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.

Spousal bereavement is associated with more pronounced ex vivo cytokine production and lower heart rate variability: Mechanisms underlying cardiovascular risk?

The loss of a spouse is a highly stressful event that puts people at excess risk of mortality. Excess mortality among those who are widowed is highest in the first six months after the death of a spouse and decreases over time. Heart disease accounts for the largest proportion of these deaths. The psychological stress associated with stressful life events can enhance inflammation and lower heart rate variability (HRV). Both lower HRV and higher inflammation are risk factors for cardiovascular morbidity and mortality. Thirty-two recently bereaved individuals (Mean = 89.68 days since death, SD = 17.09) and 33 age-matched comparisons completed a blood draw, EKG, and self-report questionnaires. In both adjusted and unadjusted models, spousal bereavement was associated with enhanced pro-inflammatory cytokine production by in vitro lipolysaccharide-stimulated peripheral blood leukocytes. Moreover, spousal bereavement was associated with lower HRV in comparison to the comparison group. This study is the first to demonstrate that bereavement is associated with a more pronounced ex vivo cytokine production and lower HRV in a population that exclusively consisted of widows and widowers. These findings add to the growing literature revealing the mechanisms that underlie bereavement-related cardiovascular problems. Future longitudinal studies are needed to determine the temporal relation between these risks. Understanding the biological mechanisms that underlie this stressful life event could allow researchers to create therapeutic targets for interventions to reduce or prevent the toll of a "broken heart."

Epac1 interacts with importin ß1 and controls neurite outgrowth independently of cAMP and Rap1.

Exchange protein directly activated by cAMP-1 (Epac1) is a cAMP sensor that regulates multiple cellular functions including cellular migration, proliferation and differentiation. Classically, Epac1 is thought to exert its effects through binding of cAMP leading to a conformational change in Epac1 and its accumulation at the plasma membrane (PM) where it activates Rap1. In search for regulators of Epac1 activity, we show here that importin ß1 (impß1) is an Epac1 binding partner that prevents PM accumulation of Epac1. We demonstrate that in the absence of impß1, endogenous as well as overexpressed Epac1 accumulate at the PM. Moreover, agonist-induced PM translocation of Epac1 leads to dissociation of Epac1 from impß1. Localization of Epac1 at the PM in the absence of impß1, requires residue R82 in its DEP domain. Notably, the PM accumulation of Epac1 in the absence of impß1 does not require binding of cAMP to Epac1 and does not result in Rap1 activation. Functionally, PM accumulation of Epac1, an Epac1 mutant deficient in cAMP binding, or an Epac1 mutant tethered to the PM, is sufficient to inhibit neurite outgrowth. In conclusion, we uncover a cAMP-independent function of Epac1 at the PM and demonstrate that impß1 controls subcellular localization of Epac1.

Critical role for Epac1 in inflammatory pain controlled by GRK2-mediated phosphorylation of Epac1.

cAMP signaling plays a key role in regulating pain sensitivity. Here, we uncover a previously unidentified molecular mechanism in which direct phosphorylation of the exchange protein directly activated by cAMP 1 (EPAC1) by G protein kinase 2 (GRK2) suppresses Epac1-to-Rap1 signaling, thereby inhibiting persistent inflammatory pain. Epac1(-/-) mice are protected against inflammatory hyperalgesia in the complete Freund's adjuvant (CFA) model. Moreover, the Epac-specific inhibitor ESI-09 inhibits established CFA-induced mechanical hyperalgesia without affecting normal mechanical sensitivity. At the mechanistic level, CFA increased activity of the Epac target Rap1 in dorsal root ganglia of WT, but not of Epac1(-/-), mice. Using sensory neuron-specific overexpression of GRK2 or its kinase-dead mutant in vivo, we demonstrate that GRK2 inhibits CFA-induced hyperalgesia in a kinase activity-dependent manner. In vitro, GRK2 inhibits Epac1-to-Rap1 signaling by phosphorylation of Epac1 at Ser-108 in the Disheveled/Egl-10/pleckstrin domain. This phosphorylation event inhibits agonist-induced translocation of Epac1 to the plasma membrane, thereby reducing Rap1 activation. Finally, we show that GRK2 inhibits Epac1-mediated sensitization of the mechanosensor Piezo2 and that Piezo2 contributes to inflammatory mechanical hyperalgesia. Collectively, these findings identify a key role of Epac1 in chronic inflammatory pain and a molecular mechanism for controlling Epac1 activity and chronic pain through phosphorylation of Epac1 at Ser-108. Importantly, using the Epac inhibitor ESI-09, we validate Epac1 as a potential therapeutic target for chronic pain.

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.

Salmeterol Efficacy and Bias in the Activation and Kinase-Mediated Desensitization of ß2-Adrenergic Receptors.

Salmeterol is a long-acting ß2-adrenergic receptor (ß2AR) agonist that is widely used as a bronchodilator for the treatment of persistent asthma and chronic obstructive pulmonary disease in conjunction with steroids. Previous studies demonstrated that salmeterol showed weak efficacy for activation of adenylyl cyclase; however, its efficacy in the complex desensitization of the ß2AR remains poorly understood. In this work, we provide insights into the roles played by the G protein-coupled receptor kinase/arrestin and protein kinase A in salmeterol-mediated desensitization through bioluminescence resonance energy transfer (BRET) studies of liganded-ß2AR binding to arrestin and through kinetic studies of cAMP turnover. First, BRET demonstrated a much reduced efficacy for salmeterol recruitment of arrestin to ß2AR relative to isoproterenol. The ratio of BRETISO/BRETSALM after 5-minute stimulation was 20 and decreased to 5 after 35 minutes, reflecting a progressive decline in BRETISO and a stable BRETSALM. Second, to assess salmeterol efficacy for functional desensitization, we examined the kinetics of salmeterol-induced cAMP accumulation (0-30 minutes) in human airway smooth muscle cells in the presence and absence of phosphodiesterase inhibition. Analysis of shaping of cAMP turnover for both agonists demonstrated significant salmeterol desensitization, although it was reduced relative to isoproterenol. Using an isoproterenol rescue protocol after either short-term (10 minutes) or long-term (2 and 14 hours) salmeterol pretreatments, we found that salmeterol progressively depressed isoproterenol stimulation but did not prevent subsequent rescue by isoproterenol and additional isoproterenol-mediated desensitization. Our findings reveal a complex efficacy for functional desensitization, demonstrating that although salmeterol shows weak efficacy for adenylyl cyclase activation and G protein-coupled receptor kinase/arrestin-mediated desensitization, it acts as a strong agonist in highly amplified protein kinase A-mediated events.

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 phototransduction 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.

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 ß2-adrenergic receptor (ß2AR), 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 ß2AR; 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.

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.

Critical role of the central 139-loop in stability and binding selectivity of arrestin-1.

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.

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 ß(2)-adrenergic receptors (ß(2)AR) 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 ß(2)AR, much more than to other receptors. The combination D260K/Q262P virtually eliminated ß(2)AR binding while preserving the interactions with M2R, D1R, and D2R. Conversely, Y239T enhanced arrestin-3 binding to ß(2)AR 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 ß(2)AR 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.

AKAP79 interacts with multiple adenylyl cyclase (AC) isoforms and scaffolds AC5 and -6 to alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptors.

Spatiotemporal specificity of cAMP action is best explained by targeting protein kinase A (PKA) to its substrates by A-kinase-anchoring proteins (AKAPs). At synapses in the brain, AKAP79/150 incorporates PKA and other regulatory enzymes into signal transduction networks that include beta-adrenergic receptors, alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA), and N-methyl-d-aspartic acid receptors. We previously showed that AKAP79/150 clusters PKA with type 5 adenylyl cyclase (AC5) to assemble a negative feedback loop in which the anchored kinase phosphorylates AC5 to dynamically suppress cAMP synthesis. We now show that AKAP79 can associate with multiple AC isoforms. The N-terminal regions of AC5, -6, and -9 mediate this protein-protein interaction. Mapping studies located a reciprocal binding surface between residues 77-108 of AKAP79. Intensity- and lifetime-based fluorescence resonance energy transfer demonstrated that deletion of AKAP79(77-108) region abolished AC5-AKAP79 interaction in living cells. The addition of the AKAP79(77-153) polypeptide fragment uncouples AC5/6 interactions with the anchoring protein and prevents PKA-mediated inhibition of AC activity in membranes. Use of the AKAP79(77-153) polypeptide fragment in brain extracts from wild-type and AKAP150(-/-) mice reveals that loss of the anchoring protein results in decreased AMPA receptor-associated AC activity. Thus, we propose that AKAP79/150 mediates protein-protein interactions that place AC5 in proximity to synaptic AMPA receptors.

Role for the regulator of G-protein signaling homology domain of G protein-coupled receptor kinases 5 and 6 in beta 2-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 beta(2)-adrenergic receptor (beta 2AR), 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 beta2AR 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 IC(50) of approximately 30 microM. 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.

Characterization of beta2-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 beta2-adrenergic receptor (beta2AR) 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 t(1/2) 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 microM isoproterenol for 5 min exhibited a lag phase of approximately 5 min, after which dephosphorylation proceeded slowly with a t(1/2) 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 beta2AR 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 beta2AR 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; t(1/2) = 1.6 min), than PKA and GRK site dephosphorylation, respectively, clearly dissociating the rapid phase of resensitization (0-5 min) from dephosphorylation.

Characterization of agonist stimulation of cAMP-dependent protein kinase and G protein-coupled receptor kinase phosphorylation of the beta2-adrenergic receptor using phosphoserine-specific antibodies.

Agonist-stimulated desensitization of the beta2-adrenergic receptor (beta2AR) is caused by both a potent cAMP-dependent protein kinase (PKA)-mediated phosphorylation and a less potent, occupancy-dependent, G protein-coupled receptor kinase (GRK)-mediated phosphorylation that leads to beta-arrestin binding and internalization. In this study the kinetics of phosphorylation of the third intracellular loop PKA site Ser262 and the putative C-tail GRK sites Ser355, Ser356 of the human beta2AR overexpressed in human embryonic kidney (HEK) 293 cells were characterized using phosphoserine-specific antibodies. Specificity of the antibodies was shown by their lack of reactivity with mutant beta2ARs lacking the respective sites. In addition, overexpression of GRK2 and GRK5 increased basal levels of phosphorylation of the GRK sites Ser355, Ser356 in both COS-7 and HEK 293 cells. Epinephrine, prostaglandin E1, and forskolin at maximum concentrations stimulated phosphorylation of the beta2AR PKA site (Ser262) by 4-fold, whereas PMA stimulated it by 2-fold. Epinephrine stimulated PKA site phosphorylation with an EC50 of 20 to 40 pM. In contrast, epinephrine stimulated GRK site phosphorylation (Ser355,Ser356) with an EC50 of 200 nM (1-min treatments), which is more than 4000-fold higher relative to PKA site phosphorylation, consistent with an occupancy-driven process. After 10 to 30 min, the EC50 for epinephrine stimulation of GRK site phosphorylation was reduced to 10 to 20 nM but was still approximately 200-fold greater than for the PKA site. The EC50 for internalization correlated with GRK site phosphorylation and showed a similar shift with time of epinephrine stimulation. The kinetics of epinephrine-stimulated GRK site phosphorylation were not altered in a mutant of the beta2AR lacking the PKA consensus sites. The initial levels (2 min) of a range of agonist-stimulated GRK site phosphorylations were correlated with their efficacy for activation of adenylyl cyclase, namely epinephrine > or = formoterol = fenoterol > terbutaline = zinterol = albuterol > salmeterol > dobutamine > or = ephedrine. However, after 20 to 30 min of treatment, agonists with intermediate strengths, such as albuterol and salmeterol, stimulate GRK site phosphorylations that are approximately equal to that produced by epinephrine, and the correlation breaks down. The GRK and PKA site antibodies were also effective in detecting phosphorylation of the endogenous beta2AR expressed in A431 human epidermoid carcinoma cells. To summarize, our results show a remarkable amplification of PKA site phosphorylation relative to the putative GRK site phosphorylation, heterologous stimulation of the PKA site phosphorylation, no dependence of GRK site phosphorylation on PKA sites, and a reasonable correlation of initial levels of GRK site phosphorylation with the strength of a range of agonists.