Community guidelines for GPCR ligand bias: IUPHAR review 32.

GPCRs modulate a plethora of physiological processes and mediate the effects of one-third of FDA-approved drugs. Depending on which ligand activates a receptor, it can engage different intracellular transducers. This 'biased signalling' paradigm requires that we now characterize physiological signalling not just by receptors but by ligand-receptor pairs. Ligands eliciting biased signalling may constitute better drugs with higher efficacy and fewer adverse effects. However, ligand bias is very complex, making reproducibility and description challenging. Here, we provide guidelines and terminology for any scientists to design and report ligand bias experiments. The guidelines will aid consistency and clarity, as the basic receptor research and drug discovery communities continue to advance our understanding and exploitation of ligand bias. Scientific insight, biosensors, and analytical methods are still evolving and should benefit from and contribute to the implementation of the guidelines, together improving translation from in vitro to disease-relevant in vivo models.

The European Research Network on Signal Transduction (ERNEST): Toward a Multidimensional Holistic Understanding of G Protein-Coupled Receptor Signaling.

G protein-coupled receptors (GPCRs) are intensively studied due to their therapeutic potential as drug targets. Members of this large family of transmembrane receptor proteins mediate signal transduction in diverse cell types and play key roles in human physiology and health. In 2013 the research consortium GLISTEN (COST Action CM1207) was founded with the goal of harnessing the substantial growth in knowledge of GPCR structure and dynamics to push forward the development of molecular modulators of GPCR function. The success of GLISTEN, coupled with new findings and paradigm shifts in the field, led in 2019 to the creation of a related consortium called ERNEST (COST Action CA18133). ERNEST broadens focus to entire signaling cascades, based on emerging ideas of how complexity and specificity in signal transduction are not determined by receptor-ligand interactions alone. A holistic approach that unites the diverse data and perspectives of the research community into a single multidimensional map holds great promise for improved drug design and therapeutic targeting.

Distinct G protein-coupled receptor phosphorylation motifs modulate arrestin affinity and activation and global conformation.

Cellular functions of arrestins are determined in part by the pattern of phosphorylation on the G protein-coupled receptors (GPCRs) to which arrestins bind. Despite high-resolution structural data of arrestins bound to phosphorylated receptor C-termini, the functional role of each phosphorylation site remains obscure. Here, we employ a library of synthetic phosphopeptide analogues of the GPCR rhodopsin C-terminus and determine the ability of these peptides to bind and activate arrestins using a variety of biochemical and biophysical methods. We further characterize how these peptides modulate the conformation of arrestin-1 by nuclear magnetic resonance (NMR). Our results indicate different functional classes of phosphorylation sites: 'key sites' required for arrestin binding and activation, an 'inhibitory site' that abrogates arrestin binding, and 'modulator sites' that influence the global conformation of arrestin. These functional motifs allow a better understanding of how different GPCR phosphorylation patterns might control how arrestin functions in the cell.

Molecular mechanism of GPCR-mediated arrestin activation.

Despite intense interest in discovering drugs that cause G-protein-coupled receptors (GPCRs) to selectively stimulate or block arrestin signalling, the structural mechanism of receptor-mediated arrestin activation remains unclear1,2. Here we reveal this mechanism through extensive atomic-level simulations of arrestin. We find that the receptor's transmembrane core and cytoplasmic tail-which bind distinct surfaces on arrestin-can each independently stimulate arrestin activation. We confirm this unanticipated role of the receptor core, and the allosteric coupling between these distant surfaces of arrestin, using site-directed fluorescence spectroscopy. The effect of the receptor core on arrestin conformation is mediated primarily by interactions of the intracellular loops of the receptor with the arrestin body, rather than the marked finger-loop rearrangement that is observed upon receptor binding. In the absence of a receptor, arrestin frequently adopts active conformations when its own C-terminal tail is disengaged, which may explain why certain arrestins remain active long after receptor dissociation. Our results, which suggest that diverse receptor binding modes can activate arrestin, provide a structural foundation for the design of functionally selective ('biased') GPCR-targeted ligands with desired effects on arrestin signalling.

Structural mechanism of arrestin activation.

The large and multifunctional family of G protein-coupled receptors (GPCRs) are regulated by a small family of structurally conserved arrestin proteins. In order to bind an active GPCR, arrestin must first be activated by interaction with the phosphorylated receptor C-terminus. Recent years have witnessed major developments in high-resolution crystal structures of pre-active arrestins and arrestin or arrestin-derived peptides in complex with an active GPCR. Although each structure individually offers only a limited snapshot, taken together and interpreted in light of recent complementary functional data, they offer valuable insight into how arrestin is activated by and couples to a phosphorylated active GPCR.

C-edge loops of arrestin function as a membrane anchor.

G-protein-coupled receptors are membrane proteins that are regulated by a small family of arrestin proteins. During formation of the arrestin-receptor complex, arrestin first interacts with the phosphorylated receptor C terminus in a pre-complex, which activates arrestin for tight receptor binding. Currently, little is known about the structure of the pre-complex and its transition to a high-affinity complex. Here we present molecular dynamics simulations and site-directed fluorescence experiments on arrestin-1 interactions with rhodopsin, showing that loops within the C-edge of arrestin function as a membrane anchor. Activation of arrestin by receptor-attached phosphates is necessary for C-edge engagement of the membrane, and we show that these interactions are distinct in the pre-complex and high-affinity complex in regard to their conformation and orientation. Our results expand current knowledge of C-edge structure and further illuminate the conformational transitions that occur in arrestin along the pathway to tight receptor binding.

Functional map of arrestin binding to phosphorylated opsin, with and without agonist.

Arrestins desensitize G protein-coupled receptors (GPCRs) and act as mediators of signalling. Here we investigated the interactions of arrestin-1 with two functionally distinct forms of the dim-light photoreceptor rhodopsin. Using unbiased scanning mutagenesis we probed the individual contribution of each arrestin residue to the interaction with the phosphorylated apo-receptor (Ops-P) and the agonist-bound form (Meta II-P). Disruption of the polar core or displacement of the C-tail strengthened binding to both receptor forms. In contrast, mutations of phosphate-binding residues (phosphosensors) suggest the phosphorylated receptor C-terminus binds arrestin differently for Meta II-P and Ops-P. Likewise, mutations within the inter-domain interface, variations in the receptor-binding loops and the C-edge of arrestin reveal different binding modes. In summary, our results indicate that arrestin-1 binding to Meta II-P and Ops-P is similarly dependent on arrestin activation, although the complexes formed with these two receptor forms are structurally distinct.

Formation and decay of the arrestin·rhodopsin complex in native disc membranes.

In the G protein-coupled receptor rhodopsin, light-induced cis/trans isomerization of the retinal ligand triggers a series of distinct receptor states culminating in the active Metarhodopsin II (Meta II) state, which binds and activates the G protein transducin (Gt). Long before Meta II decays into the aporeceptor opsin and free all-trans-retinal, its signaling is quenched by receptor phosphorylation and binding of the protein arrestin-1, which blocks further access of Gt to Meta II. Although recent crystal structures of arrestin indicate how it might look in a precomplex with the phosphorylated receptor, the transition into the high affinity complex is not understood. Here we applied Fourier transform infrared spectroscopy to monitor the interaction of arrestin-1 and phosphorylated rhodopsin in native disc membranes. By isolating the unique infrared signature of arrestin binding, we directly observed the structural alterations in both reaction partners. In the high affinity complex, rhodopsin adopts a structure similar to Gt-bound Meta II. In arrestin, a modest loss of ß-sheet structure indicates an increase in flexibility but is inconsistent with a large scale structural change. During Meta II decay, the arrestin-rhodopsin stoichiometry shifts from 1:1 to 1:2. Arrestin stabilizes half of the receptor population in a specific Meta II protein conformation, whereas the other half decays to inactive opsin. Altogether these results illustrate the distinct binding modes used by arrestin to interact with different functional forms of the receptor.

Structure-based biophysical analysis of the interaction of rhodopsin with G protein and arrestin.

In this chapter, we describe a set of complementary techniques that we use to study the activation of rhodopsin, a G protein-coupled receptor (GPCR), and its functional interactions with G protein and arrestin. The protein reagents used for these studies come from native disc membranes or heterologous expression, and G protein and arrestin are often replaced with less complex synthetic peptides derived from key interaction sites of these binding partners (BPs). We first report on our approach to protein X-ray crystallography and describe how protein crystals from native membranes are obtained. The crystal structures provide invaluable resolution, but other techniques are required to assess the dynamic equilibria characteristic for active GPCRs. The simplest approach is "Extra Meta II," which uses UV/Vis absorption spectroscopy to monitor the equilibrium of photoactivated states. Site-specific information about the BPs (e.g., arrestin) is added by fluorescence techniques employing mutants labeled with reporter groups. All functional changes in both the receptor and interacting proteins or peptides are seen with highest precision using Fourier transform infrared (FTIR) difference spectroscopy. In our approach, the lack of site-specific information in FTIR is overcome by parallel molecular dynamics simulations, which are employed to interpret the results and to extend the timescale down to the range of conformational substates.

Quantification of arrestin-rhodopsin binding stoichiometry.

We have developed several methods to quantify arrestin-1 binding to rhodopsin in the native rod disk membrane. These methods can be applied to study arrestin interactions with all functional forms of rhodopsin, including dark-state rhodopsin, light-activated metarhodopsin II (Meta II), and the products of Meta II decay, opsin and all-trans-retinal. When used in parallel, these methods report both the actual amount of arrestin bound to the membrane surface and the functional aspects of arrestin binding, such as which arrestin loops are engaged and whether Meta II is stabilized. Most of these methods can also be applied to recombinant receptor reconstituted into liposomes, bicelles, and nanodisks.

Crystal structure of a common GPCR-binding interface for G protein and arrestin.

G-protein-coupled receptors (GPCRs) transmit extracellular signals to activate intracellular heterotrimeric G proteins (Gαßγ) and arrestins. For G protein signalling, the Gα C-terminus (GαCT) binds to a cytoplasmic crevice of the receptor that opens upon activation. A consensus motif is shared among GαCT from the Gi/Gt family and the 'finger loop' region (ArrFL1-4) of all four arrestins. Here we present a 2.75 Å crystal structure of ArrFL-1, a peptide analogue of the finger loop of rod photoreceptor arrestin, in complex with the prototypical GPCR rhodopsin. Functional binding of ArrFL to the receptor was confirmed by ultraviolet-visible absorption spectroscopy, competitive binding assays and Fourier transform infrared spectroscopy. For both GαCT and ArrFL, binding to the receptor crevice induces a similar reverse turn structure, although significant structural differences are seen at the rim of the binding crevice. Our results reflect both the common receptor-binding interface and the divergent biological functions of G proteins and arrestins.

Not just signal shutoff: the protective role of arrestin-1 in rod cells.

The retinal rod cell is an exquisitely sensitive single-photon detector that primarily functions in dim light (e.g., moonlight). However, rod cells must routinely survive light intensities more than a billion times greater (e.g., bright daylight). One serious challenge to rod cell survival in daylight is the massive amount of all-trans-retinal that is released by Meta II, the light-activated form of the photoreceptor rhodopsin. All-trans-retinal is toxic, and its condensation products have been implicated in disease. Our recent work has developed the concept that rod arrestin (arrestin-1), which terminates Meta II signaling, has an additional role in protecting rod cells from the consequences of bright light by limiting free all-trans-retinal. In this chapter we will elaborate upon the molecular mechanisms by which arrestin-1 serves as both a single-photon response quencher as well as an instrument of rod cell survival in bright light. This discussion will take place within the framework of three distinct functional modules of vision: signal transduction, the retinoid cycle, and protein translocation.

Crystal structure of pre-activated arrestin p44.

Arrestins interact with G-protein-coupled receptors (GPCRs) to block interaction with G proteins and initiate G-protein-independent signalling. Arrestins have a bi-lobed structure that is stabilized by a long carboxy-terminal tail (C-tail), and displacement of the C-tail by receptor-attached phosphates activates arrestins for binding active GPCRs. Structures of the inactive state of arrestin are available, but it is not known how C-tail displacement activates arrestin for receptor coupling. Here we present a 3.0 Å crystal structure of the bovine arrestin-1 splice variant p44, in which the activation step is mimicked by C-tail truncation. The structure of this pre-activated arrestin is profoundly different from the basal state and gives insight into the activation mechanism. p44 displays breakage of the central polar core and other interlobe hydrogen-bond networks, leading to a ∼21° rotation of the two lobes as compared to basal arrestin-1. Rearrangements in key receptor-binding loops in the central crest region include the finger loop, loop 139 (refs 8, 10, 11) and the sequence Asp 296-Asn 305 (or gate loop), here identified as controlling the polar core. We verified the role of these conformational alterations in arrestin activation and receptor binding by site-directed fluorescence spectroscopy. The data indicate a mechanism for arrestin activation in which C-tail displacement releases critical central-crest loops from restricted to extended receptor-interacting conformations. In parallel, increased flexibility between the two lobes facilitates a proper fitting of arrestin to the active receptor surface. Our results provide a snapshot of an arrestin ready to bind the active receptor, and give an insight into the role of naturally occurring truncated arrestins in the visual system.

Distinct loops in arrestin differentially regulate ligand binding within the GPCR opsin.

G-protein-coupled receptors are universally regulated by arrestin binding. Here we show that rod arrestin induces uptake of the agonist all-trans-retinal [corrected] in only half the population of phosphorylated opsin in the native membrane. Agonist uptake blocks subsequent entry of the inverse agonist 11-cis-retinal (that is, regeneration of rhodopsin), but regeneration is not blocked in the other half of aporeceptors. Environmentally sensitive fluorophores attached to arrestin reported that conformational changes in loop(V-VI) (N-domain) are coupled to the entry of agonist, while loop(XVIII-XIX) (C-domain) engages the aporeceptor even before agonist is added. The data are most consistent with a model in which each domain of arrestin engages its own aporeceptor, and the different binding preferences of the domains lead to asymmetric ligand binding by the aporeceptors. Such a mechanism would protect the rod cell in bright light by concurrently sequestering toxic all-trans-retinal [corrected] and allowing regeneration with 11-cis-retinal.

Alkylated hydroxylamine derivatives eliminate peripheral retinylidene Schiff bases but cannot enter the retinal binding pocket of light-activated rhodopsin.

Besides Lys-296 in the binding pocket of opsin, all-trans-retinal forms adducts with peripheral lysine residues and phospholipids, thereby mimicking the spectral and chemical properties of metarhodopsin species. These pseudophotoproducts composed of nonspecific retinylidene Schiff bases have long plagued the investigation of rhodopsin deactivation and identification of decay products. We discovered that, while hydroxylamine can enter the retinal binding pocket of light-activated rhodopsin, the modified hydroxylamine compounds o-methylhydroxylamine (mHA), o-ethylhydroxylamine (eHA), o-tert-butylhydroxylamine (t-bHA), and o-(carboxymethyl)hydroxylamine (cmHA) are excluded. However, the alkylated hydroxylamines react quickly and efficiently with exposed retinylidene Schiff bases to form their respective retinal oximes. We further investigated how t-bHA affects light-activated rhodopsin and its interaction with binding partners. We found that both metarhodopsin II (Meta II) and Meta III are resistant to t-bHA, and neither arrestin nor transducin binding is affected by t-bHA. This discovery suggests that the hypothetical solvent channel that opens in light-activated rhodopsin is extremely stringent with regard to size and/or polarity. We believe that alkylated hydroxylamines will prove to be extremely useful reagents for the investigation of rhodopsin activation and decay mechanisms. Furthermore, the use of alkylated hydroxylamines should not be limited to in vitro studies and could help elucidate visual signal transduction mechanisms in the living cells of the retina.

Arrestin-rhodopsin binding stoichiometry in isolated rod outer segment membranes depends on the percentage of activated receptors.

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 arrestin-Rho*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.

Dynamics of arrestin-rhodopsin interactions: loop movement is involved in arrestin activation and receptor binding.

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.

Arrestin can act as a regulator of rhodopsin photochemistry.

We report that visual arrestin can regulate retinal release and late photoproduct formation in rhodopsin. Our experiments, which employ a fluorescently labeled arrestin and rhodopsin solubilized in detergent/phospholipid micelles, indicate that arrestin can trap a population of retinal in the binding pocket with an absorbance characteristic of Meta II with the retinal Schiff-base intact. Furthermore, arrestin can convert Metarhodopsin III (formed either by thermal decay or blue-light irradiation) to a Meta II-like absorbing species. Together, our results suggest arrestin may be able to play a more complex role in the rod cell besides simply quenching transducin activity. This possibility may help explain why arrestin deficiency leads to problems like stationary night blindness (Oguchi disease) and retinal degeneration.

Dynamics of arrestin-rhodopsin interactions: acidic phospholipids enable binding of arrestin to purified rhodopsin in detergent.

We report that acidic phospholipids can restore the binding of visual arrestin to purified rhodopsin solubilized in n-dodecyl-beta-d-maltopyranoside. We used this finding to investigate the interplay between arrestin binding and the status of the retinal chromophore ligand in the receptor binding pocket. Our results showed that arrestin can interact with the late photoproduct Meta III and convert it to a Meta II-like species. Interestingly in these mixed micelles, the release of retinal and arrestin was no longer directly coupled as it is in the native rod disk membrane. For example, up to approximately 50% of the retinal could be released even though arrestin remains bound to the receptor in a long lived complex. We anticipate that this new ability to study these proteins in a defined, purified system will facilitate further structural and dynamic studies of arrestin-rhodopsin interactions.