Activation-dependent Conformational Changes in β-Arrestin 2

Kunhong Xiao,Sudha K Shenoy,Kelly Nobles,Robert J Lefkowitz

doi:10.1074/jbc.m409785200

Kunhong Xiao, Sudha K Shenoy + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m409785200

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Abstract

Beta-arrestins are multifunctional adaptor proteins, which mediate desensitization, endocytosis, and alternate signaling pathways of seven membrane-spanning receptors (7MSRs). Crystal structures of the basal inactive state of visual arrestin (arrestin 1) and beta-arrestin 1 (arrestin 2) have been resolved. However, little is known about the conformational changes that occur in beta-arrestins upon binding to the activated phosphorylated receptor. Here we characterize the conformational changes in beta-arrestin 2 (arrestin 3) by comparing the limited tryptic proteolysis patterns and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) profiles of beta-arrestin 2 in the presence of a phosphopeptide (V(2)R-pp) derived from the C terminus of the vasopressin type II receptor (V(2)R) or the corresponding nonphosphopeptide (V(2)R-np). V(2)R-pp binds to beta-arrestin 2 specifically, whereas V(2)R-np does not. Activation of beta-arrestin 2 upon V(2)R-pp binding involves the release of its C terminus, as indicated by exposure of a previously inaccessible cleavage site, one of the polar core residues Arg(394), and rearrangement of its N terminus, as indicated by the shielding of a previously accessible cleavage site, residue Arg(8). Interestingly, binding of the polyanion heparin also leads to release of the C terminus of beta-arrestin 2; however, heparin and V(2)R-pp have different binding site(s) and/or induce different conformational changes in beta-arrestin 2. Release of the C terminus from the rest of beta-arrestin 2 has functional consequences in that it increases the accessibility of a clathrin binding site (previously demonstrated to lie between residues 371 and 379) thereby enhancing clathrin binding to beta-arrestin 2 by 10-fold. Thus, the V(2)R-pp can activate beta-arrestin 2 in vitro, most likely mimicking the effects of an activated phosphorylated 7MSR. These results provide the first direct evidence of conformational changes associated with the transition of beta-arrestin 2 from its basal inactive conformation to its biologically active conformation and establish a system in which receptor-beta-arrestin interactions can be modeled in vitro.

Highlights

In stark contrast to the multiplicity of 7MSRs, there are only four known isoforms of arrestin: visual arrestin, cone arrestin, ␤-arrestin 1, and ␤-arrestin 2 [3, 8]
We characterize the conformational changes in ␤-arrestin 2 by comparing the limited tryptic proteolysis patterns and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) profiles of ␤-arrestin 2 in the presence of a phosphopeptide (V2R-pp) derived from the C terminus of the vasopressin type II receptor (V2R) or the corresponding nonphosphopeptide (V2R-np)
The V2R-pp can activate ␤-arrestin 2 in vitro, most likely mimicking the effects of an activated phosphorylated 7MSR. These results provide the first direct evidence of conformational changes associated with the transition of ␤-arrestin 2 from its basal inactive conformation to its biologically active conformation and establish a system in which receptor-␤-arrestin interactions can be modeled in vitro

Summary

EXPERIMENTAL PROCEDURES

Peptide Synthesis—The phosphopeptide (V2R-pp) and the corresponding nonphosphopeptide (V2R-np) derived from the C terminus of the human V2R were synthesized by the Protein Chemistry Core Laboratory of Baylor College of Medicine. The fragments with more than one possible theoretical mass (m/z), which could not be distinguished by MALDI-TOF MS, were designated by coupling the MS data with N-terminal sequencing, Western blot analysis with antibodies that recognize different domains of ␤-arrestin 2, and limited tryptic proteolysis of truncated ␤-arrestin 2 mutants. Clathrin Binding—To measure clathrin binding to ␤-arrestin 2, 25 ␮l of ␤-arrestin 2 S-protein beads (containing 20 ␮g of S-␤-arrestin 2), in the absence or presence of 5:1 (ligand:␤-arrestin 2) molar ratio of ligand (V2R-pp, V2R-np, 28-mer peptide or heparin) were incubated in binding buffer (25 mM Tris-HCl, pH 8.5, 150 mM NaCl, 2 mM EDTA, 1 mM DTT) at room temperature for 30 min. Blots were re-probed with an anti-␤-arrestin 2 antibody (A2CT) to ensure equal loading of ␤-arrestin 2 for each reaction

RESULTS AND DISCUSSION

We next performed a competition binding experiment of

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