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 neuronspecific 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.
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.
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.
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.
Triple negative breast cancer (TNBC) accounts for 15-20% of breast cancer cases in the United States. Systemic neoadjuvant chemotherapy (NACT), with or without immunotherapy, is the current standard ...of care for patients with early-stage TNBC. However, up to 70% of TNBC patients have significant residual disease once NACT is completed, which is associated with a high risk of developing recurrence within two to three years of surgical resection. To identify targetable vulnerabilities in chemoresistant TNBC, we generated longitudinal patient-derived xenograft (PDX) models from TNBC tumors before and after patients received NACT. We then compiled transcriptomes and drug response profiles for all models. Transcriptomic analysis identified the enrichment of aberrant protein homeostasis pathways in models from post-NACT tumors relative to pre-NACT tumors. This observation correlated with increased sensitivity in vitro to inhibitors targeting the proteasome, heat shock proteins, and neddylation pathways. Pevonedistat, a drug annotated as a NEDD8-activating enzyme (NAE) inhibitor, was prioritized for validation in vivo and demonstrated efficacy as a single agent in multiple PDX models of TNBC. Pharmacotranscriptomic analysis identified a pathway-level correlation between pevonedistat activity and post-translational modification (PTM) machinery, particularly involving neddylation and sumoylation targets. Elevated levels of both NEDD8 and SUMO1 were observed in models exhibiting a favorable response to pevonedistat compared to those with a less favorable response in vivo. Moreover, a correlation emerged between the expression of neddylation-regulated pathways and tumor response to pevonedistat, indicating that targeting these PTM pathways may prove effective in combating chemoresistant TNBC.
•Those who were spousally bereaved had lower HRV than those who were not bereaved.•Widowers had higher ex vivo cytokine production than non-bereaved age-matched controls.•These findings add to the ...growing literature revealing the mechanisms that underlie bereavement-related cardiovascular problems.
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.”
•Inflammatory markers can distinguish bereaved spouses higher on grief severity compared with bereaved spouses with lower grief severity.•Even in a population high on depressive symptoms, there is a ...positive relationship between depression and inflammation.•Similar to the general population, inflammation is an important marker of elevated levels of depression among bereaved spouses.
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.
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.
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.
Salmeterol is a long-acting β2-adrenergic receptor (β2AR) 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 β2AR 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-β2AR binding to arrestin and through kinetic studies of cAMP turnover. First, BRET demonstrated a much reduced efficacy for salmeterol recruitment of arrestin to β2AR 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.
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 β-adrenergic receptors, α-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 AKAP7977–108 region abolished AC5-AKAP79 interaction in living cells. The addition of the AKAP7977–153 polypeptide fragment uncouples AC5/6 interactions with the anchoring protein and prevents PKA-mediated inhibition of AC activity in membranes. Use of the AKAP7977–153 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.
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 β1 (impβ1) is an Epac1 binding partner that prevents PM accumulation of Epac1. We demonstrate that in the absence of impβ1, endogenous as well as overexpressed Epac1 accumulate at the PM. Moreover, agonist-induced PM translocation of Epac1 leads to dissociation of Epac1 from impβ1. Localization of Epac1 at the PM in the absence of impβ1, requires residue R82 in its DEP domain. Notably, the PM accumulation of Epac1 in the absence of impβ1 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 impβ1 controls subcellular localization of Epac1.
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