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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 a 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 native residues in these positions act as binding suppressors, specifically inhibiting the arrestin-1 binding to Rh* and thereby increasing arrestin-1 selectivity for P-Rh*. This calls for the modification of a widely accepted model of the arrestin–receptor interactions.

Deactivation of G-protein-coupled receptors (GPCRs) involves multiple phosphorylations followed by arrestin binding, which uncouples the GPCR from G-protein activation. Some GPCRs, such as rhodopsin, are reused many times. Arrestin dissociation and GPCR dephosphorylation are key steps in the recycling process.In vitroevidence suggests that visual arrestin (ARR1) binding to light-activated, phosphorylated rhodopsin hinders dephosphorylation. Whether ARR1 binding also affects rhodopsin dephosphorylationin vivois not known. We investigated this using both male and female mice lacking ARR1. Mice were exposed to bright light and placed in darkness for different periods of time, and differently phosphorylated species of rhodopsin were assayed by isoelectric focusing. For WT mice, rhodopsin dephosphorylation was nearly complete by 1 h in darkness. Surprisingly, we observed that, in theArr1KO rods, rhodopsin remained phosphorylated even after 3 h. Delayed dephosphorylation inArr1KO rods cannot be explained by cell stress induced by persistent signaling, since it is not prevented by the removal of transducin, the visual G-protein, nor can it be explained by downregulation of protein phosphatase 2A, the putative rhodopsin phosphatase. We further show that cone arrestin (ARR4), which binds light-activated, phosphorylated rhodopsin poorly, had little effect in enhancing rhodopsin dephosphorylation, whereas mice expressing binding-competent mutant ARR1-3A showed a similar time course of rhodopsin dephosphorylation as WT. Together, these results reveal a novel role of ARR1 in facilitating rhodopsin dephosphorylationin vivo.SIGNIFICANCE STATEMENTG-protein-coupled receptors (GPCRs) are transmembrane proteins used by cells to receive and respond to a broad range of extracellular signals that include neurotransmitters, hormones, odorants, and light (photons). GPCR signaling is terminated by two sequential steps: phosphorylation and arrestin binding. Both steps must be reversed when GPCRs are recycled and reused. Dephosphorylation, which is required for recycling, is an understudied process. Using rhodopsin as a prototypical GPCR, we discovered that arrestin facilitated rhodopsin dephosphorylation in living mice.

cAMP signaling in the nucleus leads to the expression of immediate early genes in neurons and learning and memory. In this issue of Science Signaling , Martinez et al . found that activation of the β 2 -adrenergic receptor enhances nuclear cAMP signaling that supports learning and memory in mice by removing the phosphodiesterase PDE4D5 from the nucleus through arrestin3 bound to the internalized receptor.

Arrestins were first discovered as suppressors of G protein-mediated signaling by G protein-coupled receptors. It was later demonstrated that arrestins also initiate several signaling branches, including mitogen-activated protein kinase cascades. Arrestin-3-dependent activation of the JNK family can be recapitulated with peptide fragments, which are monofunctional elements distilled from this multi-functional arrestin protein. Here, we use maltose-binding protein fusions of arrestin-3-derived peptides to identify arrestin elements that bind kinases of the ASK1-MKK4/7-JNK3 cascade and the shortest peptide facilitating JNK signaling. We identified a 16-residue arrestin-3-derived peptide expressed as a Venus fusion that leads to activation of JNK3α2 in cells. The strength of the binding to the kinases does not correlate with peptide activity. The ASK1-MKK4/7-JNK3 cascade has been implicated in neuronal apoptosis. While inhibitors of MAP kinases exist, short peptides are the first small molecule tools that can activate MAP kinases.

The finger loop in the central crest of the receptor-binding side of arrestins engages the cavity between the transmembrane helices of activated G protein-coupled receptors. Therefore, it was hypothesized to serve as the sensor that detects the activation state of the receptor. We performed comprehensive mutagenesis of the finger loop in bovine visual arrestin-1, generated mutant radiolabeled proteins by cell-free translation, and determined the effects of mutations on the in vitro binding of arrestin-1 to purified phosphorylated light-activated rhodopsin. This interaction is driven by two factors, rhodopsin activation and rhodopsin-attached phosphates. Therefore, the binding of arrestin-1 to light-activated unphosphorylated rhodopsin is low. To evaluate the role of the finger loop specifically in the recognition of the active receptor conformation, we tested the effects of these mutations in the context of truncated arrestin-1 that demonstrates much higher binding to unphosphorylated activated and phosphorylated inactive rhodopsin. The majority of finger loop residues proved important for arrestin-1 binding to light-activated rhodopsin, with six mutations affecting the binding exclusively to this form. Thus, the finger loop is the key element of arrestin-1 activation sensor. The data also suggest that arrestin-1 and its enhanced mutant bind various functional forms of rhodopsin differently.

The multifaceted adaptor protein β-arr1 (β-arrestin1) promotes activation of focal adhesion kinase (FAK) by the chemokine receptor CXCR4, facilitating chemotaxis. This function of β-arr1 requires the assistance of the adaptor protein STAM1 (signal-transducing adaptor molecule 1) because disruption of the interaction between STAM1 and β-arr1 reduces CXCR4-mediated activation of FAK and chemotaxis. To begin to understand the mechanism by which β-arr1 together with STAM1 activates FAK, we used site-directed spin-labeling EPR spectroscopy-based studies coupled with bioluminescence resonance energy transfer–based cellular studies to show that STAM1 is recruited to activated β-arr1 by binding to a novel surface on β-arr1 at the base of the finger loop, at a site that is distinct from the receptor-binding site. Expression of a STAM1-deficient binding β-arr1 mutant that is still able to bind to CXCR4 significantly reduced CXCL12-induced activation of FAK but had no impact on ERK-1/2 activation. We provide evidence of a novel surface at the base of the finger loop that dictates non-GPCR interactions specifying β-arrestin–dependent signaling by a GPCR. This surface might represent a previously unidentified switch region that engages with effector molecules to drive β-arrestin signaling.

Dynamic structural transitions within the seven‐transmembrane bundle represent the mechanism by which G‐protein‐coupled receptors convert an extracellular chemical signal into an intracellular biological function. Here, the conformational dynamics of the neuropeptide Y receptor type 2 (Y2R) during activation was investigated. The apo, full agonist‐, and arrestin‐bound states of Y2R were prepared by cell‐free expression, functional refolding, and reconstitution into lipid membranes. To study conformational transitions between these states, all six tryptophans of Y2R were 13C‐labeled. NMR‐signal assignment was achieved by dynamic‐nuclear‐polarization enhancement and the individual functional states of the receptor were characterized by monitoring 13C NMR chemical shifts. Activation of Y2R is mediated by molecular switches involving the toggle switch residue Trp2816.48 of the highly conserved SWLP motif and Trp3277.55 adjacent to the NPxxY motif. Furthermore, a conformationally preserved “cysteine lock”‐Trp11623.50 was identified., Structural transitions of crucial molecular switches of the activation of the human neuropeptide Y receptor type 2 are reported from the 13C‐tryptophan labeled molecule by solid‐state NMR spectroscopy in lipid membranes. The molecule shows high structural dynamics in the apo and agonist‐bound states, which is reduced by arrestin binding.

Arrestins demonstrate strong preference for phosphorylated over unphosphorylated receptors, but how arrestins “sense” receptor phosphorylation is unclear. A conserved lysine in the lariat loop of arrestins directly binds the phosphate in crystal structures of activated arrestin-1, −2, and −3. The lariat loop supplies two negative charges to the central polar core, which must be disrupted for arrestin activation and high-affinity receptor binding. Therefore, we hypothesized that receptor-attached phosphates pull the lariat loop via this lysine, thus removing the negative charges and destabilizing the polar core. We tested the role of this lysine by introducing charge elimination (Lys->Ala) and reversal (Lys->Glu) mutations in arrestin-1, −2, and −3. These mutations in arrestin-1 only moderately reduced phospho-rhodopsin binding and had no detectable effect on arrestin-2 and −3 binding to cognate non-visual receptors in cells. The mutations of Lys300 in bovine and homologous Lys301 in mouse arrestin-1 on the background of pre-activated mutants had variable effects on the binding to light-activated phosphorylated rhodopsin, while affecting the binding to unphosphorylated rhodopsin to a greater extent. Thus, conserved lysine in the lariat loop participates in receptor binding, but does not play a critical role in phosphate-induced arrestin activation.

Arrestin binding to active phosphorylated G protein-coupled receptors terminates G protein coupling and initiates another wave of signaling. Among the effectors that bind directly to receptor-associated arrestins are extracellular signal-regulated kinases 1/2 (ERK1/2), which promote cellular proliferation and survival. Arrestins may also engage ERK1/2 in isolation in a pre- or post-signaling complex that is likely in equilibrium with the full signal initiation complex. Molecular details of these binary complexes remain unknown. Here, we investigate the molecular mechanisms whereby arrestin-2 and arrestin-3 (a.k.a. β-arrestin1 and β-arrestin2, respectively) engage ERK1/2 in pairwise interactions. We find that purified arrestin-3 binds ERK2 more avidly than arrestin-2. A combination of biophysical techniques and peptide array analysis demonstrates that the molecular basis in this difference of binding strength is that the two non-visual arrestins bind ERK2 via different parts of the molecule. We propose a structural model of the ERK2- arrestin-3 complex in solution using size-exclusion chromatography coupled to small angle X-ray scattering (SEC-SAXS). This binary complex exhibits conformational heterogeneity. We speculate that this drives the equilibrium either toward the full signaling complex with receptor- bound arrestin at the membrane or toward full dissociation in the cytoplasm. As ERK1/2 regulates cell migration, proliferation, and survival, understanding complexes that relate to its activation could be exploited to control cell fate.

Three classes of G-protein-coupled receptor (GPCR) partners - G proteins, GPCR kinases, and arrestins - preferentially bind active GPCRs. Our analysis suggests that the structures of GPCRs bound to these interaction partners available today do not reveal a clear conformational basis for signaling bias, which would have enabled the rational design of biased GRCR ligands. In view of this, three possibilities are conceivable: (i) there are no generalizable GPCR conformations conducive to binding a particular type of partner; (ii) subtle differences in the orientation of individual residues and/or their interactions not easily detectable in the receptor-transducer structures determine partner preference; or (iii) the dynamics of GPCR binding to different types of partners rather than the structures of the final complexes might underlie transducer bias.

Many receptors for neurotransmitters, such as dopamine, norepinephrine, acetylcholine, and neuropeptides, belong to the superfamily of G protein-coupled receptors (GPCRs). A general model posits that GPCRs undergo two-step homologous desensitization: the active receptor is phosphorylated by kinases of the G protein-coupled receptor kinase (GRK) family, whereupon arrestin proteins specifically bind active phosphorylated receptors, shutting down G protein-mediated signaling, facilitating receptor internalization, and initiating distinct signaling pathways via arrestin-based scaffolding. Here, we review the mechanisms of GRK-dependent regulation of neurotransmitter receptors, focusing on the diverse modes of GRK-mediated phosphorylation of receptor subtypes. The immediate signaling consequences of GRK-mediated receptor phosphorylation, such as arrestin recruitment, desensitization, and internalization/resensitization, are equally diverse, depending not only on the receptor subtype but also on phosphorylation by GRKs of select receptor residues. We discuss the signaling outcome as well as the biological and behavioral consequences of the GRK-dependent phosphorylation of neurotransmitter receptors where known.

Arrestins are a small family of proteins that bind G protein-coupled receptors (GPCRs). Arrestin binds to active phosphorylated GPCRs with higher affinity than to all other functional forms of the receptor, including inactive phosphorylated and active unphosphorylated. The selectivity of arrestins suggests that they must have two sensors, which detect receptor-attached phosphates and the active receptor conformation independently. Simultaneous engagement of both sensors enables arrestin transition into a high-affinity receptor-binding state. This transition involves a global conformational rearrangement that brings additional elements of the arrestin molecule, including the middle loop, in contact with a GPCR, thereby stabilizing the complex. Here, we review structural and mutagenesis data that identify these two sensors and additional receptor-binding elements within the arrestin molecule. While most data were obtained with the arrestin-1-rhodopsin pair, the evidence suggests that all arrestins use similar mechanisms to achieve preferential binding to active phosphorylated GPCRs.

Receptor adaptation mechanisms involve regulation of the number, sensitivity and subcellular localization of receptors that detect environmental cues and convert extracellular signals into a change of intracellular events. Keywords: regulation of receptor desensitization; downregulation and gene expression; pharmacology; G protein-coupled receptors; β-adrenergic receptor; rhodopsin; G protein-coupled receptor kinase; arrestins; nicotinic acetylcholine receptor

Extracellular signal-regulated kinases (ERK1/2) are mitogen-activated protein kinases (MAPKs) that play a pro-tumorigenic role in numerous cancers. ERK1/2 possess two protein-docking sites that are distinct from the active site: the D-recruitment site (DRS) and the F-recruitment site. These docking sites facilitate substrate recognition, intracellular localization, signaling specificity, and protein complex assembly. Targeting these sites on ERK in a therapeutic context may overcome many problems associated with traditional ATP-competitive inhibitors. Here, we identified a new class of inhibitors that target the ERK DRS by screening a synthetic combinatorial library of more than 30 million compounds. The screen detects the competitive displacement of a fluorescent peptide from the DRS of ERK2. The top molecular scaffold from the screen was optimized for structure–activity relationship by positional scanning of different functional groups. This resulted in 10 compounds with similar binding affinities and a shared core structure consisting of a tertiary amine hub with three functionalized cyclic guanidino branches. Compound 2507–1 inhibited ERK2 from phosphorylating a DRS-targeting substrate and prevented the phosphorylation of ERK2 by a constitutively active MEK1 (MAPK/ERK kinase 1) mutant. Interaction between an analogue, 2507–8, and the ERK2 DRS was confirmed by nuclear magnetic resonance and X-ray crystallography. 2507–8 forms critical interactions at the common docking domain residue Asp319 via an arginine-like moiety that is shared by all 10 hits, suggesting a common binding mode. The structural and biochemical insights reported here provide the basis for developing new ERK inhibitors that are not ATP-competitive but instead function by disrupting critical protein–protein interactions.

Arrestins were initially identified for their role in homologous desensitization and internalization of G protein-coupled receptors. Receptor-bound arrestins also initiate signaling by interacting with other signaling proteins. Arrestins scaffold MAPK signaling cascades, MAPK kinase kinase (MAP3K), MAPK kinase (MAP2K), and MAPK. In particular, arrestins facilitate ERK1/2 activation by scaffolding ERK1/2 (MAPK), MEK1 (MAP2K), and Raf (MAPK3). However, the structural mechanism underlying this scaffolding remains unknown. Here, we investigated the mechanism of arrestin-2 scaffolding of cRaf, MEK1, and ERK2 using hydrogen/deuterium exchange-mass spectrometry, tryptophan-induced bimane fluorescence quenching, and NMR. We found that basal and active arrestin-2 interacted with cRaf, while only active arrestin-2 interacted with MEK1 and ERK2. The ATP binding status of MEK1 or ERK2 affected arrestin-2 binding; ATP-bound MEK1 interacted with arrestin-2, whereas only empty ERK2 bound arrestin-2. Analysis of the binding interfaces suggested that the relative positions of cRaf, MEK1, and ERK2 on arrestin-2 likely facilitate sequential phosphorylation in the signal transduction cascade.

Members of the arrestin superfamily have great propensity of self-association, but the physiological significance of this phenomenon is unclear. To determine the biological role of visual arrestin-1 oligomerization in rod photoreceptors, we expressed mutant arrestin-1 with severely impaired self-association in mouse rods and analyzed mice of both sexes. We show that the oligomerization-deficient mutant is capable of quenching rhodopsin signaling normally, as judged by electroretinography and single-cell recording. Like wild type, mutant arrestin-1 is largely excluded from the outer segments in the dark, proving that the normal intracellular localization is not due the size exclusion of arrestin-1 oligomers. In contrast to wild type, supraphysiological expression of the mutant causes shortening of the outer segments and photoreceptor death. Thus, oligomerization reduces the cytotoxicity of arrestin-1 monomer, ensuring long-term photoreceptor survival.SIGNIFICANCE STATEMENTVisual arrestin-1 forms dimers and tetramers. The biological role of its oligomerization is unclear. To test the role of arrestin-1 self-association, we expressed oligomerization-deficient mutant in arrestin-1 knock-out mice. The mutant quenches light-induced rhodopsin signaling like wild type, demonstrating thatin vivomonomeric arrestin-1 is necessary and sufficient for this function. In rods, arrestin-1 moves from the inner segments and cell bodies in the dark to the outer segments in the light. Nonoligomerizing mutant undergoes the same translocation, demonstrating that the size of the oligomers is not the reason for arrestin-1 exclusion from the outer segments in the dark. High expression of oligomerization-deficient arrestin-1 resulted in rod death. Thus, oligomerization reduces the cytotoxicity of high levels of arrestin-1 monomer.

Retinal rods function as accurate photon counters to provide for vision under very dim light. To do so, rods must generate highly amplified, reproducible responses to single photons, yet outer segment architecture and randomness in the location of rhodopsin photoisomerization on the surface of an internal disk introduce variability to the rising phase of the photon response. Soon after a photoisomerization at a disk rim, depletion of cGMP near the plasma membrane closes ion channels and hyperpolarizes the rod. But with a photoisomerization in the center of a disk, local depletion of cGMP is distant from the channels in the plasma membrane. Thus, channel closure is delayed by the time required for the reduction of cGMP concentration to reach the plasma membrane. Moreover, the local fall in cGMP dissipates over a larger volume before affecting the channels, so response amplitude is reduced. This source of variability increases with disk radius. Using a fully space-resolved biophysical model of rod phototransduction, we quantified the variability attributable to randomness in the location of photoisomerization as a function of disk structure. In mouse rods that have small disks bearing a single incisure, this variability was negligible in the absence of the incisure. Variability was increased slightly by the incisure, but randomness in the shutoff of rhodopsin emerged as the main source of single photon response variability at all but the earliest times. Variability arising from randomness in the transverse location of photoisomerization increased in magnitude and persisted over a longer period in the photon response of large salamander rods. A symmetric arrangement of multiple incisures in the disks of salamander rods greatly reduced this variability during the rising phase, but the incisures had the opposite effect on variability arising from randomness in rhodopsin shutoff at later times.

The adhesion GPCR latrophilin 3 (ADGRL3) has been associated with increased risk of attention-deficit/hyperactivity disorder (ADHD) and substance use in human genetic studies. Knockdown in multiple species leads to hyperlocomotion and altered dopamine signaling. Thus, ADGRL3 is a potential target for treatment of neuropsychiatric disorders that involve dopamine dysfunction, but its basic signaling properties are poorly understood. Identification of adhesion GPCR signaling partners has been limited by lack of tools to acutely activate these receptors in living cells. Here, we designed a novel acute activation strategy to characterize ADGRL3 signaling by engineering a receptor construct in which we could trigger acute activation enzymatically. Using this assay, we found that ADGRL3 signals through G12/G13 and Gq, with G12/13 the most robustly activated. Gα12/13 is a new player in ADGRL3 biology, opening up unexplored roles for ADGRL3 in the brain. Our methodological advancements should be broadly useful in adhesion GPCR research.

β‐arrestins (βarr1 and βarr2) are ubiquitous regulators of G protein‐coupled receptor (GPCR) signaling. Available data suggest that β‐arrestins dock to different receptors in different ways. However, the structural characterization of GPCR‐arrestin complexes is challenging and alternative approaches to study GPCR‐arrestin complexes are needed. Here, starting from the finger loop as a major site for the interaction of arrestins with GPCRs, we genetically incorporate non‐canonical amino acids for photo‐ and chemical crosslinking into βarr1 and βarr2 and explore binding topologies to GPCRs forming either stable or transient complexes with arrestins: the vasopressin receptor 2 (rhodopsin‐like), the corticotropin‐releasing factor receptor 1, and the parathyroid hormone receptor 1 (both secretin‐like). We show that each receptor leaves a unique footprint on arrestins, whereas the two β‐arrestins yield quite similar crosslinking patterns. Furthermore, we show that the method allows defining the orientation of arrestin with respect to the GPCR. Finally, we provide direct evidence for the formation of arrestin oligomers in the cell., Chemical and photo‐crosslinkers genetically incorporated into β‐arrestins allow the mapping of footprints of different receptors on the arrestin surface in living cells, and to identify intermolecular pairs of proximal amino acids in GPCR‐arrestin complexes.

Three out of four subtypes of arrestin proteins expressed in mammals self-associate, each forming oligomers of a distinct kind. Monomers and oligomers have different subcellular localization and distinct biological functions. Here we summarize existing evidence regarding arrestin oligomerization and discuss specific functions of monomeric and oligomeric forms, although too few of the latter are known. The data on arrestins highlight biological importance of oligomerization of signaling proteins. Distinct modes of oligomerization might be an important contributing factor to the functional differences among highly homologous members of the arrestin protein family.

Scaffold proteins tether and orient components of a signaling cascade to facilitate signaling. Although much is known about how scaffolds colocalize signaling proteins, it is unclear whether scaffolds promote signal amplification. Here, we used arrestin-3, a scaffold of the ASK1-MKK4/7-JNK3 cascade, as a model to understand signal amplification by a scaffold protein. We found that arrestin-3 exhibited >15-fold higher affinity for inactive JNK3 than for active JNK3, and this change involved a shift in the binding site following JNK3 activation. We used systems biochemistry modeling and Bayesian inference to evaluate how the activation of upstream kinases contributed to JNK3 phosphorylation. Our combined experimental and computational approach suggested that the catalytic phosphorylation rate of JNK3 at Thr-221 by MKK7 is two orders of magnitude faster than the corresponding phosphorylation of Tyr-223 by MKK4 with or without arrestin-3. Finally, we showed that the release of activated JNK3 was critical for signal amplification. Collectively, our data suggest a “conveyor belt” mechanism for signal amplification by scaffold proteins. This mechanism informs on a long-standing mystery for how few upstream kinase molecules activate numerous downstream kinases to amplify signaling.

Until the late 1990s class A G protein-coupled receptors (GPCRs) were believed to function as monomers. Then indirect evidence that they might internalize or even signal as dimers has emerged, along with proof that class C GPCRs are obligatory dimers. Crystal structures of GPCRs and their much larger binding partners were consistent with the idea that two receptors might engage a single G protein, GRK, or arrestin. However, recent biophysical, biochemical, and structural evidence invariably suggested that a single GPCR binds G proteins, GRKs, and arrestins. Here we review existing evidence of the stoichiometry of GPCR interactions with signal transducers and discuss potential biological roles of class A GPCR oligomers, including proposed homo- and heterodimers.

Arrestins are a small family of proteins with four isoforms in humans. Remarkably, two arrestins regulate signaling from >800 G protein-coupled receptors (GPCRs) or non-receptor activators by simultaneously binding an activator and one out of hundreds of other signaling proteins. When arrestins are bound to GPCRs or other activators, the affinity for these signaling partners changes. Thus, it is proposed that an activator alters arrestin’s ability to transduce a signal. The comparison of all available arrestin structures identifies several common conformational rearrangements associated with activation. In particular, it identifies elements that are directly involved in binding to GPCRs or other activators, elements that likely engage distinct downstream effectors, and elements that likely link the activator-binding sites with the effector-binding sites.

The structure of rhodopsin, an archetypal member of the G-protein-coupled family of receptors, in complex with its specific kinase enzyme, reveals the molecular mechanism of the first step of receptor inactivation. A view of a G-protein-coupled receptor bound to its kinase.

Arrestins regulate a wide range of signaling events, most notably when bound to active G protein-coupled receptors (GPCRs). Among the known effectors recruited by GPCR-bound arrestins are Src family kinases, which regulate cellular growth and proliferation. Here, we focus on arrestin-3 interactions with Fgr kinase, a member of the Src family. Previous reports demonstrated that Fgr exhibits high constitutive activity, but can be further activated by both arrestin-dependent and arrestin-independent pathways. We report that arrestin-3 modulates Fgr activity with a hallmark bell-shaped concentration-dependence, consistent with a role as a signaling scaffold. We further demonstrate using NMR spectroscopy that a polyproline motif within arrestin-3 interacts directly with the SH3 domain of Fgr. To provide a framework for this interaction, we determined the crystal structure of the Fgr SH3 domain at 1.9 Å resolution and developed a model for the GPCR-arrestin-3-Fgr complex that is supported by mutagenesis. This model suggests that Fgr interacts with arrestin-3 at multiple sites and is consistent with the locations of disease-associated Fgr mutations. Collectively, these studies provide a structural framework for arrestin-dependent activation of Fgr.

Purpose The purpose of this study was to identify the molecular defect in the disease-causing human arrestin-1 C147F mutant. Methods The binding of wild-type (WT) human arrestin-1 and several mutants with substitutions in position 147 (including C147F, which causes dominant retinitis pigmentosa in humans) to phosphorylated and unphosphorylated light-activated rhodopsin was determined. Thermal stability of WT and mutant human arrestin-1, as well as unfolded protein response in 661W cells, were also evaluated. Results WT human arrestin-1 was selective for phosphorylated light-activated rhodopsin. Substitutions of Cys-147 with smaller side chain residues, Ala or Val, did not substantially affect binding selectivity, whereas residues with bulky side chains in the position 147 (Ile, Leu, and disease-causing Phe) greatly increased the binding to unphosphorylated rhodopsin. Functional survival of mutant proteins with bulky substitutions at physiological and elevated temperature was also compromised. C147F mutant induced unfolded protein response in cultured cells. Conclusions Bulky Phe substitution of Cys-147 in human arrestin-1 likely causes rod degeneration due to reduced stability of the protein, which induces unfolded protein response in expressing cells.