Role and modulation of G protein-coupled receptor signaling in inflammatory processes

Many extracellular stimuli, such as neurotransmitters, hormones, chemokines, proteinases, inflammatory mediators, odorants, and light, are recognized by the superfamily of G protein-coupled receptors (GPCRs). Immune cells express GPCRs for classical chemoattractants, chemokines, neuropeptides, and neurotransmitters. GPCRs transmit information by interacting with heterotrimeric G proteins, resulting in rapid and transient signaling. The signal given by GPCRs is terminated rapidly by the activity of regulators of G protein signaling (RGS). In addition, GPCR responsiveness diminishes after repeated or prolonged exposure to the agonist. This process of homologous desensitization of GPCRs is dependent on receptor phosphorylation by G protein-coupled receptor kinases (GRKs). In this review, we describe the role of RGS and GRKs in the regulation of GPCR signaling in the immune system, with special emphasis on the role of changes in GRKs and RGS expression during (auto) immune processes. Since altered regulation of GPCR signaling can influence disease states, the molecules involved in this process can also represent attractive therapeutic targets.     

Suppression of immunoglobulin E-mediated allergic responses by regulator of G protein signaling 13

Mast cells elicit allergic responses through degranulation and release of proinflammatory mediators after antigen crosslinking of the immunoglobulin E receptor FcepsilonRI. Proteins of the 'regulator of G protein signaling' (RGS) family negatively control signaling mediated by G protein-coupled receptors through GTPase-accelerating protein activity. Here we show that RGS13 inhibited allergic responses by physically interacting with the regulatory p85alpha subunit of phosphatidylinositol-3-OH kinase in mast cells and disrupting its association with an FcepsilonRI-activated scaffolding complex. Rgs13-/- mice had enhanced immunoglobulin E-mediated mast cell degranulation and anaphylaxis. Thus, RGS13 inhibits the assembly of immune receptor-induced signalosomes in mast cells. Abnormal RGS13 expression or function may contribute to disorders of amplified mast cell activity, such as idiopathic anaphylaxis.     

Lsc is required for marginal zone B cells, regulation of lymphocyte motility and immune responses

Lsc (the murine homolog of human p115 Rho GEF) is a member of the Dbl-homology family of GTP exchange factors and is a specific activator of Rho. Lsc is activated by the G alpha(13) subunit of heterotrimeric G proteins and contains a regulator of G protein signaling domain that downmodulates G alpha(12) and G alpha(13). Lsc is expressed primarily in the hematopoietic system and links the activation of G alpha(12) and G alpha(13)-coupled receptors to actin polymerization in B and T cells. Lsc is essential for marginal zone B (MZB) cell homeostasis and for the generation of immune responses. Although Lsc-deficient lymphocytes show reduced basal motility, MZB cells show enhanced migration after serum activation. Thus, Lsc is a critical regulator of MZB cells and immune functions.     

Evidence for association of GABAB receptors with Kir3 channels and regulators of G protein signalling (RGS4) proteins

Many neurotransmitters and hormones signal by stimulating G protein-coupled neurotransmitter receptors (GPCRs), which activate G proteins and their downstream effectors. Whether these signalling proteins diffuse freely within the plasma membrane is not well understood. Recent studies have suggested that direct protein–protein interactions exist between GPCRs, G proteins and G protein-gated inwardly rectifying potassium (GIRK or Kir3) channels. Here, we used fluorescence resonance energy transfer (FRET) combined with total internal reflection fluorescence microscopy to investigate whether proteins within this signalling pathway move within 100 Å of each other in the plasma membrane of living cells. GABA_B R1 and R2 receptors, Kir3 channels, Gαo subunits and regulators of G protein signalling (RGS4) proteins were each fused to cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP) and first assessed for functional expression in HEK293 cells. The presence of the fluorophore did not significantly alter the signalling properties of these proteins. Possible FRET was then investigated for different protein pair combinations. As a positive control, FRET was measured between tagged GABA_B R1 and R2 subunits (∼12% FRET), which are known to form heterodimers. We measured significant FRET between tagged RGS4 and GABA_B R1 or R2 subunits (∼13% FRET), and between Gαo and GABA_B R1 or R2 subunits (∼10% FRET). Surprisingly, FRET also occurred between tagged Kir3.2a/Kir3.4 channels and GABA_B R1 or R2 subunits (∼10% FRET). FRET was not detected between Kir3.2a and RGS4 nor between Kir3.2a and Gαo. These data are discussed in terms of a model in which GABA_B receptors, G proteins, RGS4 proteins and Kir3 channels are closely associated in a signalling complex.     

Expression of regulator of G protein signalling proteins in natural killer cells, and their modulation by Ly49A and Ly49D

The small GTPase accelerators regulator of G protein signalling (RGS) proteins are important regulators of proximal signalling from G protein coupled receptors. Although natural killer (NK) cells express a number of G-protein coupled receptors, expression of RGS proteins has not been investigated. We analysed the expression of RGS proteins in rat NK cells, and detected mRNA for RGS1, RGS2, RGS5, RGS8, RGS16, and RGS18. Interestingly, when we included a panel of different leucocyte subsets, we found that RGS8 was selectively expressed by NK cells. NK cells are under control of both activating and inhibitory receptors and, utilizing a xenogeneic system where the mouse activating Ly49D or inhibitory Ly49A receptors were transfected into the rat RNK-16 cell line, the potential regulation of RGS proteins by single NK cell receptors was studied. We found that ligation of Ly49D led to a rapid and transient increase in message for RGS2, while Ly49A ligation up-regulated RGS2, RGS16, and RGS18 mRNA. Both receptors also induced a prolonged increase in RGS2 endogenous protein levels. These findings suggest that RGS proteins may be influenced by or involved in NK cell receptor events, suggesting a crosstalk between G-protein coupled receptors and NK cell receptors.     

Postnatal induction and localization of R7BP, a membrane-anchoring protein for regulator of G protein signaling 7 family-Gbeta5 complexes in brain

Members of the regulator of G protein signaling 7 (RGS7) (R7) family and Gbeta5 form obligate heterodimers that are expressed predominantly in the nervous system. R7-Gbeta5 heterodimers are GTPase-activating proteins (GAPs) specific for Gi/o-class Galpha subunits, which mediate phototransduction in retina and the action of many modulatory G protein-coupled receptors (GPCRs) in brain. Here we have focused on the R7-family binding protein (R7BP), a recently identified palmitoylated protein that can bind R7-Gbeta5 complexes and is hypothesized to control the intracellular localization and function of the resultant heterotrimeric complexes. We show that: 1) R7-Gbeta5 complexes are obligate binding partners for R7BP in brain because they co-immunoprecipitate and exhibit similar expression patterns. Furthermore, R7BP and R7 protein accumulation in vivo requires Gbeta5. 2) Expression of R7BP in Neuro2A cells at levels approximating those in brain recruits endogenous RGS7-Gbeta5 complexes to the plasma membrane. 3) R7BP immunoreactivity in brain concentrates in neuronal soma, dendrites, spines or unmyelinated axons, and is absent or low in glia, myelinated axons, or axon terminals. 4) RGS7-Gbeta5-R7BP complexes in brain extracts associate inefficiently with detergent-resistant lipid raft fractions with or without G protein activation. 5) R7BP and Gbeta5 protein levels are upregulated strikingly during the first 2-3 weeks of postnatal brain development. Accordingly, we suggest that R7-Gbeta5-R7BP complexes in the mouse or rat could regulate signaling by modulatory Gi/o-coupled GPCRs in the developing and adult nervous systems.     

RGS16 Restricts the Pro‐Inflammatory Response of Monocytes

Immune cells express powerful and harmful effectors that require tight regulation. Heterotrimeric G proteins are critical mediators in translating extracellular signals into cell responses, which need a fine‐tuned regulation for the control of cell activation. Regulator of G‐protein signalling 16 (RGS16) has been identified as a key factor of G protein‐mediated activation in lymphocytes, modulating inflammatory and survival responses of various cell types. However, data about the expression of this regulatory protein in monocytes are scarce, and it has remained unclear whether activation and migration of these cells are regulated by RGS16. In this study, the impact of RGS16 on the production of inflammatory cytokines by activated human monocytes was investigated in vitro using the human promonocytic cell line THP‐1 as a model. Gain and loss of function experiments showed that RGS16 overexpression reduces the expression of pro‐inflammatory cytokines IL‐1β, IL‐6, IL‐8 and TNFα, while RGS16 knockdown by RNAi upregulates IL‐1β, IL‐6 and TNFα but not IL‐8. RGS16 knockdown was also shown to enhance Pam3‐mediated induction of the anti‐inflammatory cytokine IL‐10. Our results indicate that RGS16 restricts the activation‐induced pro‐inflammatory profile in myeloid cells.     

RGS1 and RGS13 mRNA silencing in a human B lymphoma line enhances responsiveness to chemoattractants and impairs desensitization

Chemokines bind receptors that are members of the G-protein-coupled receptor family. Chemokine receptors transduce intracellular signals by activating heterotrimeric G-proteins. Acting to limit and modulate heterotrimeric G-protein signaling is a family of proteins, termed regulator of G-protein signaling (RGS). Two of these proteins, RGS1 and RGS13, are well-expressed in germinal center B cells and many Burkitt's lymphoma cell lines. Reducing RGS13 and to a lesser extent RGS1 expression in a Burkitt's lymphoma cell line enhances responsiveness to two chemokines, CXC chemokine ligand 12 (CXCL12) and CXCL13, and reducing both mRNAs augments the responses more dramatically. The double knock-down (KD) cells respond better to restimulation with CXCL12 or CXCL13 after a primary stimulation with CXCL12 than do the control cells. The double-KD cells also exhibit a greater propensity to polarize and to develop multiple small lamellipodia. These results indicate that RGS1 and RGS13 act together to regulate chemokine receptor signaling in human germinal center B lymphocytes and provide evidence that they contribute significantly to the rapid desensitization of the signaling pathway.     

Mammalian G proteins and their cell type specific functions

Heterotrimeric G proteins are key players in transmembrane signaling by coupling a huge variety of receptors to channel proteins, enzymes, and other effector molecules. Multiple subforms of G proteins together with receptors, effectors, and various regulatory proteins represent the components of a highly versatile signal transduction system. G protein-mediated signaling is employed by virtually all cells in the mammalian organism and is centrally involved in diverse physiological functions such as perception of sensory information, modulation of synaptic transmission, hormone release and actions, regulation of cell contraction and migration, or cell growth and differentiation. In this review, some of the functions of heterotrimeric G proteins in defined cells and tissues are described.     

Distribution and phenotype of rotavirus-specific B cells induced during the antigen-driven primary response to 2/6 virus-like particles administered by the intrarectal and the intranasal routes

Selection of mucosal sites is an important step in mucosal vaccine development. The intrarectal (IR) route represents an alternative to the oral route of immunization; nevertheless, immune responses induced by this route are not well defined. Here, we studied the early primary B cell response (induction, homing, and phenotype) induced by IR immunization with rotavirus (RV)-2/6 virus-like particles (VLP). Using flow cytometry, we traced RV-specific B cells in different lymphoid tissues and analyzed the expression of alpha4beta7 and CCR9, which are important receptors for homing to the gut, as well as CD5, a marker expressed by B1-a cells, which are a major source of natural antibodies. We observed a massive, specific B cell response in rectal follicles, lumbar, and mesenteric lymph nodes but not in Peyer's patches or cervical lymph nodes. A minority of cells expressed alpha4beta7, suggesting a probable lack of migration to the gut, whereas CCR9 and CD5 were expressed by 30-50% and 30-75% of specific B cells, respectively. Then, we compared the intranasal route of immunization and observed similar B cell frequency and phenotype but in respiratory lymphoid tissues. These results confirm the high compartmentalization of B cell responses within the mucosal system. They show that CCR9 expression, conversely to alpha4beta7, is not restricted to B cells induced in the gut. Finally, an important part of the RV-specific B cell response induced at the mucosal level during the primary response to VLP is most likely a result of B1-a cells.     

Expression of regulator of G protein signalling protein 5 (RGS5) in the tumour vasculature of human renal cell carcinoma

The gene expression profiles of tumour and normal vasculature are distinctively different. The altered expression of various angiogenesis-related genes in tumour-derived endothelial cells has been investigated intensively, but there may be as yet unidentified molecules that regulate tumour angiogenesis. In the present study, the distinctive expression of regulator of G protein signalling protein 5 (RGS5) in tumour vessels in human renal cell carcinoma (RCC) has been clarified. RGS5 is a member of the RGS superfamily and acts as a negative regulator of heterotrimeric G protein-mediated signalling through G protein-coupled receptors (GPCRs). RT-PCR showed strong expression of RGS5 in all RCCs examined, but expression was very weak or undetectable in normal kidneys. By real-time RT-PCR, the ratio of RGS5 mRNA in RCC to that in normal kidney was 6.6 : 1 (p = 0.0012). In situ hybridization showed strong expression of RGS5 in vessels within tumour cell nests. It was expressed neither in tumour cells nor in normal renal capillaries. Immunohistochemical staining using serial sections for endothelial cell markers (CD31 and CD34) and smooth muscle cell markers (alpha-SMA and desmin), as well as fluorescence double staining, strongly suggested that tumour endothelial cells were the main location of RGS5 in RCC. These findings suggest that RGS5 may be involved in G protein-mediated signalling in tumour vessels in human RCC.     

G protein coupled pattern recognition receptors expressed in neutrophils: Recognition,activation/modulation,signaling and receptor regulated functions

Neutrophils, the most abundant white blood cell in human blood, express receptors that recognize damage/microbial associated pattern molecules of importance for cell recruitment to sites of inflammation. Many of these receptors belong to the family of G protein coupled receptors (GPCRs). These receptor-proteins span the plasma membrane in expressing cells seven times and the down-stream signaling rely in most cases on an activation of heterotrimeric G proteins. The GPCRs expressed in neutrophils recognize a number of structurally diverse ligands (activating agonists, allosteric modulators, and inhibiting antagonists) and share significant sequence homologies. Studies of receptor structure and function have during the last 40 years generated important information on GPCR biology in general; this knowledge aids in the overall understanding of general pharmacological principles, governing regulation of neutrophil function and inflammatory processes, including novel leukocyte receptor activities related to ligand recognition, biased/functional selective signaling, allosteric modulation, desensitization, and reactivation mechanisms as well as communication (receptor transactivation/cross-talk) between GPCRs. This review summarizes the recent discoveries and pharmacological hallmarks with focus on some of the neutrophil expressed pattern recognition GPCRs. In addition, unmet challenges, including recognition by the receptors of diverse ligands and how biased signaling mediate different biological effects are described/discussed.     

Chemokines in the systemic organization of immunity

Summary: Directed cellular migrations underlie immune system organization. Chemokines and their receptors (along with surface‐adhesion molecules) are central to these migrations, targeting developing and mature leukocytes to tissues and microenvironments suitable for their differentiation and function. The chemokine CXCL12 and its receptor CXCR4 play a central role in the migration of hematopoietic stem cells, and several chemokine receptors are transiently expressed during distinct stages of B‐ and T‐cell development. In the periphery, mature naïve B and T cells utilize the receptors CCR7, CXCR4, and CXCR5 to recirculate through specialized microenvironments within the secondary lymphoid tissues, while effector and memory lymphocytes express bewildering patterns of adhesion molecules and chemokine receptors that allow them to function within microenvironments and non‐lymphoid tissues inaccessible to naïve cells. Here, we summarize the role of chemokines and their receptors in the spatial organization of the immune system and consider the implications for immune function.     

G protein coupled receptors signaling pathways implicate in inflammatory and immune response of rheumatoid arthritis

Introduction

G protein-coupled receptors (GPCRs) are transmembrane receptor proteins, which allow the transfer of signals across the membrane. Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovitis and accompanied with inflammatory and abnormal immune response. GPCRs signaling pathways play a significant role in inflammatory and immune response processes including RA.

Findings

In this review, we have focused on the advances in GPCRs signaling pathway implicating the inflammatory and immune response of RA. The signaling pathways of GPCRs–adenylyl cyclase (AC)–cyclic adenosine 3′, 5′-monophosphate (cAMP) include β₂ adrenergic receptors (β₂-ARs)–AC–cAMP signaling pathways, E-prostanoid2/4 (EP2/4)–AC–cAMP signaling pathways and so on. Regulatory proteins, such as G protein-coupled receptor kinases (GRKs) and β-arrestins, play important modulatory roles in GPCRs signaling pathway. GPCRs signaling pathway and regulatory proteins implicate the pathogenesis process of inflammatory and immune response.

Conclusion

GPCRs–AC–cAMP signal pathways involve in the inflammatory and immune response of RA. Different signaling pathways are mediated by different receptors, such as β₂-AR, PGE2 receptor, chemokines receptor, and adenosine receptor. GRKs and β-arrestins are crucial proteins in the regulation of GPCRs signaling pathways. The potential therapeutic targets as well as strategies to modulate GPCRs signaling pathway are new development trends.

     

Translating Inhibitory Fc Receptor Biology into Novel Therapeutic Approaches

Innate and adaptive immune responses represent well balanced reactions aimed at resolving microbial infections without causing major collateral damage to the host. Disturbances in this system either due to enhanced activating or decreased inhibitory signaling pathways may lead to excessive immune activation resulting in tissue damage, the induction of autoimmune disease and/or chronic inflammation. On the molecular level this balance is achieved by the integration of inhibitory and activating signals, which are delivered by pairs of activating and inhibitory cell surface receptors expressed on innate and adaptive immune cells. The regulation of immunoglobulin G activity through cellular Fc receptors is a prime example for this type of regulation. This is not only relevant for the regulation of antibody-mediated effector functions through innate immune effector cells but also for the regulation of B cell activation and antibody production itself.     

Heterotrimeric G protein signaling: role in asthma and allergic inflammation

Asthma and rhinitis are pathophysiologic conditions associated with a prototypical allergic response to inhaled allergens consisting of both neuromechanical and inflammatory components. Heptahelical receptors that bind guanosine triphosphate-binding proteins (G proteins), referred to as G protein-coupled receptors (GPCRs), have been intimately linked with asthma and allergic inflammation for many years. G protein signaling mediates responses throughout the immune, nervous, and muscular systems that might contribute to the pathogenesis of allergic processes and asthma. For example, GPCR agonists or antagonists are used as therapies for asthma either by promoting airway smooth muscle relaxation (beta2 adrenergic receptor agonists) or by inhibiting inflammation in the nasal mucosa and airways (cysteinyl leukotriene receptor antagonists). The focus of this review is to explore how downstream signaling cascades elicited by GPCR activation contribute to the allergic phenotype and the mechanism by which pharmaceuticals alter signaling to generate a therapeutic effect. We also discuss physiologic modulators of G protein signaling, such as regulator of G protein signaling proteins and G protein receptor kinases, inasmuch as they represent potential new therapeutic targets in the treatment of atopy and other inflammatory conditions.     

G‐protein coupled receptors and ligands that organize humoral immune responses

B‐cell responses are dynamic processes that depend on multiple types of interactions. Rare antigen‐specific B cells must encounter antigen and specialized systems are needed—unique to each lymphoid tissue type—to ensure this happens efficiently. Lymphoid tissue barrier cells act to ensure that pathogens, while being permitted entry for B‐cell recognition, are blocked from replication or dissemination. T follicular helper (Tfh) cells often need to be primed by dendritic cells before supporting B‐cell responses. For most responses, antigen‐specific helper T cells and B cells need to interact, first to initiate clonal expansion and the plasmablast response, and later to support the germinal center (GC) response. Newly formed plasma cells need to travel to supportive niches. GC B cells must become confined to the follicle center, organize into dark and light zones, and interact with Tfh cells. Memory B cells need to be positioned for rapid responses following reinfection. Each of these events requires the actions of multiple G‐protein coupled receptors (GPCRs) and their ligands, including chemokines and lipid mediators. This review will focus on the guidance cue code underlying B‐cell immunity, with an emphasis on findings from our laboratory and on newer advances in related areas. We will discuss our recent identification of geranylgeranyl‐glutathione as a ligand for P2RY8. Our goal is to provide the reader with a focused knowledge about the GPCRs guiding B‐cell responses and how they might be therapeutic targets, while also providing examples of how multiple types of GPCRs can cooperate or act iteratively to control cell behavior.     

Lymphoid stroma in the initiation and control of immune responses

Summary: The lymphoid tissues are characterized by a complex organized architecture that is supported by a network of stromal cells. These stromal cells play many important roles in addition to serving as structural support. Lymphoid stromal cells, including the specialized fibroblastic reticular cells and follicular dendritic cells, express chemokines, cytokines, adhesion molecules, as well as other factors required for the migration, homeostasis, and survival of immune cells. Studies have demonstrated the dynamic role that lymphoid stromal cells and the chemokines they produce play in directing lymphocyte migration within the spleen and lymph nodes. The stromal network may also play roles in influencing antigen presentation, via adherence of antigen-presenting cells to the network, and cell survival, via provision of survival factors such as interleukin-7. Recently, we have shown that dramatic changes in lymphoid chemokine expression occur during many immune responses. This can alter the trafficking and localization of immune cells in lymphoid organs and has many implications for the regulation and shut-down of immune responses. Here we briefly summarize the roles of stromal cells and lymphoid architecture in the control of immune responses.