Enhanced Mutant Compensates for Defects in Rhodopsin Phosphorylation in the Presence of Endogenous Arrestin-1 (original) (raw)
Related papers
Enhanced arrestin facilitates recovery and protects rods lacking rhodopsin phosphorylation
Current biology : CB, 2009
G protein-coupled receptors (GPCRs) are the largest family of signaling proteins expressed in every cell in the body and targeted by the majority of clinically used drugs . GPCR signaling, including rhodopsin-drive phototransduction, is terminated by receptor phosphorylation followed by arrestin binding . Genetic defects in receptor phosphorylation and excessive signaling by overactive GPCR mutants result in a wide variety of diseases, from retinal degeneration to cancer . Here we tested whether arrestin1 mutants with enhanced ability to bind active unphosphorylated rhodopsin [7-10] can suppress uncontrolled signaling, bypassing receptor phosphorylation by rhodopsin kinase (RK) and replacing this two-step mechanism with a single step deactivation in rod photoreceptors. We show that in this precisely timed signaling system with single photon sensitivity , an "enhanced" arrestin1 mutant partially compensates for defects in rhodopsin phosphorylation, promoting photoreceptor survival, improving functional performance, and facilitating photoresponse recovery. These proof-of-principle experiments demonstrate the feasibility of functional compensation in vivo for the first time, which is a promising approach for correcting genetic defects associated with gain-of-function mutations. Successful modification of protein-protein interactions by appropriate mutations paves the way to targeted redesign of signaling pathways to achieve desired functional outcomes.
PLoS ONE, 2011
Light-induced rhodopsin signaling is turned off with sub-second kinetics by rhodopsin phosphorylation followed by arrestin-1 binding. To test the availability of the arrestin-1 pool in dark-adapted outer segment (OS) for rhodopsin shutoff, we measured photoresponse recovery rates of mice with arrestin-1 content in the OS of 2.5%, 5%, 60%, and 100% of wild type (WT) level by two-flash ERG with the first (desensitizing) flash at 160, 400, 1000, and 2500 photons/rod. The time of half recovery (t half ) in WT retinas increases with the intensity of the initial flash, becoming ,2.5-fold longer upon activation of 2500 than after 160 rhodopsins/rod. Mice with 60% and even 5% of WT arrestin-1 level recovered at WT rates. In contrast, the mice with 2.5% of WT arrestin-1 had a dramatically slower recovery than the other three lines, with the t half increasing ,28 fold between 160 and 2500 rhodopsins/rod. Even after the dimmest flash, the rate of recovery of rods with 2.5% of normal arrestin-1 was two times slower than in other lines, indicating that arrestin-1 level in the OS between 100% and 5% of WT is sufficient for rapid recovery, whereas with lower arrestin-1 the rate of recovery dramatically decreases with increased light intensity. Thus, the OS has two distinct pools of arrestin-1: cytoplasmic and a separate pool comprising ,2.5% that is not immediately available for rhodopsin quenching. The observed delay suggests that this pool is localized at the periphery, so that its diffusion across the OS rate-limits the recovery. The line with very low arrestin-1 expression is the first where rhodopsin inactivation was made rate-limiting by arrestin manipulation.
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.
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.
Regulation of Arrestin Binding by Rhodopsin Phosphorylation Level
Journal of Biological Chemistry, 2007
Arrestins ensure the timely termination of receptor signaling. The role of rhodopsin phosphorylation in visual arrestin binding was established more than 20 years ago, but the effects of the number of receptor-attached phosphates on this interaction remain controversial. Here we use purified rhodopsin fractions with carefully quantified content of individual phosphorylated rhodopsin species to elucidate the impact of phosphorylation level on arrestin interaction with three biologically relevant functional forms of rhodopsin: light-activated and dark phosphorhodopsin and phospho-opsin. We found that a single receptor-attached phosphate does not facilitate arrestin binding, two are necessary to induce high affinity interaction, and three phosphates fully activate arrestin. Higher phosphorylation levels do not increase the stability of arrestin complex with light-activated rhodopsin but enhance its binding to the dark phosphorhodopsin and phospho-opsin. The complex of arrestin with hyperphosphorylated light-activated rhodopsin is less sensitive to high salt and appears to release retinal faster. These data suggest that arrestin likely quenches rhodopsin signaling after the third phosphate is added by rhodopsin kinase. The complex of arrestin with heavily phosphorylated rhodopsin, which appears to form in certain disease states, has distinct characteristics that may contribute to the phenotype of these visual disorders.
Cellular Signalling, 2013
The effects of activating mutations associated with night blindness on the stoichiometry of rhodopsin interactions with G protein-coupled receptor kinase 1 (GRK1) and arrestin-1 have not been reported. Here we show that the monomeric form of WT rhodopsin and its constitutively active mutants M257Y, G90D, and T94I, reconstituted into HDL particles are effectively phosphorylated by GRK1, as well as two more ubiquitously expressed subtypes, GRK2 and GRK5. All versions of arrestin-1 tested (WT, pre-activated, and constitutively monomeric mutants) bind to monomeric rhodopsin and show the same selectivity for different functional forms of rhodopsin as in native disc membranes. Rhodopsin phosphorylation by GRK1 and GRK2 promotes arrestin-1 binding to a comparable extent, whereas similar phosphorylation by GRK5 is less effective, suggesting that not all phosphorylation sites on rhodopsin are equivalent in promoting arrestin-1 binding. The binding of WT arrestin-1 to phospho-opsin is comparable to the binding to its preferred target, P-Rh*, suggesting that in photoreceptors arrestin-1 only dissociates after opsin regeneration with 11-cis-retinal, which converts phospho-opsin into inactive phosphorhodopsin that has lower affinity for arrestin-1. Reduced binding of arrestin-1 to the phospho-opsin form of G90D mutant likely contributes to night blindness caused by this mutation in humans. (V.V. Gurevich). We use systematic names of arrestin proteins: arrestin-1 (historic names S-antigen, 48 kDa protein, visual or rod arrestin), arrestin-2 (β-arrestin or β-arrestin1), arrestin-3 (β-arrestin2 or hTHY-ARRX), and arrestin-4 (cone or X-arrestin; for reasons that are not clear its gene is called "arrestin 3" in HUGO database).
Prolonged photoresponses in transgenic mouse rods lacking arrestin
…, 1997
Arrestins are soluble cytoplasmic proteins that bind to G-proteincoupled receptors, thus switching off activation of the G protein and terminating the signalling pathway that triggers the cellular response 1,2. Although visual arrestin has been shown to quench the catalytic activity of photoexcited, phosphorylated rhodopsin in a reconstituted system 3 , its role in the intact rod cell remains unclear because phosphorylation alone reduces the catalytic activity of rhodopsin 4-6. Here we have recorded photocurrents of rods from transgenic mice in which one or both copies of the arrestin gene were disrupted. Photoresponses were unaffected when arrestin expression was halved, indicating that arrestin binding is not rate limiting for recovery of the rod photoresponse, as it is in Drosophila 7,8. With arrestin absent, the flash response displayed a rapid partial recovery followed by a prolonged final phase. This behaviour indicates that an arrestin-independent mechanism initiates the quench of rhodopsin's catalytic activity and that arrestin completes the quench. The intensity dependence of the photoresponse in rods lacking arrestin further suggests that, although arrestin is required for normal signal termination, it does not participate directly in light adaptation. One or both copies of the arrestin gene were disrupted by homologous recombination in transgenic mice (Fig. 1a). The disruption is expected to prevent expression of both the fulllength and the truncated splice variant of rod arrestin 9. Retinas hemizygous for arrestin (+/−) contained approximately 30% of the normal amount of arrestin, whereas retinas from knockout mice (−/−) contained no arrestin (Fig. 1b). Immunoblots revealed that the levels of rod and cone transducin-␣, transducin- 1 , transducin- 2 , phosphodiesterase-␥, phosducin and recoverin were similar in control (+/+), +/− and −/− retinas (data not shown), indicating that the absence of arrestin did not lead to substantial changes in the amounts of these visual transduction proteins. Gross retinal morphology was little affected by the absence of rod arrestin. The number of photoreceptor nuclei in four-week-old −/− mice raised in cyclic light (12 h light : 12 h dark) was normal, but the rod outer segments were about 25% shorter than those of +/+ and +/− mice, and were somewhat disorganized (Fig. 2). These abnormalities were less evident in retinas from −/− mice born and raised in constant darkness (not shown). Furthermore, the rhodopsin content was lowered by as much as 20-fold in −/− retinas of four-weekold mice raised in cyclic light, but it was normal in −/− mice reared in darkness. There was thus a light-dependent reduction in the length and rhodopsin content of −/− outer segments as well as a disorganization of outer-segment structure. These effects are like letters to nature
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 ...
Neuron, 2000
and second messengers differ between the two systems (Stryer, 1986). In vertebrates, the effector molecule is not PLC, as in the Drosophila visual system, but instead is a cGMP phosphodiesterase that is activated upon Summary light stimulation. This results in a transient decrease in the levels of intracellular cGMP. In addition, the verte-Although many different mutations in humans and Drobrate cation-specific channel is a cGMP-gated channel. sophila cause retinal degeneration, in most cases, a These channels are open in the dark and close in remolecular mechanism for the degeneration has not sponse to light stimulation and lower cGMP levels. In been found. We now demonstrate the existence of spite of these fundamental differences, the activation stable, persistent complexes between rhodopsin and of rhodopsin and the G protein as well as the inactivation its regulatory protein arrestin in several different of rhodopsin are essentially identical between verteretinal degeneration mutants. Elimination of these rhobrates and invertebrates. dopsin-arrestin complexes by removing either rho-Although many of the factors involved in the activation dopsin or arrestin rescues the degeneration phenoof the visual transduction cascade have been described, type. Furthermore, we show that the accumulation of the proteins that deactivate each intermediate in the these complexes triggers apoptotic cell death and that cascade have yet to be identified. However, the steps the observed retinal degeneration requires the endoinvolved in "shutting off" the activated receptor have cytic machinery. This suggests that the endocytosis been well studied. The inactivation of photoactivated of rhodopsin-arrestin complexes is a molecular mechrhodopsin involves the concerted action of two proteins. anism for the initiation of retinal degeneration. We First, rhodopsin kinase phosphorylates photoactivated propose that an identical mechanism may be responsirhodopsin on numerous serine and threonine residues ble for the pathology found in a subset of human retinal at the C terminus. The photoactivated, phosphorylated degenerative disorders. rhodopsin is then a substrate for arrestin binding, which interacts stoichiometrically with rhodopsin and inactivates it, presumably by competing for the same active