Protein-coupled Receptor Internalization Research Articles

Abstract 16000: β-arrestins Mediate Cardiac Infiltration of Cytotoxic CD8+ T Cells and Macrophages in Acute CVB3 Viral Myocarditis

Introduction: In response to Coxsackievirus B3 (CVB3) infection, immune cells infiltrate the heart affecting viral clearance, tissue damage, and cardiac remodeling. However, the molecular mechanisms regulating the immune response during acute CVB3-induced myocarditis are not well understood. β arrestins (βarrs), scaffolding proteins that mediate G protein-coupled receptor internalization, desensitization, and signaling are known to regulate immune responses. Hypothesis: We hypothesized that the immune populations infiltrating the heart during acute CVB3 infection are dependent on βarrs. Methods: To test our hypothesis, 5- to 7-week-old mice lacking βarr1 (βarr1 KO; n=8), βarr2 (βarr2 KO; n=4), or C57BL/6 controls (WT; n=7) were injected intraperitoneally with 1x10 4 plaque forming units (PFU) of CVB3 or saline and immune cell populations were measured in the heart at 7 days post infection (dpi) using flow cytometry. Results: At 7 dpi, there was a marked increase in cytotoxic CD8+ T cells in hearts of WT mice that positively correlated with the level of viral infection as assessed by heart tissue PFU (slope= 1.34, Y-intercept= -3.77). In contrast, the degree of infiltration of CD8+ T cells was significantly blunted in βarr KO hearts over a broad range of heart PFU (βarr1 KO: Slope=-0.3, Y-intercept=1.65; p=0.03 vs WT; βarr2 KO: Slope=-0.32, Y-intercept= 1.3; p=0.003 vs WT). Helper CD4+ T cell populations remained unaltered at 7 dpi among all groups. Macrophages at 7 dpi were significantly reduced in βarr KO hearts over a range of PFU (WT: Slope= 18.6, Y-intercept -52.32; βarr1 KO: Slope= -5, Y-intercept=32; βarr2 KO: Slope=3.46, Y-intercept=-8.4; p=0.04 vs WT). To assess cardiac injury, we measured the level of apoptosis by TUNEL staining and found that CVB3 infected βarr KO hearts showed significantly fewer TUNEL positive cells than WT CVB3 infected controls (WT 8.7%± 2.74; βarr1 KO 1.7%±0.51; βarr2 KO 1.7%±0.37; p=0.04 vs. WT). Conclusions: These data indicate that βarrs participate in the cardiac infiltration of CD8+ T cells and macrophages in response to CVB3 infection and that blocking the action of βarrs may diminish the cardiotoxicity with the acute phase of CVB3-induced myocarditis.

Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy.

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent fusion proteins, such as green fluorescent protein (GFP), to determine their expression, localization, and function. The subcellular localization of target proteins is important for identification, characterization, and functional analyses. Internalization is one of the predominant mechanisms controlling G protein-coupled receptor (GPCR) signaling to ensure the appropriate cellular responses to stimuli. Here, we describe an experimental method to detect the subcellular localization and internalization of GPCR in HEK293 cells with confocal microscopy. In addition, this experiment provides some details about cell culture and transfection. This protocol is compatible with a variety of widely available fluorescent markers and is applicable to the visualization of the subcellular localization of a majority of proteins, as well as of the internalization of GPCR. This technique should enable researchers to efficiently manipulate GPCR gene expression in mammalian cell lines and should facilitate studies on GPCR subcellular localization and internalization.

Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy

Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy

A Broad G Protein-Coupled Receptor Internalization Assay that Combines SNAP-Tag Labeling, Diffusion-Enhanced Resonance Energy Transfer, and a Highly Emissive Terbium Cryptate.

Although G protein-coupled receptor (GPCR) internalization has long been considered as a major aspect of the desensitization process that tunes ligand responsiveness, internalization is also involved in receptor resensitization and signaling, as well as the ligand scavenging function of some atypical receptors. Internalization thus contributes to the diversity of GPCR-dependent signaling, and its dynamics and quantification in living cells has generated considerable interest. We developed a robust and sensitive assay to follow and quantify ligand-induced and constitutive-induced GPCR internalization but also receptor recycling in living cells. This assay is based on diffusion-enhanced resonance energy transfer (DERET) between cell surface GPCRs labeled with a luminescent terbium cryptate donor and a fluorescein acceptor present in the culture medium. GPCR internalization results in a quantifiable reduction of energy transfer. This method yields a high signal-to-noise ratio due to time-resolved measurements. For various GPCRs belonging to different classes, we demonstrated that constitutive and ligand-induced internalization could be monitored as a function of time and ligand concentration, thus allowing accurate quantitative determination of kinetics of receptor internalization but also half-maximal effective or inhibitory concentrations of compounds. In addition to its selectivity and sensitivity, we provided evidence that DERET-based internalization assay is particularly suitable for characterizing biased ligands. Furthermore, the determination of a Z′-factor value of 0.45 indicates the quality and suitability of DERET-based internalization assay for high-throughput screening (HTS) of compounds that may modulate GPCRs internalization.

The cytoskeletal regulatory scaffold protein GIT2 modulates mesenchymal stem cell differentiation and osteoblastogenesis

G protein-coupled receptor kinase interacting protein 2 (GIT2) is a signaling scaffold protein involved in the regulation of cytoskeletal structure, membrane trafficking, and G protein-coupled receptor internalization. Since dynamic cytoskeletal reorganization plays key roles both in osteoblast differentiation and in the maintenance of osteoclast polarity during bone resorption, we hypothesized that skeletal physiology would be altered in GIT2−/− mice. We found that adult GIT2−/− mice have decreased bone mineral density and bone volume in both the trabecular and cortical compartments. This osteopenia was associated with decreased numbers of mature osteoblasts, diminished osteoblastic activity, and increased marrow adiposity, suggesting a defect in osteoblast maturation. In vitro, mesenchymal stem cells derived from GIT2−/− mice exhibited impaired differentiation into osteoblasts and increased adipocyte differentiation, consistent with a role for GIT2 in mesenchymal stem cell fate determination. Despite elevated osteoclast inducing cytokines and osteoclast numbers, GIT2−/− mice also exhibit impaired bone resorption, consistent with a further role for GIT2 in regulating osteoclast function. Collectively, these findings underscore the importance of the cytoskeleton in both osteoblast and osteoclast function and demonstrate that GIT2 plays essential roles in skeletal metabolism, affecting both bone formation and bone resorption in vivo.

Acute depletion of plasma membrane Phosphatidylinositol 4,5-bisphosphate impairs specific steps in G protein-coupled receptor endocytosis

Receptor endocytosis plays an important role in regulating the responsiveness of cells to specific ligands. Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] has been shown to be crucial for endocytosis of some cell surface receptors, such as EGF and transferrin receptors, but its role in G-protein-coupled receptor internalization has not been investigated. By using luciferase-labeled type 1 angiotensin II (AT1R), type 2C serotonin (5HT2CR) or β(2) adrenergic (β2AR) receptors and fluorescently tagged proteins (β-arrestin-2, plasma-membrane-targeted Venus, Rab5) we were able to follow the sequence of molecular interactions along the endocytic route of the receptors in HEK293 cells using the highly sensitive method of bioluminescence resonance energy transfer and confocal microscopy. To study the role of plasma membrane PtdIns(4,5)P(2) in receptor endocytosis, we used our previously developed rapamycin-inducible heterodimerization system, in which the recruitment of a 5-phosphatase domain to the plasma membrane degrades PtdIns(4,5)P(2). Here we show that ligand-induced interaction of AT1, 5HT2C and β(2)A receptors with β-arrestin-2 was unaffected by PtdIns(4,5)P(2) depletion. However, trafficking of the receptors to Rab5-positive early endosomes was completely abolished in the absence of PtdIns(4,5)P(2). Remarkably, removal of the receptors from the plasma membrane was reduced but not eliminated after PtdIns(4,5)P(2) depletion. Under these conditions, stimulated AT1 receptors clustered along the plasma membrane, but did not enter the cells. Our data suggest that in the absence of PtdIns(4,5)P(2), these receptors move into clathrin-coated membrane structures, but these are not cleaved efficiently and hence cannot reach the early endosomal compartment.

Signalosomes: delivering cardioprotective signals from GPCRs to mitochondria

Signalosomes: delivering cardioprotective signals from GPCRs to mitochondria

The yin and yang of increased β-adrenergic signaling: β1-adrenergic genetic polymorphism and protection against acute myocardial ischemic injury

significant improvements in the treatment and prevention of cardiac diseases have led to declining morbidity and mortality over the past three decades ([2][1]), and drugs targeting adrenergic signaling have played a key role in the beneficial effects recently afforded to these patients. However,

Lipid Raft-dependent Glucagon-like Peptide-2 Receptor Trafficking Occurs Independently of Agonist-induced Desensitization

The intestinotrophic and cytoprotective actions of glucagon-like peptide-2 (GLP-2) are mediated by the GLP-2 receptor (GLP-2R), a member of the class II glucagon-secretin G protein-coupled receptor superfamily. Although native GLP-2 exhibits a short circulating half-life, long-acting degradation-resistant GLP-2 analogues are being evaluated for therapeutic use in human subjects. Accordingly, we examined the mechanisms regulating signaling, internalization, and trafficking of the GLP-2R to identify determinants of receptor activation and desensitization. Heterologous cells expressing the transfected rat or human GLP-2R exhibited a rapid, dose-dependent, and prolonged desensitization of the GLP-2-stimulated cAMP response and a sustained GLP-2-induced decrease in levels of cell surface receptor. Surprisingly, inhibitors of clathrin-dependent endocytosis failed to significantly decrease GLP-2R internalization, whereas cholesterol sequestration inhibited ligand-induced receptor internalization and potentiated homologous desensitization. The hGLP-2R localized to both Triton X-100-soluble and -insoluble (lipid raft) cellular fractions and colocalized transiently with the lipid raft marker caveolin-1. Although GLP-2R endocytosis was dependent on lipid raft integrity, the receptor transiently associated with green fluorescent protein tagged-early endosome antigen 1-positive vesicles and inhibitors of endosomal acidification attenuated the reappearance of the GLP-2R on the cell surface. Our data demonstrate that GLP-2R desensitization and raft-dependent trafficking represent distinct and independent cellular mechanisms and provide new evidence implicating the importance of a clathrin- and dynamin-independent, lipid raft-dependent pathway for homologous G protein-coupled receptor internalization.

G protein-coupled receptor internalization signaling is required for cardioprotection in ischemic preconditioning.

The present study is designed to explore the role of G protein-coupled receptors (GPCRs) in the protection afforded by ischemic preconditioning (PC). We used TG mice with cardiac-specific overexpression of a Gbetagamma-sequestering peptide, betaARKct (TG betaARKct mice), to test whether the protection of PC is Gbetagamma-dependent. To test the role of G(i) protein, we used wild-type mice pretreated with the G(i) inhibitor pertussis toxin. Recovery of left ventricular developed pressure and infarct size were measured as indices of protection. PC induced protection in wild-type mice, but this protection was blocked by pertussis toxin treatment and was also blocked in TG betaARKct mice. To determine the mechanism of Gbetagamma-induced protection in PC, we investigated one of the downstream targets of Gbetagamma, the PI3K/p70S6K pathway. PC-induced phosphorylation of p70S6K was not blocked in TG betaARKct hearts; therefore, we investigated other targets of Gbetagamma. Recent studies suggest a role for Gbetagamma in GPCR internalization. We found that betaARKct, a specific PI3K inhibitor wortmannin, and bafilomycin A1, which all block receptor recycling, all blocked the protective effect of PC. To additionally test whether PI3K is involved in PC-activated receptor internalization and endosomal signaling, we used TG mice with cardiac-specific overexpression of a catalytically inactive mutant PI3Kgamma, which disrupts the recruitment of functional PI3K to agonist-activated GPCRs in vivo. We found that the catalytically inactive mutant of PI3Kgamma blocks the protection of PC. In summary, these data suggest the novel finding that the cardioprotective effect of PC requires receptor internalization.

Substrate-induced regulation of gamma-aminobutyric acid transporter trafficking requires tyrosine phosphorylation.

Neurotransmitter transporters regulate synaptic transmitter levels and are themselves functionally regulated by a number of different signal transduction cascades. A common theme in transporter regulation is redistribution of transporter protein between intracellular stores and the plasma membrane. The triggers and mechanisms underlying this regulation are important in the control of extracellular transmitter concentrations and hence synaptic signaling. Previously, we demonstrated that the gamma-aminobutyric acid transporter GAT1 is regulated by direct tyrosine phosphorylation, resulting in an up-regulation of transporter expression on the plasma membrane. In the present report, we show that two tyrosine residues on GAT1 contribute to the phosphorylation and transporter redistribution. Tyrosine phosphorylation is concomitant with a decrease in the rate of transporter internalization from the plasma membrane. A decrease in GAT internalization rates also occurs in the presence of GAT1 substrates, suggesting the hypothesis that tyrosine phosphorylation is required for the substrate-induced up-regulation of GAT1 surface expression. In support of this hypothesis, incubation of GAT1-expressing cells with transporter ligands alters the amount of GAT1 tyrosine phosphorylation, and substrate-induced surface expression is unchanged in a GAT1 mutant lacking tyrosine phosphorylation sites. These data suggest a model in which substrates permit the phosphorylation of GAT1 on tyrosine residues and that the phosphorylated state of the transporter is refractory for internalization.

Multiple Pathways of Dynamin-regulated G protein-coupled Receptor Internalization

Multiple Pathways of Dynamin-regulated G protein-coupled Receptor Internalization

Differential roles of the NPXXY motif in formyl peptide receptor signaling.

The NPXXY motif (X represents any amino acid) in the seventh transmembrane domain of the chemotactic formyl peptide receptor (FPR) is highly conserved among G protein-coupled receptors. Recent work suggested that this motif contributes to G protein-coupled receptor internalization and signal transduction; however, its role in FPR signaling remains unclear. In this study we replaced Asn(297) and Tyr(301) in the NPXXY motif of the human FPR with Ala (N297A) and Ala/Phe (Y301A/Y301F), respectively, and determined the effects of the substitutions on FPR functions in transfected rat basophilic leukemia cells. Whereas all the mutant receptors were expressed on the cell surface, the N297A receptor exhibited reduced binding affinity and was unable to mediate activation of phospholipase C-beta and the p42/44 mitogen-activated protein kinase (MAP kinase). The Y301F receptor displayed significantly decreased ligand-stimulated internalization and MAP kinase activation, suggesting that the hydrogen bonding at Tyr(301) is critical for these functions. The Y301F receptor showed a chemotactic response similar to that of wild-type FPR, indicating that cell chemotaxis does not require receptor internalization and hydrogen bonding at the Tyr(301) position. In contrast, the Y301A receptor displayed a left-shifted, but overall reduced, chemotaxis response that peaked at 0.1-1 nM. Finally, using a specific MAP kinase kinase inhibitor, we found that activation of MAP kinase is required for efficient FPR internalization, but is not essential for chemotaxis. These findings demonstrate that residues within the NPXXY motif differentially regulate the functions of FPR.

Stimulation of Mitogen-activated Protein Kinase by G Protein-coupled α2-Adrenergic Receptors Does Not Require Agonist-elicited Endocytosis

Agonist-elicited receptor sequestration is strikingly different for the alpha(2A)- versus alpha(2B)-adrenergic receptor (alpha(2)-AR) subtypes; the alpha(2B)-AR undergoes rapid and extensive disappearance from the HEK 293 cell surface, whereas the alpha(2A)-AR does not (Daunt, D. A., Hurt, C., Hein, L., Kallio, J., Feng, F., and Kobilka, B. K. (1997) Mol. Pharmacol. 51, 711-720; Eason, M. G., and Liggett, S. B. (1992) J. Biol. Chem. 267, 25473-25479). Since recent reports suggest that endocytosis is required for some G protein-coupled receptors to stimulate the mitogen-activated protein (MAP) kinase cascade (Daaka, Y., Luttrell, L. M., Ahn, S., Della Rocca, G. J., Ferguson, S. S., Caron, M. G., and Lefkowitz, R. J. (1998) J. Biol. Chem. 273, 685-688; Luttrell, L. M., Daaka, Y., Della Rocca, G. J., and Lefkowitz, R. J. (1997) J. Biol. Chem. 272, 31648-31656; Ignatova, E. G., Belcheva, M. M., Bohn, L. M., Neuman, M. C., and Coscia, C. J. (1999) J. Neurosci. 19, 56-63), we evaluated the differential ability of these two subtypes to activate MAP kinase. We observed no correlation between subtype-dependent agonist-elicited receptor redistribution and receptor activation of the MAP kinase cascade. Furthermore, incubation of cells with K(+)-depleted medium eliminated alpha(2B)-AR internalization but did not eliminate MAP kinase activation, suggesting that receptor internalization is not a general prerequisite for activation of the MAP kinase cascade via G(i)-coupled receptors. We also noted that neither dominant negative dynamin (K44A) nor concanavalin A treatment dramatically altered MAP kinase activation or receptor redistribution, indicating that these experimental tools do not universally block G protein-coupled receptor internalization.

Identification of NSF as a β-Arrestin1-binding Protein: IMPLICATIONS FOR β2-ADRENERGIC RECEPTOR REGULATION

Previous studies have demonstrated that beta-arrestin1 serves to target G protein-coupled receptors for internalization via clathrin-coated pits and that its endocytic function is regulated by dephosphorylation at the plasma membrane. Using the yeast two-hybrid system, we have identified a novel beta-arrestin1-binding protein, NSF (N-ethylmaleimide-sensitive fusion protein), an ATPase essential for many intracellular transport reactions. We demonstrate that purified recombinant beta-arrestin1 and NSF interact in vitro and that these proteins can be coimmunoprecipitated from cells. beta-Arrestin1-NSF complex formation exhibits a conformational dependence with beta-arrestin1 preferentially interacting with the ATP bound form of NSF. In contrast to the beta-arrestin1-clathrin interaction, however, the phosphorylation state of beta-arrestin1 does not affect NSF binding. Functionally, overexpression of NSF in HEK 293 cells significantly enhances agonist-mediated beta2-adrenergic receptor (beta2-AR) internalization. Furthermore, when coexpressed with a beta-arrestin1 mutant (betaarr1S412D) that mimics a constitutively phosphorylated form of beta-arrestin1 and that acts as a dominant negative with regards to beta2-AR internalization, NSF rescues the betaarr1S412D-mediated inhibition of beta2-AR internalization. The demonstration of beta-arrestin1-NSF complex formation and the functional consequences of NSF overexpression suggest a hitherto unappreciated role for NSF in facilitating clathrin coat-mediated G protein-coupled receptor internalization.

Role of beta-arrestin in mediating agonist-promoted G protein-coupled receptor internalization.

beta-Arrestins are proteins that bind phosphorylated heterotrimeric GTP-binding protein (G protein)-coupled receptors (GPCRs) and contribute to the desensitization of GPCRs by uncoupling the signal transduction process. Resensitization of GPCR responsiveness involves agonist-mediated receptor sequestration. Overexpression of beta-arrestins in human embryonic kidney cells rescued the sequestration of beta 2-adrenergic receptor (beta 2AR) mutants defective in their ability to sequester, an effect enhanced by simultaneous overexpression of beta-adrenergic receptor kinase 1. Wild-type beta 2AR sequestration was inhibited by the overexpression of two beta-arrestin mutants. These findings suggest that beta-arrestins play an integral role in GPCR internalization and thus serve a dual role in the regulation of GPCR function.