An additional phosphate-binding element in arrestin molecule - Implications for the mechanism of arrestin activation

Arrestins quench the signaling of a wide variety of G protein-coupled recep tors by virtue of high-affinity binding to phosphorylated activated recepto rs, The high selectivity of arrestins for this particular functional form o f receptor ensures their timely binding and dissociation. In a continuing e ffort to elucidate the molecular mechanisms responsible for arrestin's sele ctivity, 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) a nd Lys(15). Charge elimination and reversal in positions 14 and 15 dramatic ally reduce arrestin binding to phosphorylated light-activated rhodopsin (P -Rh*). The same mutations in the context of various constitutively active a rrestin 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 result s 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 hydrophobi c side chains of residues 11-13 (triple mutation V11A, I12A, and F13A) mode rately enhances arrestin binding to P-Rh and Rh*, The effects of triple mut ation V11A, I12A, and F13A in the context of phosphorylation-independent mu tants 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 wi th alternative partners. In the context of the recently solved crystal stru cture of arrestin's basal state, these findings allow us to propose a model of initial phosphate-driven structural rearrangements in arrestin that ult imately result in its transition into the active receptor-binding state.