Faiza Baameur

Critical Role of the Central 139-Loop in Stability and Binding Selectivity of Arrestin-1

Background: Arrestin-1 selectively binds light-activated phosphorhodopsin. Results: Deletions in the 139-loop or disruptions of its interactions with the body of arrestin-1 greatly reduce arrestin-1 stability and selectivity. Conclusion: The central 139-loop supports basal arrestin-1 conformation and reduces its binding to non-preferred forms of rhodopsin. Significance: The central 139-loop is an earlier unappreciated element contributing to the thermal stability and binding selectivity of arrestins. Arrestin-1 selectively binds active phosphorylated rhodopsin (P-Rh*), demonstrating much lower affinity for inactive phosphorylated (P-Rh) and unphosphorylated active (Rh*) forms. Receptor interaction induces significant conformational changes in arrestin-1, which include large movement of the previously neglected 139-loop in the center of the receptor binding surface, away from the incoming receptor. To elucidate the functional role of this loop, in mouse arrestin-1 we introduced deletions of variable lengths and made several substitutions of Lys-142 in it and Asp-72 in the adjacent loop. Several mutants with perturbations in the 139-loop demonstrate increased binding to P-Rh*, dark P-Rh, Rh*, and phospho-opsin. Enhanced binding of arrestin-1 mutants to non-preferred forms of rhodopsin correlates with decreased thermal stability. The 139-loop perturbations increase P-Rh* binding of arrestin-1 at low temperatures and further change its binding profile on the background of 3A mutant, where the C-tail is detached from the body of the molecule by triple alanine substitution. Thus, the 139-loop stabilizes basal conformation of arrestin-1 and acts as a brake, preventing its binding to non-preferred forms of rhodopsin. Conservation of this loop in other subtypes suggests that it has the same function in all members of the arrestin family.

Manipulation of Very Few Receptor Discriminator Residues Greatly Enhances Receptor Specificity of Non-visual Arrestins

Background: WT non-visual arrestins are promiscuous, binding numerous GPCRs. Results: Mutations of very few receptor discriminator residues greatly increase receptor specificity of arrestin-3. Conclusion: Targeted manipulation of key residues that determine receptor preference is a viable approach to the construction of arrestins with high specificity for particular GPCR subtypes. Significance: Non-visual arrestins with high receptor specificity make therapeutic use of signaling-biased arrestin mutants feasible. Based on the identification of residues that determine receptor selectivity of arrestins and the analysis of the evolution in the arrestin family, we introduced 10 mutations of “receptor discriminator” residues in arrestin-3. The recruitment of these mutants to M2 muscarinic (M2R), D1 (D1R) and D2 (D2R) dopamine, and β2-adrenergic receptors (β2AR) was assessed using bioluminescence resonance energy transfer-based assays in cells. Seven of 10 mutations differentially affected arrestin-3 binding to individual receptors. D260K and Q262P reduced the binding to β2AR, much more than to other receptors. The combination D260K/Q262P virtually eliminated β2AR binding while preserving the interactions with M2R, D1R, and D2R. Conversely, Y239T enhanced arrestin-3 binding to β2AR and reduced the binding to M2R, D1R, and D2R, whereas Q256Y selectively reduced recruitment to D2R. The Y239T/Q256Y combination virtually eliminated the binding to D2R and reduced the binding to β2AR and M2R, yielding a mutant with high selectivity for D1R. Eleven of 12 mutations significantly changed the binding to light-activated phosphorhodopsin. Thus, manipulation of key residues on the receptor-binding surface modifies receptor preference, enabling the construction of non-visual arrestins specific for particular receptor subtypes. These findings pave the way to the construction of signaling-biased arrestins targeting the receptor of choice for research or therapeutic purposes.

Characterization of β2-adrenergic receptor dephosphorylation: comparison with the rate of resensitization

Dephosphorylation of the cyclic AMP-dependent protein kinase (PKA) site phosphoserine 262 and the G protein-coupled receptor kinase (GRK) site phosphoserines 355 and 356 of the β2-adrenergic receptor (β2AR) were characterized in both intact human embryonic kidney 293 cells and subcellular fractions and were correlated with the rate of resensitization of isoproterenol stimulation of adenylyl cyclase after treatment with isoproterenol and blockade by antagonist. Dephosphorylation of the PKA site after stimulation with 300 pM isoproterenol occurred with a t½ of 9 min (k = 0.08 ± 0.016/min) in intact cells in the absence of internalization. Dephosphorylation of the GRK sites in intact cells after treatment with 1.0 μM isoproterenol for 5 min exhibited a lag phase of ≈ 5 min, after which dephosphorylation proceeded slowly with a t½ of 18 min (k = 0.039 ± 0.006/min). Consistent with the slow rate of GRK site dephosphorylation, the phosphatase inhibitors calyculin A and okadaic acid failed to augment phosphorylation in intact cells during continuous agonist stimulation indicating that GRK site dephosphorylation was minimal. However, both inhibited dephosphorylation of the GRK sites after the addition of antagonist. Slow GRK site dephosphorylation after antagonist treatment was also demonstrated by the relative stability of internalized phosphorylated β2AR in cells as observed both by immunofluorescence microscopy using a phospho-site-specific antibody and by studies of the subcellular localization of the GRK-phosphorylated β2AR on sucrose gradients that revealed nearly equivalent levels of GRK site phosphorylation in the plasma membrane and vesicular fractions. In addition, dephosphorylation of the GRK sites by intrinsic phosphatase activity occurred only in the heavy vesicle fractions. In contrast to the slow rates of dephosphorylation, the rate of resensitization of isoproterenol stimulation of adenylyl cyclase was 5- and 10-fold faster (k = 0.43 ± 0.009/min; t½ = 1.6 min), than PKA and GRK site dephosphorylation, respectively, clearly dissociating the rapid phase of resensitization (0-5 min) from dephosphorylation.

Role for the Regulator of G-Protein Signaling Homology Domain of G Protein-Coupled Receptor Kinases 5 and 6 in β2-Adrenergic Receptor and Rhodopsin Phosphorylation

Phosphorylation of G protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) is a major mechanism of desensitization of these receptors. GPCR activation of GRKs involves an allosteric site on GRKs distinct from the catalytic site. Although recent studies have suggested an important role of the N- and C-termini and domains surrounding the kinase active site in allosteric activation, the nature of that site and the relative roles of the RH domain in particular remain unknown. Based on evolutionary trace analysis of both the RH and kinase domains of the GRK family, we identified an important cluster encompassing helices 3, 9, and 10 in the RH domain in addition to sites in the kinase domain. To define its function, a panel of GRK5 and -6 mutants was generated and screened by intact-cell assay of constitutive GRK phosphorylation of the β2-adrenergic receptor (β2AR), in vitro GRK phosphorylation of light-activated rhodopsin, and basal catalytic activity measured by tubulin phosphorylation and autophosphorylation. A number of double mutations within helices 3, 9, and 10 reduced phosphorylation of the β2AR and rhodopsin by 50 to 90% relative to wild-type GRK, as well as autophosphorylation and tubulin phosphorylation. Based on these results, helix 9 peptide mimetics were designed, and several were found to inhibit rhodopsin phosphorylation by GRK5 with an IC50 of ∼30 μM. In summary, our studies have uncovered previously unrecognized functionally important sites in the regulator of G-protein signaling homology domain of GRK5 and -6 and identified a peptide inhibitor with potential for specific blockade of GRK-mediated phosphorylation of receptors.

C-terminal threonines and serines play distinct roles in the desensitization of rhodopsin, a G protein-coupled receptor

Rod photoreceptors generate measurable responses to single-photon activation of individual molecules of the G protein-coupled receptor (GPCR), rhodopsin. Timely rhodopsin desensitization depends on phosphorylation and arrestin binding, which quenches G protein activation. Rhodopsin phosphorylation has been measured biochemically at C-terminal serine residues, suggesting that these residues are critical for producing fast, low-noise responses. The role of native threonine residues is unclear. We compared single-photon responses from rhodopsin lacking native serine or threonine phosphorylation sites. Contrary to expectation, serine-only rhodopsin generated prolonged step-like single-photon responses that terminated abruptly and randomly, whereas threonine-only rhodopsin generated responses that were only modestly slower than normal. We show that the step-like responses of serine-only rhodopsin reflect slow and stochastic arrestin binding. Thus, threonine sites play a privileged role in promoting timely arrestin binding and rhodopsin desensitization. Similar coordination of phosphorylation and arrestin binding may more generally permit tight control of the duration of GPCR activity. DOI: http://dx.doi.org/10.7554/eLife.05981.001

Rapid degeneration of rod photoreceptors expressing self-association-deficient arrestin-1 mutant.

Arrestin-1 binds light-activated phosphorhodopsin and ensures timely signal shutoff. We show that high transgenic expression of an arrestin-1 mutant with enhanced rhodopsin binding and impaired oligomerization causes apoptotic rod death in mice. Dark rearing does not prevent mutant-induced cell death, ruling out the role of arrestin complexes with light-activated rhodopsin. Similar expression of WT arrestin-1 that robustly oligomerizes, which leads to only modest increase in the monomer concentration, does not affect rod survival. Moreover, WT arrestin-1 co-expressed with the mutant delays retinal degeneration. Thus, arrestin-1 mutant directly affects cell survival via binding partner(s) other than light-activated rhodopsin. Due to impaired self-association of the mutant its high expression dramatically increases the concentration of the monomer. The data suggest that monomeric arrestin-1 is cytotoxic and WT arrestin-1 protects rods by forming mixed oligomers with the mutant and/or competing with it for the binding to non-receptor partners. Thus, arrestin-1 self-association likely serves to keep low concentration of the toxic monomer. The reduction of the concentration of harmful monomer is an earlier unappreciated biological function of protein oligomerization.

Development of an MRI biomarker sensitive to tetrameric visual arrestin 1 and its reduction via light-evoked translocation in vivo

Rod tetrameric arrestin 1 (tet‐ARR1), stored in the outer nuclear layer/inner segments in the dark, modulates photoreceptor synaptic activity; light exposure stimulates a reduction via translocation to the outer segments for terminating G‐protein coupled photo‐transduction signaling. Here, we test the hypothesis that intraretinal spin‐lattice relaxation rate in the rotating frame (1/T1ρ), an endogenous MRI contrast mechanism, has high potential for evaluating rod tet‐ARR1 and its reduction via translocation. Dark‐ and light‐exposed mice (null for the ARR1 gene, overexpressing ARR1, diabetic, or wild type with or without treatment with Mn2+, a calcium channel probe) were studied using 1/T1ρ MRI. Immunohistochemistry and single‐cell recordings of the retinas were also performed. In wild‐type mice with or without treatment with Mn2+, 1/T1ρ of avascular outer retina (64% to 72% depth) was significantly (P < 0.05) greater in the dark than in the light; a significant (P < 0.05) but opposite pattern was noted in the inner retina (<50% depth). Light‐evoked outer retina Δ1/T1ρ was absent in ARR1‐null mice and supernormal in overexpressing mice. In diabetic mice, the outer retinal Δ1/T1ρ pattern suggested normal dark‐to‐light tet‐ARR1 translocation and chromophore content, conclusions confirmed ex vivo. Light‐stimulated Δ1/T1ρ in inner retina was linked to changes in blood volume. Our data support 1/T1ρ MRI for noninvasively assessing rod tet‐ARR1 and its reduction via protein translocation, which can be combined with other metrics of retinal function in vivo.—Berkowitz, B. A., Gorgis, J., Patel, A., Baameur, F., Gurevich, V. V., Craft, C. M., Kefalov, V. J., Roberts, R. Development of an MRI biomarker sensitive to tetrameric visual arrestin 1 and its reduction via light‐evoked translocation in vivo. FASEB J. 29, 554‐564 (2015). www.fasebj.org

Biochemical and Cellular Specificity of Peptide Inhibitors of G Protein-Coupled Receptor Kinases

Identifying novel allosteric inhibitors of G protein-coupled receptor kinases (GRKs) would be of considerable use in limiting both the extent of desensitization of GPCRs as well as downstream positive regulation through GRKs. Several peptides have previously been identified as inhibitors of specific GRKs, but to date there have been few comparisons of the selectivities of these materials on the seven GRKs, modifications to allow cell penetration, or off-target activities. The goal of this study was to determine if a panel of peptides mimicking domains on either GPCRs or GRKs would exhibit selective inhibition of GRKs 2, 5, 6 and 7 phosphorylation of rhodopsin. Peptides included sequences from GRK5; helices 3, 9, and 10 (α3, α9, and α10) in the RH domain, and the N-terminal peptide (N-Ter), as well as the intracellular loop 1 (iL1) of the β2-adrenergic receptor (β2AR), and the Gα transducin C-tail (TCT). While some selectivity for individual GRKs was found, overall selectivity was limited and often not reflective of structural predictions. Off-target effects were probed by determining peptide inhibition of adenylyl cyclase (AC) and PKA, and while peptides had no effect on AC activity, N-Ter, iL1, and α10 were potent inhibitors of PKA. To probe inhibition of GRK activity in intact cells, we synthesized TAT-tagged peptides, and found that TAT-α9-R169A and TAT–TCT inhibited isoproterenol-stimulated GRK phosphorylation of the β2AR; however, the TAT peptides also inhibited isoproterenol and forskolin stimulation of AC activity. Our findings demonstrate potent peptide inhibition of GRK activities in vitro, highlight the differences in the environments of biochemical and cell-based assays, and illustrate the care that must be exercised in interpreting results of either assay alone.

Molecular dynamics simulation of solid C60 under progressively raised pressure

Recent molecular dynamics investigations have shown that C 60 fullerites can be investigated by means of simple potentials and few calculations have focused on the pressure dependence of thermodynamic properties and the induced order-disorder phase transition. The aim of this work is to perform molecular dynamics simulation of the C 60 crystal lattice in the face centred cubic structure under progressively raised pressure, to illustrate the limits of the crude pseudo atom approach and the validity of the potential in the rigid molecule, when seeking for possible phase transitions. For that purpose, several thermodynamic and structural properties are calculated with different potential models in the pseudo atom approach and as a rigid freely rotating C 60 molecule. Calculated properties are compared to experimental data. It will be demonstrated that though the pseudo atom approach is reasonable in the low-pressure region (< 4 kbar), the rigid molecule is in better agreement with experiments depending on the choice of the model potential and the pressure applied. Orientational transition pressures are observed at 5 and 260 K, the distorted lattice and the cell contraction are measured and the density of phonon states is reported at different pressures.

Grief, depressive symptoms, and inflammation in the spousally bereaved

Grief is conceptualized by strong negative emotions, which include longing, sadness, and preoccupations with thoughts, recollections, and images of the spouse. In the initial months after the loss of a spouse, those who are widowed are at risk for cardiovascular problems and premature mortality. In the general population, depression is characterized by chronic low-grade inflammation, a key predictor of cardiovascular problems, morbidity, and mortality. Although depression and grief share similarities, they are distinct constructs. We aimed to identify if grief was related to inflammation among those who had a spouse recently die. We also sought to determine if those who are widowed and already experience elevated levels of depressive symptoms compared with the general population had higher levels of inflammation compared with those who are widowed who report fewer depressive symptoms. Ninety-nine recently bereaved individuals (M = 84.74 days since passing, SD = 18.17) completed a blood draw and psychological assessments. Proinflammatory T cell-derived cytokines were assessed, which included interferon gamma (IFN-γ), interleukin (IL)-6, tumor necrosis factor alpha (TNF-α), IL17-A, and IL-2. Bereaved individuals with a higher grief severity (using an established cut-score) had higher levels of the proinflammatory cytokines IFN-γ, IL-6, and TNF-α than those with less grief severity. Those who experienced higher levels of depression exhibited elevated levels of proinflammatory cytokines compared with those who had lower levels of depression (using a continuous measure of depressive symptoms, as well as an established cut score). This is the first study to demonstrate that inflammatory markers can distinguish those who are widowed based on grief severity such that those who are higher on grief severity have higher levels of inflammation compared with those who are lower on grief severity. These findings also add to the broader literature on depression and inflammation by showing that even in a population with high levels of depressive symptoms, there is a positive relationship between depression and inflammation.