title: An additional phosphate-binding element in arrestin molecule. Implications for the mechanism of arrestin activation.
abstract: Arrestins quench the signaling of a wide variety of G protein-coupled receptors by virtue of high-affinity binding to phosphorylated activated receptors. The high selectivity of arrestins for this particular functional form of receptor ensures their timely binding and dissociation. In a continuing effort to elucidate the molecular mechanisms responsible for arrestin's selectivity, we used the visual arrestin model to probe the functions of its N-terminal beta-strand I comprising the highly conserved hydrophobic element Val-Ile-Phe (residues 11-13) and the adjacent positively charged Lys(14) and Lys(15). Charge elimination and reversal in positions 14 and 15 dramatically reduce arrestin binding to phosphorylated light-activated rhodopsin (P-Rh*). The same mutations in the context of various constitutively active arrestin mutants (which bind to P-Rh*, dark phosphorylated rhodopsin (P-Rh), and unphosphorylated light-activated rhodopsin (Rh*)) have minimum impact on P-Rh* and Rh* binding and virtually eliminate P-Rh binding. These results suggest that the two lysines "guide" receptor-attached phosphates toward the phosphorylation-sensitive trigger Arg(175) and participate in phosphate binding in the active state of arrestin. The elimination of the hydrophobic side chains of residues 11-13 (triple mutation V11A, I12A, and F13A) moderately enhances arrestin binding to P-Rh and Rh*. The effects of triple mutation V11A, I12A, and F13A in the context of phosphorylation-independent mutants suggest that residues 11-13 play a dual role. They stabilize arrestin's basal conformation via interaction with hydrophobic elements in arrestin's C-tail and alpha-helix I as well as its active state by interactions with alternative partners. In the context of the recently solved crystal structure of arrestin's basal state, these findings allow us to propose a model of initial phosphate-driven structural rearrangements in arrestin that ultimately result in its transition into the active receptor-binding state.
authors.affiliation: Ralph & Muriel Roberts Laboratory for Vision Science, Sun Health Research Institute, Sun City, Arizona 85372, USA.
authors.forename: S A
authors.initials: SA
authors.lastname: Vishnivetskiy
authors.forename: C
authors.initials: C
authors.lastname: Schubert
authors.forename: G C
authors.initials: GC
authors.lastname: Climaco
authors.forename: Y V
authors.initials: YV
authors.lastname: Gurevich
authors.forename: M G
authors.initials: MG
authors.lastname: Velez
authors.forename: V V
authors.initials: VV
authors.lastname: Gurevich
mesh_terms: D000595:Amino Acid Sequence; D019393:Arrestin / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000378:metabolism*; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship
chemical_list: D019393:Arrestin; D010710:Phosphates