Activation-dependent conformational changes in {beta}-arrestin 2.
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.
Xiao
Kunhong
K
Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA.
Shenoy
Sudha K
SK
Nobles
Kelly
K
Lefkowitz
Robert J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D019390:Arrestins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002966:Clathrin / Q000737:chemistry; D018360:Crystallography, X-Ray; D005982:Glutathione Transferase / Q000378:metabolism; D006493:Heparin / Q000737:chemistry; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D015398:Signal Transduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000737:chemistry / Q000494:pharmacology; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
C000605256:Arrb1 protein, rat; C000605257:Arrb2 protein, rat; D019390:Arrestins; D002966:Clathrin; D010455:Peptides; D010750:Phosphoproteins; D011994:Recombinant Proteins; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D006493:Heparin; D001120:Arginine; D005982:Glutathione Transferase; D014357:Trypsin