Arrestins are key negative regulators of G Protein-Coupled Receptors (GPCRs) through mediation of G protein desensitisation and receptor internalisation. Arrestins can also contribute to signal transduction by scaffolding downstream signalling effectors for activation. GPCR kinase (GRK) enzymes phosphorylate the intracellular C-terminal domain, or intracellular loop regions of GPCRs to promote arrestin interaction. There are seven different GRK subtypes, which may uniquely phosphorylate the C-terminal tail in a type of \'phosphorylation barcode,\' potentially differentially contributing to arrestin translocation and arrestin-dependent signalling. Such contributions may be exploited to develop arrestin-biased ligands. Here, we examine the effect of different GRK subtypes on the ability to promote translocation of arrestin-2 and arrestin-3 to the cannabinoid CB1 receptor (CB1) with a range of ligands. We find that most GRK subtypes (including visual GRK1) can enhance arrestin-2 and -3 translocation to CB1, and that GRK-dependent changes in arrestin-2 and arrestin-3 translocation were broadly shared for most agonists tested. GRK2/3 generally enhanced arrestin translocation more than the other GRK subtypes, with some small differences between ligands. We also explore the interplay between G protein activity and GRK2/3-dependent arrestin translocation, highlighting that high-efficacy G protein agonists will cause GRK2/3 dependent arrestin translocation. This study supports the hypothesis that arrestin-biased ligands for CB1 must engage GRK5/6 rather than GRK2/3, and G protein-biased ligands must have inherently low efficacy.

Efficient induction of humoral immune responses depends on the orchestrated migration of B cells within lymphoid organs, which is governed by G protein-coupled receptors (GPCRs) responding to chemoattractants, represented by chemokines. After ligand binding, GPCRs are phosphorylated by different GPCR kinases (GRKs) at distinct sites on the receptor C termini, which dictates functional outcomes of β-arrestin-mediated signaling, ranging from receptor inactivation to effector molecule activation. However, the molecular mechanisms by which individual GRKs are selectively targeted to GPCRs have been poorly understood. Our recent study revealed that a protein complex consisting of copper metabolism MURR1 domain-containing (COMMD) 3 and 8 (the COMMD3/8 complex) functions as an adaptor that recruits a specific GRK to chemoattractant receptors and plays an important role in the control of B-cell migration during humoral immune responses. In this review, we summarize the current understanding of chemoattractant receptor signaling in the context of humoral immunity and discuss the potential of the COMMD3/8 complex as a therapeutic target for autoimmune diseases.

The D2 dopamine receptor (D2R) signals through both G proteins and β-arrestins to regulate important physiological processes, such as movement, reward circuitry, emotion, and cognition. β-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 β-arrestin recruitment. However, GRK-mediated receptor phosphorylation is not required for β-arrestin recruitment to the D2R, and the role of GRKs in D2R-β-arrestin interactions remains largely unexplored. In this study, we used GRK knockout cells engineered using CRISPR-Cas9 technology to determine the extent to which β-arrestin recruitment to the D2R is GRK-dependent. Genetic elimination of all GRK expression decreased, but did not eliminate, agonist-stimulated β-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 β-arrestin recruitment and receptor internalization, highlighting the importance of this GRK subfamily for D2R-β-arrestin interactions. These results were recapitulated using a phosphorylation-deficient D2R mutant, emphasizing that GRKs can enhance β-arrestin recruitment and activation independently of receptor phosphorylation.

G protein-coupled receptors (GPCRs) utilize complex cellular systems to respond to diverse ligand concentrations. By taking BLT1, a GPCR for leukotriene B4 (LTB4 ), as a model, our previous work elucidated that this system functions through the modulation of phosphorylation status on two specific residues: Thr308 and Ser310 . Ser310 phosphorylation occurs at a lower LTB4 concentration than Thr308 , leading to a shift in ligand affinity from a high-to-low state. However, the implications of BLT1 phosphorylation in signal transduction processes or the underlying mechanisms have remained unclear. Here, we identify the sequential BLT1-engaged conformations of β-arrestin and subsequent alterations in signal transduction. Stimulation of the high-affinity BLT1 with LTB4 induces phosphorylation at Ser310 via the ERK1/2-GRK pathway, resulting in a β-arrestin-bound low-affinity state. This configuration, referred to as the \"low-LTB4 -induced complex,\" necessitates the finger loop region and the phosphoinositide-binding motif of β-arrestins to interact with BLT1 and deactivates the ERK1/2 signaling. Under high LTB4 concentrations, the low-affinity BLT1 again binds to the ligand and triggers the generation of the low-LTB4 -induced complex into a different form termed \"high-LTB4 -induced complex.\" This change is propelled by The308 -phosphorylation-dependent basal phosphorylation by PKCs. Within the high-LTB4 -induced complex, β-arrestin adapts a unique configuration that involves additional N domain interaction to the low-affinity BLT1 and stimulates the PI3K/AKT pathway. We propose that the stepwise phosphorylation of BLT1 defines the formation of complex assemblies, wherein β-arrestins perform distinct functions.

Introduction: Dysfunction of the endothelium impairs its\' protective role and promotes inflammation and progression of vascular diseases. Activated Protein C (APC) elicits endothelial cytoprotective responses including barrier stabilization, anti-inflammatory and anti-apoptotic responses through the activation of the G protein-coupled receptor (GPCR) protease-activated receptor-1 (PAR1) and is a promising therapeutic. Despite recent advancements in developing new Activated protein C variants with clinical potential, the mechanism by which APC/PAR1 promotes different cytoprotective responses remains unclear and is important to understand to advance Activated protein C and new targets as future therapeutics. Here we examined the mechanisms by which APC/PAR1 attenuates cytokine-induced pro-inflammatory vascular cell adhesion molecule (VCAM-1) expression, a key mediator of endothelial inflammatory responses. Methods: Quantitative multiplexed mass spectrometry analysis of Activated protein C treated endothelial cells, endothelial cell transcriptomics database (EndoDB) online repository queries, biochemical measurements of protein expression, quantitative real-time polymerase chain reaction (RT-qPCR) measurement of mRNA transcript abundance, pharmacological inhibitors and siRNA transfections of human cultured endothelial cells. Results: Here we report that Activated Protein C modulates phosphorylation of tumor necrosis factor (TNF)-α signaling pathway components and attenuates of TNF-α induced VCAM-1 expression independent of mRNA stability. Unexpectedly, we found a critical role for the G protein-coupled receptor co-receptor sphingosine-1 phosphate receptor-1 (S1PR1) and the G protein receptor kinase-2 (GRK2) in mediating APC/PAR1 anti-inflammatory responses in endothelial cells. Discussion: This study provides new knowledge of the mechanisms by which different APC/PAR1 cytoprotective responses are mediated through discrete β-arrestin-2-driven signaling pathways modulated by specific G protein-coupled receptor co-receptors and GRKs.

Retinal rods evolved to be able to detect single photons. Despite their exquisite sensitivity, rods operate over many log units of light intensity. Several processes inside photoreceptor cells make this incredible light adaptation possible. Here, we added to our previously developed, fully space resolved biophysical model of rod phototransduction, some of the mechanisms that play significant roles in shaping the rod response under high illumination levels: the function of RGS9 in shutting off G protein transducin, and calcium dependences of the phosphorylation rates of activated rhodopsin, of the binding of cGMP to the light-regulated ion channel, and of two membrane guanylate cyclase activities. A well stirred version of this model captured the responses to bright, saturating flashes in WT and mutant mouse rods and was used to explain \"Pepperberg plots,\" that graph the time during which the response is saturated against the natural logarithm of flash strength for bright flashes. At the lower end of the range, saturation time increases linearly with the natural logarithm of flash strength. The slope of the relation (τD) is dictated by the time constant of the rate-limiting (slowest) step in the shutoff of the phototransduction cascade, which is the hydrolysis of GTP by transducin. We characterized mathematically the X-intercept ( Φ o ) which is the number of photoisomerizations that just saturates the rod response. It has been observed that for flash strengths exceeding a few thousand photoisomerizations, the curves depart from linearity. Modeling showed that the \"upward bend\" for very bright flash intensities could be explained by the dynamics of RGS9 complex and further predicted that there would be a plateau at flash strengths giving rise to more than ~107 photoisomerizations due to activation of all available PDE. The model accurately described alterations in saturation behavior of mutant murine rods resulting from transgenic perturbations of the cascade targeting membrane guanylate cyclase activity, and expression levels of GRK, RGS9, and PDE. Experimental results from rods expressing a mutant light-regulated channel purported to lack calmodulin regulation deviated from model predictions, suggesting that there were other factors at play.

Viruses rely on host-cell machinery in order to invade host cells and carry out a successful infection. G-protein coupled receptor (GPCR)-mediated signaling pathways are master regulators of cellular physiological processing and are an attractive target for viruses to rewire cells during infection. In particular, the GPCR-associated scaffolding proteins β-arrestins and GPCR signaling effectors G-protein receptor kinases (GRKs) have been identified as key cellular factors that mediate viral entry and orchestrate signaling pathways that reprogram cells for viral replication. Interestingly, a broad range of viruses have been identified to activate and/or require GPCR-mediated pathways for infection, including polyomaviruses, flaviviruses, influenza virus, and SARS-CoV-2, demonstrating that these viruses may have conserved mechanisms of host-cell invasion. Thus, GPCR-mediated pathways highlight an attractive target for the development of broad antiviral therapies.

G-protein coupled receptor kinases (GRKs) participate in the regulation of chemokine receptors by mediating receptor desensitization. They can be recruited to agonist-activated G-protein coupled receptors (GPCRs) and phosphorylate their intracellular parts, which eventually blocks signal propagation and often induces receptor internalization. However, there is growing evidence that GRKs can also control cellular functions beyond GPCR regulation. Immune cells commonly express two to four members of the GRK family (GRK2, GRK3, GRK5, GRK6) simultaneously, but we have very limited knowledge about their interplay in primary immune cells. In particular, we are missing comprehensive studies comparing the role of this GRK interplay for (a) multiple GPCRs within one leukocyte type, and (b) one specific GPCR between several immune cell subsets. To address this issue, we generated mouse models of single, combinatorial and complete GRK knockouts in four primary immune cell types (neutrophils, T cells, B cells and dendritic cells) and systematically addressed the functional consequences on GPCR-controlled cell migration and tissue localization. Our study shows that combinatorial depletions of GRKs have pleiotropic and cell-type specific effects in leukocytes, many of which could not be predicted. Neutrophils lacking all four GRK family members show increased chemotactic migration responses to a wide range of GPCR ligands, whereas combinatorial GRK depletions in other immune cell types lead to pro- and anti-migratory responses. Combined depletion of GRK2 and GRK6 in T cells and B cells shows distinct functional outcomes for (a) one GPCR type in different cell types, and (b) different GPCRs in one cell type. These GPCR-type and cell-type specific effects reflect in altered lymphocyte chemotaxis in vitro and localization in vivo. Lastly, we provide evidence that complete GRK deficiency impairs dendritic cell homeostasis, which unexpectedly results from defective dendritic cell differentiation and maturation in vitro and in vivo. Together, our findings demonstrate the complexity of GRK functions in immune cells, which go beyond GPCR desensitization in specific leukocyte types. Furthermore, they highlight the need for studying GRK functions in primary immune cells to address their specific roles in each leukocyte subset.

G protein-coupled receptor (GPCR) kinases (GRKs) and arrestins mediate GPCR desensitization, internalization, and signaling. The spatial pattern of GPCR phosphorylation is predicted to trigger these discrete GRK and arrestin-mediated functions. Here, we provide evidence that distal carboxyl-terminal tail (C-tail), but not proximal, phosphorylation of the chemokine receptor CXCR4 specifies βarrestin1 (βarr1)-dependent signaling. We demonstrate by pharmacologic inhibition of GRK2/3-mediated phosphorylation of the chemokine receptor CXCR4 coupled with site-directed mutagenesis and bioluminescence resonance energy transfer approaches that distal, not proximal, C-tail phosphorylation sites are required for recruitment of the adaptor protein STAM1 (signal-transducing adaptor molecule) to βarr1 and focal adhesion kinase phosphorylation but not extracellular signal-regulated kinase 1/2 phosphorylation. In addition, we show that GPCRs that have similarly positioned C-tail phosphoresidues are also able to recruit STAM1 to βarr1. However, although necessary for some GPCRs, we found that distal C-tail sites might not be sufficient to specify recruitment of STAM1 to βarr1 for other GPCRs. In conclusion, this study provides evidence that distal C-tail phosphorylation sites specify GRK-βarrestin-mediated signaling by CXCR4 and other GPCRs.

G protein-coupled receptor (GPCR) kinases (GRKs) and arrestins interact with agonist-bound GPCRs to promote receptor desensitization and downregulation. They also trigger signaling cascades distinct from those of heterotrimeric G proteins. Biased agonists for GPCRs that favor either heterotrimeric G protein or GRK/arrestin signaling are of profound pharmacological interest because they could usher in a new generation of drugs with greatly reduced side effects. One mechanism by which biased agonism might occur is by stabilizing receptor conformations that preferentially bind to GRKs and/or arrestins. In this review, we explore this idea by comparing structures of GPCRs bound to heterotrimeric G proteins with those of the same GPCRs in complex with arrestins and GRKs. The arrestin and GRK complexes all exhibit high conformational heterogeneity, which is likely a consequence of their unusual ability to adapt and bind to hundreds of different GPCRs. This dynamic behavior, along with the experimental tactics required to stabilize GPCR complexes for biophysical analysis, confounds these comparisons, but some possible molecular mechanisms of bias are beginning to emerge. We also examine if and how the recent structures advance our understanding of how arrestins parse the \"phosphorylation barcodes\" installed in the intracellular loops and tails of GPCRs by GRKs. In the future, structural analyses of arrestins in complex with intact receptors that have well-defined native phosphorylation barcodes, such as those installed by the two nonvisual subfamilies of GRKs, will be particularly illuminating.