A key GPCR phosphorylation motif discovered in arrestin2⋠CCR5 phosphopeptide complexes.

The two non-visual arrestins, arrestin2 and arrestin3, bind hundreds of GPCRs with different phosphorylation patterns, leading to distinct functional outcomes. Structural information on these interactions is available only for very few GPCRs. Here, we have characterized the interactions between the phosphorylated human CC chemokine receptor 5 (CCR5) and arrestin2. We identified several new CCR5 phosphorylation sites necessary for stable arrestin2 complex formation. Structures of arrestin2 in the apo form and complexes with CCR5 C-terminal phosphopeptides, together with NMR, biochemical, and functional assays, revealed three phosphoresidues in a pXpp motif that are essential for arrestin2 binding and activation. The identified motif appears responsible for robust arrestin2 recruitment in many other GPCRs. An analysis of receptor sequences and available structural and functional information provides hints on the molecular basis of arrestin2/arrestin3 isoform specificity. Our findings demonstrate how multi-site phosphorylation controls GPCR⋅arrestin interactions and provide a framework to probe the intricate details of arrestin signaling.

Allosteric modulation of GPCR-induced ß-arrestin trafficking and signaling by a synthetic intrabody.

Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) is a primary determinant of ß-arrestin (ßarr) recruitment and trafficking. For several GPCRs such as the vasopressin receptor subtype 2 (V2R), agonist-stimulation first drives the translocation of ßarrs to the plasma membrane, followed by endosomal trafficking, which is generally considered to be orchestrated by multiple phosphorylation sites. We have previously shown that mutation of a single phosphorylation site in the V2R (i.e., V2RT360A) results in near-complete loss of ßarr translocation to endosomes despite robust recruitment to the plasma membrane, and compromised ERK1/2 activation. Here, we discover that a synthetic intrabody (Ib30), which selectively recognizes activated ßarr1, efficiently rescues the endosomal trafficking of ßarr1 and ERK1/2 activation for V2RT360A. Molecular dynamics simulations reveal that Ib30 enriches active-like ßarr1 conformation with respect to the inter-domain rotation, and cellular assays demonstrate that it also enhances ßarr1-ß2-adaptin interaction. Our data provide an experimental framework to positively modulate the receptor-transducer-effector axis for GPCRs using intrabodies, which can be potentially integrated in the paradigm of GPCR-targeted drug discovery.