Dynamics of Arrestin-Rhodopsin Interactions

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In this study we investigate conformational changes in Loop V-VI of visual arrestin during binding to light-activated, phosphorylated rhodopsin (Rho*-P) using a combination of site-specific cysteine mutagenesis and intramolecular fluorescence quenching. Introduction of cysteines at positions in the N-domain at residues predicted to be in close proximity to Ile-72 in Loop V-VI of arrestin (i.e. Glu-148 and Lys-298) appear to form an intramolecular disulfide bond with I72C, significantly diminishing the binding of arrestin to Rho*-P. Using a fluorescence approach, we show that the steady-state emission from a monobromobimane fluorophore in Loop V-VI is quenched by tryptophan residues placed at 148 or 298. This quenching is relieved upon binding of arrestin to Rho*-P. These results suggest that arrestin Loop V-VI moves during binding to Rho*-P and that conformational flexibility of this loop is essential for arrestin to adopt a high affinity binding state.Rhodopsin activity is controlled by a multistep process whereby activated rhodopsin is phosphorylated on its C terminus by rhodopsin kinase, leading to the binding and quenching of rhodopsin activity by visual arrestin. Visual arrestin belongs to the arrestin superfamily, which includes rod and cone arrestins as well the ␤-arrestins (1). The ␤-arrestins, which are involved in the inactivation of agonist-occupied G-proteincoupled receptors (for review, see Ref. 2), have an additional role not seen in visual arrestins whereby they direct receptor endocytosis by acting as a clathrin or AP-2 adaptor; e.g. Refs. 3 and 4. Visual arrestin is an important model system not only for understanding the visual response but also for understanding the broadreaching mechanism for controlling cellular signal transduction cascades mediated by G protein-coupled receptors.The crystal structures of rod, cone, and ␤-1 arrestin are remarkably similar. All show a structure consisting of two cupshaped domains formed from a seven-stranded ␤ sandwich (5-7). These structures, of the unbound state, have provided an important framework on which to base predictions for the binding interaction between arrestin and light-activated phosphorylated rhodopsin (Rho*-P). 4 Interestingly, in the crystal structures of visual arrestin, Loop V-VI (residues 68 -78 in bovine visual arrestin (7)) is seen in two primary conformers, one in which Loop V-VI is extended away from the main bulk of the protein (Fig. 1, ␣ conformer) and one with the loop folded into the N-terminal domain of the protein (␤ conformer). Several groups have identified that Loop V-VI has an important role in receptor binding. Experimental evidence shows (i) this loop is a vital component of arrestin that directs receptor preference (8), (ii) insertion of a peptide into this loop can either directly block or be used to competitively block binding to Rho*-P (9, 10), (iii) a fluorescent label at residue 72 in this loop apparently buries itself into the rhodopsin interface upon binding (10, 11), and (iv) spin labels in this loop have reduced ...

Section: Rationale Of Mutant Design-the Arrestin Crystal Structuresmentioning

confidence: 99%

“…Bimane Labeling of Arrestin-Arrestins were labeled with excess monobromobimane as previously described (16). Labeling efficiency for all mutants was Ͼ90% and was determined as described (11).…”

Section: Preparation Of Rod Outer Segment and Recombinant Arrestinmentioning

confidence: 99%

Dynamics of Arrestin-Rhodopsin Interactions

Sommer

Farrens

McDowell

et al. 2007

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“…The importance of negatively charged lipids for arrestin-1 binding was noted previously (47,66), but the mechanism of their action was not understood. Our analysis of POPS dependence of the binding of a variety of structurally distinct arrestin-1 mutants showed that negative charges on the surface of the membrane assist in pushing the negatively charged arrestin C-tail out of the receptor-binding cavity of the arrestin N-domain, thereby facilitating arrestin-1 association with P-Rh*.…”

mentioning

confidence: 99%

Monomeric Rhodopsin Is Sufficient for Normal Rhodopsin Kinase (GRK1) Phosphorylation and Arrestin-1 Binding

Bayburt

Vishnivetskiy

McLean

et al. 2011

174

177

G-protein-coupled receptor (GPCR) oligomerization has been observed in a wide variety of experimental contexts, but the functional significance of this phenomenon at different stages of the life cycle of class A GPCRs remains to be elucidated. Rhodopsin (Rh), a prototypical class A GPCR of visual transduction, is also capable of forming dimers and higher order oligomers. The recent demonstration that Rh monomer is sufficient to activate its cognate G protein, transducin, prompted us to test whether the same monomeric state is sufficient for rhodopsin phosphorylation and arrestin-1 binding. Here we show that monomeric active rhodopsin is phosphorylated by rhodopsin kinase (GRK1) as efficiently as rhodopsin in the native disc membrane. Monomeric phosphorylated lightactivated Rh (P-Rh*) in nanodiscs binds arrestin-1 essentially as well as P-Rh* in native disc membranes. We also measured the affinity of arrestin-1 for P-Rh* in nanodiscs using a fluorescence-based assay and found that arrestin-1 interacts with monomeric P-Rh* with low nanomolar affinity and 1:1 stoichiometry, as previously determined in native disc membranes. Thus, similar to transducin activation, rhodopsin phosphorylation by GRK1 and high affinity arrestin-1 binding only requires a rhodopsin monomer.Visual phototransduction is quenched by a two-step mechanism. First, light-activated rhodopsin (Rh*) 3 is phosphorylated multiple times by GRK1. Arrestin-1 4 binding to active phosphorylated rhodopsin (P-Rh*) blocks further transducin activation (1) by steric exclusion (2). The binding of arrestin-1 to P-Rh* is an important molecular mechanism for signal shut-off (3). However, key details of the requirements for physical interaction of arrestin-1 with rhodopsin remain to be explored. Rhodopsin, which is highly concentrated in photoreceptor membranes, has been observed to form arrays of dimers, thus raising the possibility that the dimer is a functional unit (4). Although evidence of a preferred dimer interface has been reported (5), the functional role of rhodopsin oligomers remains controversial (6, 7). Accumulating evidence with other GPCRs indicates that oligomerization could be widespread (8 -10). Several models for rhodopsin dimers (11, 12) and monomers (6, 13) interacting with signaling partners transducin, rhodopsin kinase, and arrestin have been proposed. These models need rigorous experimental testing at different steps of the functional cycle of rhodopsin and other class A GPCRs (reviewed in Refs. 7 and 14). Modified high density lipoprotein particles (nanodiscs) consist of a phospholipid bilayer stabilized by a membrane scaffold protein (MSP) (15-17). These nanodiscs can be used to selectively isolate monomeric GPCRs imbedded into lipid bilayer. We (18) and others (19,20) have previously demonstrated that rhodopsin monomers incorporated in nanodiscs are highly functional in signaling to transducin. The same was shown for monomeric rhodopsin purified in detergent (dodecyl maltoside), where rhodopsin and transducin form a 1:1 complex (21). Howe...

“…1) were created using PCR and verified by DNA sequencing. Labeling of these introduced cysteine residues with IANBD was performed as described previously for bimane (31). The concentration and labeling efficiency were determined using molar extinction coefficients of 0.025 M Ϫ1 cm Ϫ1 at 500 nm for IANBD (as reported by the manufacturer) and 0.02076 M Ϫ1 cm Ϫ1 at 278 nm for arrestin (33).…”

mentioning

confidence: 99%

“…Preparation of Fluorescently Labeled Arrestin Mutants-Recombinant bovine mutant arrestin, from which the native cysteine and tryptophan residues had been removed (C63A, C128S, C143A, and W194F), was expressed and purified from Escherichia coli exactly as described previously (31). "Cysless" arrestin has been previously shown to retain wild-type function (32,33), and we verified that our mutant construct was functionally identical to native arrestin by centrifugal pulldown, light scattering, and extra Meta II analyses (data not shown).…”

mentioning

confidence: 99%

Arrestin-Rhodopsin Binding Stoichiometry in Isolated Rod Outer Segment Membranes Depends on the Percentage of Activated Receptors

Sommer

Hofmann

Heck

2011

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In the rod cell of the retina, arrestin is responsible for blocking signaling of the G-protein-coupled receptor rhodopsin. The general visual signal transduction model implies that arrestin must be able to interact with a single light-activated, phosphorylated rhodopsin molecule (Rho*P), as would be generated at physiologically relevant low light levels. However, the elongated bi-lobed structure of arrestin suggests that it might be able to accommodate two rhodopsin molecules. In this study, we directly addressed the question of binding stoichiometry by quantifying arrestin binding to Rho*P in isolated rod outer segment membranes. We manipulated the "photoactivation density," i.e. the percentage of active receptors in the membrane, with the use of a light flash or by partially regenerating membranes containing phosphorylated opsin with 11-cis-retinal. Curiously, we found that the apparent arrestinRho*P binding stoichiometry was linearly dependent on the photoactivation density, with one-to-one binding at low photoactivation density and one-to-two binding at high photoactivation density. We also observed that, irrespective of the photoactivation density, a single arrestin molecule was able to stabilize the active metarhodopsin II conformation of only a single Rho*P. We hypothesize that, although arrestin requires at least a single Rho*P to bind the membrane, a single arrestin can actually interact with a pair of receptors. The ability of arrestin to interact with heterogeneous receptor pairs composed of two different photo-intermediate states would be well suited to the rod cell, which functions at low light intensity but is routinely exposed to several orders of magnitude more light.The hundreds of different types of G-protein-coupled receptors (GPCRs) 2 and their binding partners represent versatile protein families, whose individual members have been modified by nature to accomplish many different functions while preserving a basic structure and activation mechanism (1, 2). GPCRs share a common seven-transmembrane helical structure, which, when activated by ligand or stimuli, binds and activates G-proteins to initiate cell signaling (3). Most GPCRs also share a common mechanism of signal termination, which involves receptor phosphorylation and binding of the protein arrestin. However, the fates of arrestin-bound receptors vary widely depending on the type of receptor and its bound ligand. Arrestin-bound receptors may be internalized and degraded, internalized and recycled, or even initiate Gprotein independent signaling (4).Although detailed structural information on different GPCRs (5-9) and arrestins (10 -13) has been available for some time, basic questions regarding their interaction remain unanswered. In particular, the binding stoichiometry (how many active receptors does a single arrestin bind?) is a matter of debate. Historically, one-to-one binding has always been assumed, because many GPCRs are present at low concentrations on the cell surface (1), or in the case of the photoreceptor rhodopsin, activ...