Helix Formation in Arrestin Accompanies Recognition of Photoactivated Rhodopsin (original) (raw)
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Involvement of distinct arrestin-1 elements in binding to different functional forms of rhodopsin
Proceedings of the National Academy of Sciences, 2013
Solution NMR spectroscopy of labeled arrestin-1 was used to explore its interactions with dark-state phosphorylated rhodopsin (P-Rh), phosphorylated opsin (P-opsin), unphosphorylated light-activated rhodopsin (Rh*), and phosphorylated light-activated rhodopsin (P-Rh*). Distinct sets of arrestin-1 elements were seen to be engaged by Rh* and inactive P-Rh, which induced conformational changes that differed from those triggered by binding of P-Rh*. Although arrestin-1 affinity for Rh* was seen to be low (K D > 150 μM), its affinity for P-Rh (K D ∼80 μM) was comparable to the concentration of active monomeric arrestin-1 in the outer segment, suggesting that P-Rh generated by high-gain phosphorylation is occupied by arrestin-1 under physiological conditions and will not signal upon photo-activation. Arrestin-1 was seen to bind P-Rh* and P-opsin with fairly high affinity (K D of ∼50 and 800 nM, respectively), implying that arrestin-1 dissociation is triggered only upon P-opsin regeneration with 11-cis-retinal, precluding noise generated by opsin activity. Based on their observed affinity for arrestin-1, P-opsin and inactive P-Rh very likely affect the physiological monomer-dimer-tetramer equilibrium of arrestin-1, and should therefore be taken into account when modeling photoreceptor function. The data also suggested that complex formation with either P-Rh* or P-opsin results in a global transition in the conformation of arrestin-1, possibly to a dynamic molten globule-like structure. We hypothesize that this transition contributes to the mechanism that triggers preferential interactions of several signaling proteins with receptor-activated arrestins.
The arrestin-1 finger loop interacts with two distinct conformations of active rhodopsin
The Journal of biological chemistry, 2018
Signaling of the prototypical G protein-coupled receptor (GPCR) rhodopsin through its cognate G protein transducin (G) is quenched when arrestin binds to the activated receptor. Although the overall architecture of the rhodopsin/arrestin complex is known, many questions regarding its specificity remain unresolved. Here, using FTIR difference spectroscopy and a dual pH/peptide titration assay, we show that rhodopsin maintains certain flexibility upon binding the "finger loop" of visual arrestin (prepared as synthetic peptide ArrFL-1). We found that two distinct complexes can be stabilized depending on the protonation state of E3.49 in the conserved (D)ERY motif. Both complexes exhibit different interaction modes and affinities of ArrFL-1 binding. The plasticity of the receptor within the rhodopsin/ArrFL-1 complex stands in contrast to the complex with the C terminus of the G α-subunit (GαCT), which stabilizes only one specific substate out of the conformational ensemble. Ho...
Molecular vision, 2006
The purpose of our study was to determine whether arrestin residues previously predicted by computational modeling to interact with an aspartic acid substituted rhodopsin tail are actually involved in interactions with phospho-residues on the rhodopsin cytoplasmic tail. We generated arrestin mutants with altered charges at predicted positions. These mutants were then tested for the ability to inhibit rhodopsin using both direct binding assays, as well as functional assays involving transducin inhibition assays. Our results demonstrate that the computer-predicted residues are indeed involved in both the ability of the low-affinity state of arrestin to bind to rhodopsin as well as the ability of arrestin to be induced into a higher-affinity state in a phospho-residue-dependent manner. Our results also suggest that positions K14, K15, R29, H301, and K300 on arrestin interact with the phosphorylated carboxyl tail of rhodopsin and that this translates to the efficient activation of arres...
N-terminal and C-terminal Domains of Arrestin Both Contribute in Binding to Rhodopsin†
Photochemistry and Photobiology, 2007
Visual arrestin terminates the signal amplification cascade in photoreceptor cells by blocking the interaction of light activated phosphorylated rhodopsin with the G-protein transducin. Although crystal structures of arrestin and rhodopsin are available, it is still unknown how the complex of the two proteins is formed. To investigate the interaction sites of arrestin with rhodopsin various surface regions of recombinant arrestin were sterically blocked by different numbers of fluorophores (Alexa 633). The binding was recorded by time-resolved light scattering. To accomplish site-specific shielding of protein regions, in a first step all three wild-type cysteines were replaced by alanines. Nevertheless, regarding the magnitude and specificity of rhodopsin binding, the protein is still fully active. In a second step, new cysteines were introduced at selected sites to allow covalent binding of fluorophores. Upon attachment of Alexa 633 to the recombinant cysteines we observed that these bulky labels residing in the concave area of either the Nor the C-terminal domain do not perturb the activity of arrestin. By simultaneously modifying both domains with one Alexa 633 the binding capacity was reduced. The presence of two Alexa 633 molecules in each domain prevented binding of rhodopsin to arrestin. This observation indicates that both concave sites participate in binding.
Arrestin-rhodopsin interaction. Multi-site binding delineated by peptide inhibition
Journal of Biological Chemistry, 1994
Visual arrestin modulates the intracellular response of retinal rod cells to light by specifically binding to the phosphorylated light-activated form of the photoreceptor rhodopsin (P-Rh*). In order to characterize the molecular interaction between rhodopsin and arrestin, we have studied the ability of synthetic peptides from the proposed cytoplasmic loops of rhodopsin to inhibit arrestin binding. A third cytoplasmic loop peptide competed most effectively for arrestin binding to P-Rh*, exhibiting an ICso of 34 p, while a first cytoplasmic loop peptide weakly inhibited binding with an ICso of "1100 p. The first and third cytoplasmic loop peptides also inhibited P-Rh* interaction with both ARR[A(2-18)-404], an arrestin mutant that lacks residues 2-16, and A R R [ 1-1911, a mutant that contains only the amino half of arrestin. However, the third loop peptide had an-5-fold lower affinity at inhibiting the binding of ARR[l-1911 to P-Rh*. While the first and third loop peptides also inhibited arrestin binding to light-activated rhodopsin and a truncated rhodopsin lacking its C-terminal sites of phosphorylation, the peptides modestly enhanced arrestin binding to phosphorylated dark rhodopsin. These results suggest that the third and, to a lesser extent, the first cytoplasmic loops of rhodopsin may play an important role in arrestin binding to light-activated forms of rhodopsin.
Arrestins are regulatory molecules for G-protein coupled receptor function. In visual rhodopsin, selective binding of arrestin to the cytoplasmic side of light-activated, phosphorylated rhodopsin (P-Rh*) terminates signaling via the G-protein transducin. While the "phosphate-sensor" of arrestin for the recognition of receptorattached phosphates is identified, the molecular mechanism of arrestin binding and the involvement of receptor conformations in this process are still largely hypothetic. Here we used fluorescence pump-probe and time-resolved fluorescence depolarization measurements to investigate the kinetics of arrestin conformational changes and the corresponding nanosecond dynamical changes at the receptor surface. We show that at least two sequential conformational changes of arrestin occur upon interaction with P-Rh*, thus providing a kinetic proof for the suggested multistep nature of arrestin binding. At the cytoplasmic surface of P-Rh*, the structural dynamics of the amphipathic helix 8 (H8), connecting transmembrane helix 7 and the phosphorylated C-terminal tail, depends on the arrestin interaction state. We find that a high mobility of H8 is required in the low-affinity (prebinding) but not in the high-affinity binding state. High-affinity arrestin binding is inhibited when a bulky, inflexible group is bound to H8, indicating close interaction. We further show that this close steric interaction of H8 with arrestin is mandatory for the transition from prebinding to high-affinity binding; i.e., for arrestin activation. This finding implies a regulatory role for H8 in activation of visual arrestin, which shows high selectivity to P-Rh* in contrast to the broad receptor specificity displayed by the two nonvisual arrestins. membrane receptor | protein conformational change | binding kinetics
FEBS Letters, 1995
A synthetic heptaphosphopeptide comprising the fully pbosphorylated carboxyl terminal pbosphorylation region of bovine rbodopsin, residues 330-348, was found to induce a conformational change in bovine arrestin. This caused an alteration of the pattern of limited proteolysis of arrestin similar to that induced by binding pbospborylated rbodopsin or heparin. Unlike heparin, the phosphopeptide also induced light-activated binding of arrestin to both unphospborylated rhodopsin in disk membranes as well as to endoproteinase Asp-N-treated rbodopsin (des 330-348). These findings suggest that one function of phosphorylation of rhodopsin is to activate arrestin which can then bind to other regions of the surface of the photoactivated rhodopsin.
Functional Role of Arrestin-1 Residues Interacting With Unphosphorylated Rhodopsin Elements
Arrestin-1, or visual arrestin, exhibits an exquisite selectivity for light-activated phosphorylated rhodopsin (P-Rh*) over its other functional forms. That selectivity is believed to be mediated by two well-established structural elements in the arrestin-1 molecule, the activation sensor detecting the active conformation of rhodopsin and the phosphorylation sensor responsive to the rhodopsin phosphorylation, which only active phosphorylated rhodopsin can engage simultaneously. However, in the crystal structure of the arrestin-1-rhodopsin complex there are arrestin-1 residues located close to rhodopsin, which do not belong to either sensor. Here we tested by site-directed mutagenesis the functional role of these residues in wild type arrestin-1 using direct binding assay to P-Rh* and light-activated unphosphorylated rhodopsin (Rh*). We found that many mutations either enhanced the binding only to Rh* or increased the binding to Rh* much more than to P-Rh*. The data suggest that the ...
Journal of Biological Chemistry, 1998
Arrestin plays an important role in quenching phototransduction via its ability to bind to the phosphorylated light-activated form of the visual receptor rhodopsin (P-Rh*). Remarkable selectivity of visual arrestin toward this functional form is determined by an elegant sequential multisite binding mechanism. Previous structure-function studies have suggested that the COOH-terminal region of arrestin (residues 356-404) is not directly involved in rhodopsin interaction, but instead plays a regulatory role. This region supports basal arrestin conformation and ensures arrestin's transition into a high affinity rhodopsin-binding state upon an encounter with P-Rh*. Overall, our results corroborate this hypothesis and identify three functional subregions (residues 361-368, 369-378, and 379-404) and individual amino acids involved in the control of arrestin stability and binding selectivity. Two of the most potent mutants, arrestin(1-378) and arrestin(F375A,V376A,F377A) belong to a novel class of constitutively active arrestins with high affinity for P-Rh*, dark P-Rh, and Rh* (but not dark Rh), in contrast to earlier constructed mutants arrestin(R175E) and arrestin(⌬2-16) with high affinity for light-activated forms only. The implications of these findings for the mechanism of arrestin-rhodopsin interaction are discussed in light of the recently determined crystal structure of arrestin.