RESUMEN
G protein-coupled receptors (GPCRs) constitute the largest family of transmembrane proteins and play a crucial role in regulating diverse cellular functions. They transmit their signaling via binding to intracellular signal transducers and effectors, such as G proteins, GPCR kinases, and ß-arrestins. To influence specific GPCR signaling behaviors, ß-arrestins recruit effectors to form larger signaling complexes. Intriguingly, they facilitate divergent functions for the binding to different receptors. Recent studies relying on advanced structural approaches, novel biosensors and interactome analyses bring us closer to understanding how this specificity is achieved. In this article, we share our hypothesis of how active GPCRs induce specific conformational rearrangements within ß-arrestins to reveal distinct binding interfaces, enabling the recruitment of a subset of effectors to foster specialized signaling complexes. Furthermore, we discuss methods of how to comprehensively assess ß-arrestin conformational states and present the current state of research regarding the functionality of these multifaceted scaffolding proteins.
Asunto(s)
Arrestinas , Receptores Acoplados a Proteínas G , beta-Arrestinas/metabolismo , Arrestinas/química , Arrestinas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Sepsis is characterized as life-threatening organ dysfunction caused by a dysregulated host response to an infection. Despite numerous clinical trials that addressed this syndrome, there is still no causative treatment available to dampen its severity. Curtailing the infection at an early stage with anti-infectives is the only effective treatment regime besides intensive care. In search for additional treatment options, we recently discovered the inhibition of the sphingosine 1-phosphate (S1P) lyase and subsequent activation of the S1P receptor type 3 (S1PR3) in pre-conditioning experiments as promising targets for sepsis prevention. Here, we demonstrate that treatment of septic mice with the direct S1P lyase inhibitor C31 or the S1PR3 agonist CYM5541 in the advanced phase of sepsis resulted in a significantly increased survival rate. A single dose of each compound led to a rapid decline of sepsis severity in treated mice and coincided with decreased cytokine release and increased lung barrier function with unaltered bacterial load. The survival benefit of both compounds was completely lost in S1PR3 deficient mice. Treatment of the murine macrophage cell line J774.1 with either C31 or CYM5541 resulted in decreased protein kinase B (Akt) and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) phosphorylation without alteration of the mitogen-activated protein kinase (MAPK) p38 and p44/42 phosphorylation. Thus, activation of S1PR3 in the acute phase of sepsis by direct agonism or S1P lyase inhibition dampened Akt and JNK phosphorylation, resulting in decreased cytokine release, improved lung barrier stability, rapid decline of sepsis severity and better survival in mice.
Asunto(s)
Aldehído-Liasas , Ratones Endogámicos C57BL , Sepsis , Receptores de Esfingosina-1-Fosfato , Animales , Sepsis/tratamiento farmacológico , Sepsis/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismo , Receptores de Esfingosina-1-Fosfato/antagonistas & inhibidores , Aldehído-Liasas/antagonistas & inhibidores , Aldehído-Liasas/metabolismo , Ratones , Receptores de Lisoesfingolípidos/agonistas , Receptores de Lisoesfingolípidos/metabolismo , Masculino , Modelos Animales de Enfermedad , Línea Celular , Proteínas Proto-Oncogénicas c-akt/metabolismo , Citocinas/metabolismo , Ratones NoqueadosRESUMEN
BACKGROUND: Bile salts of hepatic and microbial origin mediate interorgan cross talk in the gut-liver axis. Here, we assessed whether the newly discovered class of microbial bile salt conjugates (MBSCs) activate the main host bile salt receptors (Takeda G protein-coupled receptor 5 [TGR5] and farnesoid X receptor [FXR]) and enter the human systemic and enterohepatic circulation. METHODS: N-amidates of (chenodeoxy) cholic acid and leucine, tyrosine, and phenylalanine were synthesized. Receptor activation was studied in cell-free and cell-based assays. MBSCs were quantified in mesenteric and portal blood and bile of patients undergoing pancreatic surgery. RESULTS: MBSCs were activating ligands of TGR5 as evidenced by recruitment of Gsα protein, activation of a cAMP-driven reporter, and diminution of lipopolysaccharide-induced cytokine release from macrophages. Intestine-enriched and liver-enriched FXR isoforms were both activated by MBSCs, provided that a bile salt importer was present. The affinity of MBSCs for TGR5 and FXR was not superior to host-derived bile salt conjugates. Individual MBSCs were generally not detected (ie, < 2.5 nmol/L) in human mesenteric or portal blood, but Leu-variant and Phe-variant were readily measurable in bile, where MBSCs comprised up to 213 ppm of biliary bile salts. CONCLUSIONS: MBSCs activate the cell surface receptor TGR5 and the transcription factor FXR and are substrates for intestinal (apical sodium-dependent bile acid transporter) and hepatic (Na+ taurocholate co-transporting protein) transporters. Their entry into the human circulation is, however, nonsubstantial. Given low systemic levels and a surplus of other equipotent bile salt species, the studied MBSCs are unlikely to have an impact on enterohepatic TGR5/FXR signaling in humans. The origin and function of biliary MBSCs remain to be determined.
Asunto(s)
Ácidos y Sales Biliares , Receptores Citoplasmáticos y Nucleares , Receptores Acoplados a Proteínas G , Humanos , Bilis/química , Ácidos y Sales Biliares/farmacología , Ácidos y Sales Biliares/metabolismo , Hígado/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
The D2 dopamine receptor (D2R) signals through both G proteins and ß-arrestins to regulate important physiological processes, such as movement, reward circuitry, emotion, and cognition. ß-arrestins are believed to interact with G protein-coupled receptors (GPCRs) at the phosphorylated C-terminal tail or intracellular loops. GPCR kinases (GRKs) are the primary drivers of GPCR phosphorylation, and for many receptors, receptor phosphorylation is indispensable for ß-arrestin recruitment. However, GRK-mediated receptor phosphorylation is not required for ß-arrestin recruitment to the D2R, and the role of GRKs in D2R-ß-arrestin interactions remains largely unexplored. In this study, we used GRK knockout cells engineered using CRISPR-Cas9 technology to determine the extent to which ß-arrestin recruitment to the D2R is GRK-dependent. Genetic elimination of all GRK expression decreased, but did not eliminate, agonist-stimulated ß-arrestin recruitment to the D2R or its subsequent internalization. However, these processes were rescued upon the re-introduction of various GRK isoforms in the cells with GRK2/3 also enhancing dopamine potency. Further, treatment with compound 101, a pharmacological inhibitor of GRK2/3 isoforms, decreased ß-arrestin recruitment and receptor internalization, highlighting the importance of this GRK subfamily for D2R-ß-arrestin interactions. These results were recapitulated using a phosphorylation-deficient D2R mutant, emphasizing that GRKs can enhance ß-arrestin recruitment and activation independently of receptor phosphorylation.
Asunto(s)
Quinasas de Receptores Acoplados a Proteína-G , Receptores Dopaminérgicos , Arrestinas/metabolismo , beta-Arrestinas/metabolismo , Quinasas de Receptores Acoplados a Proteína-G/metabolismo , Fosforilación , Isoformas de Proteínas/metabolismo , Receptores Dopaminérgicos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Humanos , Células HEK293RESUMEN
ß-arrestins mediate regulatory processes for over 800 different G protein-coupled receptors (GPCRs) by adopting specific conformations that result from the geometry of the GPCR-ß-arrestin complex. However, whether ß-arrestin1 and 2 respond differently for binding to the same GPCR is still unknown. Employing GRK knockout cells and ß-arrestins lacking the finger-loop-region, we show that the two isoforms prefer to associate with the active parathyroid hormone 1 receptor (PTH1R) in different complex configurations ("hanging" and "core"). Furthermore, the utilisation of advanced NanoLuc/FlAsH-based biosensors reveals distinct conformational signatures of ß-arrestin1 and 2 when bound to active PTH1R (P-R*). Moreover, we assess ß-arrestin conformational changes that are induced specifically by proximal and distal C-terminal phosphorylation and in the absence of GPCR kinases (GRKs) (R*). Here, we show differences between conformational changes that are induced by P-R* or R* receptor states and further disclose the impact of site-specific GPCR phosphorylation on arrestin-coupling and function.
Asunto(s)
Arrestinas , Transducción de Señal , Arrestinas/metabolismo , Quinasas de Receptores Acoplados a Proteína-G/metabolismo , Luciferasas , Hormona Paratiroidea/metabolismo , Fosforilación/fisiología , Isoformas de Proteínas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/fisiología , beta-Arrestina 1/genética , beta-Arrestina 1/metabolismo , Arrestina beta 2/genética , Arrestina beta 2/metabolismo , beta-Arrestinas/metabolismoRESUMEN
G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane receptors and their signal transduction is tightly regulated by GPCR kinases (GRKs) and ß-arrestins. In this review, we discuss novel aspects of the regulatory GRK/ß-arrestin system. Therefore, we briefly revise the origin of the "barcode" hypothesis for GPCR/ß-arrestin interactions, which states that ß-arrestins recognize different receptor phosphorylation states to induce specific functions. We emphasize two important parameters which may influence resulting GPCR phosphorylation patterns: (A) direct GPCR-GRK interactions and (B) tissue-specific expression and availability of GRKs and ß-arrestins. In most studies that focus on the molecular mechanisms of GPCR regulation, these expression profiles are underappreciated. Hence we analyzed expression data for GRKs and ß-arrestins in 61 tissues annotated in the Human Protein Atlas. We present our analysis in the context of pathophysiological dysregulation of the GPCR/GRK/ß-arrestin system. This tissue-specific point of view might be the key to unraveling the individual impact of different GRK isoforms on GPCR regulation.
RESUMEN
BACKGROUND & AIMS: Retention of bile acids in the blood is a hallmark of liver failure. Recent studies have shown that increased serum bile acid levels correlate with bacterial infection and increased mortality. However, the mechanisms by which circulating bile acids influence patient outcomes still are elusive. METHODS: Serum bile acid profiles in 33 critically ill patients with liver failure and their effects on Takeda G-protein-coupled receptor 5 (TGR5), an immunomodulatory receptor that is highly expressed in monocytes, were analyzed using tandem mass spectrometry, novel highly sensitive TGR5 bioluminescence resonance energy transfer using nanoluciferase (NanoBRET, Promega Corp, Madison, WI) technology, and in vitro assays with human monocytes. RESULTS: Twenty-two patients (67%) had serum bile acids that led to distinct TGR5 activation. These TGR5-activating serum bile acids severely compromised monocyte function. The release of proinflammatory cytokines (eg, tumor necrosis factor α or interleukin 6) in response to bacterial challenge was reduced significantly if monocytes were incubated with TGR5-activating serum bile acids from patients with liver failure. By contrast, serum bile acids from healthy volunteers did not influence cytokine release. Monocytes that did not express TGR5 were protected from the bile acid effects. TGR5-activating serum bile acids were a risk factor for a fatal outcome in patients with liver failure, independent of disease severity. CONCLUSIONS: Depending on their composition and quantity, serum bile acids in liver failure activate TGR5. TGR5 activation leads to monocyte dysfunction and correlates with mortality, independent of disease activity. This indicates an active role of TGR5 in liver failure. Therefore, TGR5 and bile acid metabolism might be promising targets for the treatment of immune dysfunction in liver failure.
Asunto(s)
Ácidos y Sales Biliares/metabolismo , Fallo Hepático/metabolismo , Monocitos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Ácidos y Sales Biliares/sangre , Femenino , Células HEK293 , Humanos , Fallo Hepático/sangre , Masculino , Persona de Mediana Edad , Receptores Acoplados a Proteínas G/genéticaRESUMEN
Arrestin-1 desensitizes the activated and phosphorylated photoreceptor rhodopsin by forming transient rhodopsin-arrestin-1 complexes that eventually decay to opsin, retinal and arrestin-1. Via a multi-dimensional screening setup, we identified and combined arrestin-1 mutants that form lasting complexes with light-activated and phosphorylated rhodopsin in harsh conditions, such as high ionic salt concentration. Two quadruple mutants, D303A + T304A + E341A + F375A and R171A + T304A + E341A + F375A share similar heterologous expression and thermo-stability levels with wild type (WT) arrestin-1, but are able to stabilize complexes with rhodopsin with more than seven times higher half-maximal inhibitory concentration (IC50) values for NaCl compared to the WT arrestin-1 protein. These quadruple mutants are also characterized by higher binding affinities to phosphorylated rhodopsin, light-activated rhodopsin and phosphorylated opsin, as compared with WT arrestin-1. Furthermore, the assessed arrestin-1 mutants are still specifically associating with phosphorylated or light-activated receptor states only, while binding to the inactive ground state of the receptor is not significantly altered. Additionally, we propose a novel functionality for R171 in stabilizing the inactive arrestin-1 conformation as well as the rhodopsin-arrestin-1 complex. The achieved stabilization of the active rhodopsin-arrestin-1 complex might be of great interest for future structure determination, antibody development studies as well as drug-screening efforts targeting G protein-coupled receptors (GPCRs).