaling.

Distinct beta-arrestin coupling and intracellular trafficking of metabotropic glutamate receptor homo- and heterodimers

“…This result shows that merely cointernalizing with β-arrs is not sufficient to drive GPCR trafficking to lysosomes. A possible explanation for this is that different CTDs stabilize different β-arr conformations, consistent with the conformational complexity that has been observed in spectroscopic studies of β-arrs (48,(79)(80)(81)(82). Β-arr conformation likely controls the ability of receptors to interact with key trafficking factors such as ubiquitin, GPCR associated sorting protein-1 (GASP-1), or ALG-2 interacting protein X (ALIX), as has been proposed for other GPCRs (18,(83)(84)(85).…”

“…3A). ST1 contains three ST residues that match the proposed Px(x)PxxP/E/D phospho-codes (P, serine or threonine; E, glutamate; and D, aspartate) as identified from an arrestin-bound rhodopsin crystal structure (47), while ST2 region contains 11 ST three residues, which produce seven overlapping Px(x)PxxP/E/D phospho-codes, as well as two of the recently proposed PxPP phospho-code (48). We deleted either the ST1 or ST2 region in mGluR8 to produce "mGluR8ΔST1" and "mGluR8ΔST2" and examined the properties of each construct (fig.…”

Ligand-Dependent Mechanisms of C-C Chemokine Receptor 5 (CCR5) Trafficking Revealed by APEX2 Proximity Labeling Proteomics

“…GRKs were not observed in the proximity labeling data, consistent with the transient nature of their interactions with GPCRs (52). However, receptor phosphorylation is a pre-requisite for β-arrestin1/2 interactions (23, 53) and thus we hypothesized that the level and pattern of GRK phosphorylation may play important roles in ligand-dependent internalization of CCR5. Corroborating this, β-arrestin2 recruitment to CCR5 and CCR5 internalization showed different sensitivities to knockout of different GRK subtypes in a ligand-dependent manner.…”

Molecular insights into atypical modes of β-arrestin interaction with seven transmembrane receptors

“…Still however, considering the divergent primary sequence and phosphorylation patterns of GPCRs, the molecular mechanisms driving the broadly conserved nature of GPCR-βarr interaction and activation remains elusive to a large extent until recently. Some recent studies however have started to shed light on phosphorylation-mediated components of GPCR-βarr binding through broadly conserved phosphorylation motifs identified in a large number of GPCRs (17)(18)(19)(20). For example, structural and biophysical studies have proposed the framework of phosphorylation codes and modulatory sites in the GPCR carboxyl-terminus as a possible mechanism governing phosphorylation-mediated βarr interaction (19,20).…”

“…For example, structural and biophysical studies have proposed the framework of phosphorylation codes and modulatory sites in the GPCR carboxyl-terminus as a possible mechanism governing phosphorylation-mediated βarr interaction (19,20). More recently, two independent structural studies have identified that the presence of a P-X-P-P type phosphorylation motif in the carboxyl-terminus of a broad set of GPCRs, where P is a phosphorylation site, is a critical determinant of βarr interaction and activation (17,18). Still however, there are several key questions about this versatile interaction that remain unanswered and represent important knowledge gaps in our current understanding of this signaling and regulatory paradigm.…”