Transition of Arrestin into the Active Receptor-binding State Requires an Extended Interdomain Hinge (original) (raw)
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The Role of Arrestin α-Helix I in Receptor Binding
Journal of Molecular Biology, 2010
Arrestins rapidly bind phosphorylated activated forms of their cognate G protein-coupled receptors (GPCRs), thereby preventing G protein coupling and often switching the signaling to other pathways. Amphipathic α-helix I (residues 100-111) has been implicated in receptor binding, but the mechanism of its action is not yet determined. Here we show that several mutations in the helix itself and adjacent hydrophobic residues in the body of the N-domain reduce arrestin1 binding to phosphorylated light-activated rhodopsin (PRh*). On the background of phosphorylationindependent mutants that bind with high affinity to both P-Rh* and light-activated unphosphorylated rhodopsin (Rh*), these mutations reduce the stability of the arrestin complex with P-Rh*, but not with Rh*. Using site-directed spin labeling we found that the local structure around α helix I changes upon binding to rhodopsin. However, the intra-molecular distances between α-helix I and adjacent α-strand I, or the rest of the N-domain, measured using double electron-electron resonance, do not change, ruling out relocation of the helix due to receptor binding. Collectively, these data demonstrate that α-helix I plays an indirect role in receptor binding, likely keeping α-strand I, carrying several phosphate-binding residues, in a position favorable for its interaction with receptor-attached phosphates.
Journal of Biological Chemistry, 2003
Arrestins selectively bind to phosphorylated activated forms of their cognate G protein-coupled receptors. Arrestin binding prevents further G protein activation and often redirects signaling to other pathways. The comparison of the high-resolution crystal structures of arrestin2, visual arrestin, and rhodopsin as well as earlier mutagenesis and peptide inhibition data collectively suggest that the elements on the concave sides of both arrestin domains most likely participate in receptor binding directly, thereby dictating its receptor preference. Using comparative binding of visual arrestin/arrestin2 chimeras to the preferred target of visual arrestin, light-activated phosphorylated rhodopsin (P-Rh*), and to the arrestin2 target, phosphorylated activated m2 muscarinic receptor (P-m2 mAChR*), we identified the elements that determine the receptor specificity of arrestins. We found that residues 49 -90 (-strands V and VI and adjacent loops in the N-domain) and 237-268 (-strands XV and XVI in the C-domain) in visual arrestin and homologous regions in arrestin2 are largely responsible for their receptor preference. Only 35 amino acids (22 of which are nonconservative substitutions) in the two elements are different. Simultaneous exchange of both elements between visual arrestin and arrestin2 fully reverses their receptor specificity, demonstrating that these two elements in the two domains of arrestin are necessary and sufficient to determine their preferred receptor targets.
How Does Arrestin Respond to the Phosphorylated State of Rhodopsin?
Journal of Biological Chemistry, 1999
Here we present structure-function data, which in conjunction with the refined crystal structure of arrestin (Hirsch, J. A., Schubert, C., Gurevich, V. V., and Sigler, P. B. (1999) Cell, in press), support a model for the conversion of a basal or "inactive" conformation of free arrestin to one that can bind to and inhibit the light activated receptor. The trigger for this transition is an interaction of the phosphorylated COOH-terminal segment of the receptor with arrestin that disrupts intramolecular interactions, including a hydrogen-bonded network of buried, charged side chains, referred to as the "polar core." This disruption permits structural adjustments that allow arrestin to bind to the receptor. Our mutational survey identifies residues in arrestin (Arg 175 , Asp 30 , Asp 296 , Asp 303 , Arg 382 ), which when altered bypass the need for the interaction with the receptor's phosphopeptide, enabling arrestin to bind to activated, nonphosphorylated rhodopsin (Rh*). These mutational changes disrupt interactions and substructures which the crystallographic model and previous biochemical studies have shown are responsible for maintaining the inactive state. The molecular basis for these disruptions was confirmed by successfully introducing structure-based second site substitutions that restored the critical interactions. The nearly absolute conservation of the mutagenically sensitive residues throughout the arrestin family suggests that this mechanism is likely to be applicable to arrestin-mediated desensitization of most G-protein-coupled receptors.
Journal of Biological Chemistry, 2011
Arrestins bind active phosphorylated forms of G protein-coupled receptors, terminating G protein activation, orchestrating receptor trafficking, and redirecting signaling to alternative pathways. Visual arrestin-1 preferentially binds rhodopsin, whereas the two non-visual arrestins interact with hundreds of G protein-coupled receptor subtypes. Here we show that an extensive surface on the concave side of both arrestin-2 domains is involved in receptor binding. We also identified a small number of residues on the receptor binding surface of the N-and C-domains that largely determine the receptor specificity of arrestins. We show that alanine substitution of these residues blocks the binding of arrestin-1 to rhodopsin in vitro and of arrestin-2 and -3 to 2-adrenergic, M2 muscarinic cholinergic, and D2 dopamine receptors in intact cells, suggesting that these elements critically contribute to the energy of the interaction. Thus, in contrast to arrestin-1, where direct phosphate binding is crucial, the interaction of non-visual arrestins with their cognate receptors depends to a lesser extent on phosphate binding and more on the binding to non-phosphorylated receptor elements.
The Role of Arrestin-1 Middle Loop in Rhodopsin Binding
International Journal of Molecular Sciences
Arrestins preferentially bind active phosphorylated G protein-coupled receptors (GPCRs). The middle loop, highly conserved in all arrestin subtypes, is localized in the central crest on the GPCR-binding side. Upon receptor binding, it directly interacts with bound GPCR and demonstrates the largest movement of any arrestin element in the structures of the complexes. Comprehensive mutagenesis of the middle loop of rhodopsin-specific arrestin-1 suggests that it primarily serves as a suppressor of binding to non-preferred forms of the receptor. Several mutations in the middle loop increase the binding to unphosphorylated light-activated rhodopsin severalfold, which makes them candidates for improving enhanced phosphorylation-independent arrestins. The data also suggest that enhanced forms of arrestin do not bind GPCRs exactly like the wild-type protein. Thus, the structures of the arrestin-receptor complexes, in all of which different enhanced arrestin mutants and reengineered receptors...
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.
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.
Journal of Biological Chemistry, 2012
The relative contribution of phosphates and active GPCR conformation is unknown. Results: Using WT and mutant arrestins and receptors, we show that phosphates are critical for arrestin binding to some GPCRs but not to others. Conclusion: The role of receptor-attached phosphates in arrestin binding varies widely depending on the arrestin-receptor combination. Significance: Distinct molecular mechanisms mediate arrestin recruitment to different GPCRs.
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 ...