scholarly journals Substance P Serves as a Balanced Agonist for MRGPRX2 and a Single Tyrosine Residue Is Required for β-arrestin Recruitment and Receptor Internalization

2021 ◽  
Vol 22 (10) ◽  
pp. 5318
Author(s):  
Chalatip Chompunud Na Ayudhya ◽  
Aetas Amponnawarat ◽  
Hydar Ali
Keyword(s):  
Substance P ◽  
G Protein ◽  
G Proteins ◽  
Receptor Internalization ◽  
The Novel ◽  
Tyrosine Residue ◽  
Wild Type ◽  
Human Ortholog ◽  
G Protein Coupled ◽  
Type Receptor

The neuropeptide substance P (SP) mediates neurogenic inflammation and pain and contributes to atopic dermatitis in mice through the activation of mast cells (MCs) via Mas-related G protein-coupled receptor (GPCR)-B2 (MrgprB2, human ortholog MRGPRX2). In addition to G proteins, certain MRGPRX2 agonists activate an additional signaling pathway that involves the recruitment of β-arrestins, which contributes to receptor internalization and desensitization (balanced agonists). We found that SP caused β-arrestin recruitment, MRGPRX2 internalization, and desensitization. These responses were independent of G proteins, indicating that SP serves as a balanced agonist for MRGPRX2. A tyrosine residue in the highly conserved NPxxY motif contributes to the activation and internalization of many GPCRs. We have previously shown that Tyr279 of MRGPRX2 is essential for G protein-mediated signaling and degranulation. To assess its role in β-arrestin-mediated MRGPRX2 regulation, we replaced Tyr279 in the NPxxY motif of MRGPRX2 with Ala (Y279A). Surprisingly, we found that, unlike the wild-type receptor, Y279A mutant of MRGPRX2 was resistant to SP-induced β-arrestin recruitment and internalization. This study reveals the novel findings that activation of MRGPRX2 by SP is regulated by β-arrestins and that a highly conserved tyrosine residue within MRGPRX2’s NPxxY motif contributes to both G protein- and β-arrestin-mediated responses.

scholarly journals Ubiquitination differentially regulates clathrin-dependent internalization of protease-activated receptor-1

2007 ◽  
Vol 177 (5) ◽  
pp. 905-916 ◽  
Author(s):  
Breann L. Wolfe ◽  
Adriano Marchese ◽  
JoAnn Trejo
Keyword(s):  
G Protein ◽  
Protein Complex ◽  
Adaptor Protein ◽  
Receptor Internalization ◽  
Wild Type ◽  
Endocytic Machinery ◽  
Protease Activated Receptor 1 ◽  
Clathrin Adaptor ◽  
G Protein Coupled ◽  
Type Receptor

Protease-activated receptor-1 (PAR1), a G protein–coupled receptor (GPCR) for thrombin, is irreversibly activated by proteolysis. Consequently, PAR1 trafficking is critical for the fidelity of thrombin signaling. PAR1 displays constitutive and agonist-induced internalization, which are clathrin and dynamin dependent but are independent of arrestins. The clathrin adaptor AP2 (adaptor protein complex-2) is critical for constitutive but not for activated PAR1 internalization. In this study, we show that ubiquitination negatively regulates PAR1 constitutive internalization and specifies a distinct clathrin adaptor requirement for activated receptor internalization. PAR1 is basally ubiquitinated and deubiquitinated after activation. A PAR1 lysineless mutant signaled normally but was not ubiquitinated. Constitutive internalization of ubiquitin (Ub)-deficient PAR1 was markedly increased and inhibited by the fusion of Ub to the cytoplasmic tail. Ub-deficient PAR1 constitutive internalization was AP2 dependent like the wild-type receptor. However, unlike wild-type PAR1, AP2 was required for the internalization of activated Ub-deficient receptor, suggesting that the internalization of ubiquitinated PAR1 requires different endocytic machinery. These studies reveal a novel function for ubiquitination in the regulation of GPCR internalization.

scholarly journals Nuclear GPCRs in cardiomyocytes: an insider's view of β-adrenergic receptor signaling

2011 ◽  
Vol 301 (5) ◽  
pp. H1754-H1764 ◽  
Author(s):  
George Vaniotis ◽  
Bruce G. Allen ◽  
Terence E. Hébert
Keyword(s):  
Subcellular Localization ◽  
G Protein ◽  
G Proteins ◽  
Adrenergic Receptor ◽  
Physiological State ◽  
Signaling Cascades ◽  
Special Focus ◽  
The Novel ◽  
Receptor Signaling ◽  
G Protein Coupled

In recent years, we have come to appreciate the complexity of G protein-coupled receptor signaling in general and β-adrenergic receptor (β-AR) signaling in particular. Starting originally from three β-AR subtypes expressed in cardiomyocytes with relatively simple, linear signaling cascades, it is now clear that there are large receptor-based networks which provide a rich and diverse set of responses depending on their complement of signaling partners and the physiological state. More recently, it has become clear that subcellular localization of these signaling complexes also enriches the diversity of phenotypic outcomes. Here, we review our understanding of the signaling repertoire controlled by nuclear β-AR subtypes as well our understanding of the novel roles for G proteins themselves in the nucleus, with a special focus, where possible, on their effects in cardiomyocytes. Finally, we discuss the potential pathological implications of alterations in nuclear β-AR signaling.

scholarly journals Selective Modulation of Wild Type Receptor Functions by Mutants of G-Protein-coupled Receptors

1999 ◽  
Vol 274 (18) ◽  
pp. 12548-12554 ◽  
Author(s):  
Christian Le Gouill ◽  
Jean-Luc Parent ◽  
Carolyn-Ann Caron ◽  
Rémi Gaudreau ◽  
Léonid Volkov ◽  
...  
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptors ◽  
Wild Type ◽  
Selective Modulation ◽  
G Protein Coupled ◽  
Type Receptor

scholarly journals Inhibition of Gonadotropin-Releasing Hormone Receptor Signaling by Expression of a Splice Variant of the Human Receptor

1997 ◽  
Vol 11 (9) ◽  
pp. 1305-1318 ◽  
Author(s):  
Robert Grosse ◽  
Torsten Schöneberg ◽  
Günter Schultz ◽  
Thomas Gudermann
Keyword(s):  
Ligand Binding ◽  
G Protein ◽  
Splice Variant ◽  
Gnrh Receptor ◽  
Cell Surface Receptor ◽  
Enzyme Linked Immunosorbent Assay ◽  
Wild Type ◽  
Exon 2 ◽  
G Protein Coupled ◽  
Type Receptor

Abstract GnRH binds to a specific G protein-coupled receptor in the pituitary to regulate synthesis and secretion of gonadotropins. Using RT-PCR and human pituitary poly(A)+ RNA as a template, the full-length GnRH receptor (wild type) and a second truncated cDNA characterized by a 128-bp deletion between nucleotide positions 522 and 651 were cloned. The deletion causes a frame shift in the open reading frame, thus generating new coding sequence for further 75 amino acids. The truncated cDNA arises from alternative splicing by accepting a cryptic splicing acceptor site in exon 2. Distinct translation products of approximately 45–50 and 42 kDa were immunoprecipitated from COS-7 cells transfected with cDNA coding for wild type GnRH receptor and the truncated splice variant, respectively. Immunocytochemical and enzyme-linked immunosorbent assay studies revealed a membranous expression pattern for both receptor isoforms. Expression of the splice variant, however, occurred at a significantly lower cell surface receptor density. In terms of ligand binding and phospholipase C activation, the wild type receptor showed characteristics of a typical GnRH receptor, whereas the splice variant was incapable of ligand binding and signal transduction. Coexpression of wild type and truncated proteins in transiently or stably transfected cells, however, resulted in impaired signaling via the wild type receptor by reducing maximal agonist-induced inositol phosphate accumulation. The inhibitory effect depended on the amount of splice variant cDNA cotransfected and was specific for the GnRH receptor because signaling via other Gq/11-coupled receptors, such as the thromboxane A2, M5 muscarinic, and V1 vasopressin receptors, was not affected. Immunological studies revealed that coexpression of the wild type receptor and the truncated splice variant resulted in impaired insertion of the wild type receptor into the plasma membrane. Thus, expression of truncated receptor proteins may highlight a novel principle of specific functional inhibition of G protein-coupled receptors.

scholarly journals MRGPRX2 Activation by Rocuronium: Insights from Studies with Human Skin Mast Cells and Missense Variants

Cells ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 156 ◽  
Author(s):  
Chalatip Chompunud Na Ayudhya ◽  
Aetas Amponnawarat ◽  
Saptarshi Roy ◽  
Carole A. Oskeritzian ◽  
Hydar Ali
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Hypersensitivity Reaction ◽  
Neuromuscular Blocking ◽  
Missense Mutations ◽  
Blocking Drug ◽  
Wild Type ◽  
Human Ortholog ◽  
G Protein Coupled ◽  
Type Receptor

Perioperative hypersensitivity (POH) to the neuromuscular blocking drug (NMBD) rocuronium was previously thought to be IgE and mast cell (MC)-mediated. However, the recent seminal observation that rocuronium induces degranulation in murine peritoneal MCs (PMCs) via Mas-related G protein-coupled receptor B2 (MrgprB2) led to the idea that POH to this drug involves the activation of MRGPRX2 (human ortholog of MrgprB2). Furthermore, based on the demonstration that a patient with POH to rocuronium displayed three missense mutations (M196I, L226P and L237P) in MRGPRX2’s transmembrane domains, it was proposed that this hypersensitivity reaction resulted from aberrant activation of this receptor. We found that rocuronium at 20 µg/mL caused degranulation in mouse PMCs via MrgprB2 but required at least 500 µg/mL to induce degranulation in human MCs via MRGPRX2. Furthermore, RBL-2H3 cells transiently expressing M196I, L226P and L237P variants did not display enhanced degranulation in response to rocuronium when compared to the wild-type receptor. These findings provide the first demonstration that rocuronium induces degranulation in human MCs via MRGPRX2. Furthermore, the important differences between MrgprB2 and MRGPRX2 and the inability of rocuronium to induce enhanced response in cells expressing MRGPRX2 variants suggest that the mechanism of its POH is more complex than previously thought.

scholarly journals Manifold roles of β-arrestins in GPCR signaling elucidated with siRNA and CRISPR/Cas9

2018 ◽  
Vol 11 (549) ◽  
pp. eaat7650 ◽  
Author(s):  
Louis M. Luttrell ◽  
Jialu Wang ◽  
Bianca Plouffe ◽  
Jeffrey S. Smith ◽  
Lama Yamani ◽  
...  
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Hormone Receptors ◽  
Receptor Internalization ◽  
Protein Signaling ◽  
Mitogen Activated Protein ◽  
Cast Doubt ◽  
G Protein Coupled ◽  
Gpcr Desensitization

G protein–coupled receptors (GPCRs) use diverse mechanisms to regulate the mitogen-activated protein kinases ERK1/2. β-Arrestins (βArr1/2) are ubiquitous inhibitors of G protein signaling, promoting GPCR desensitization and internalization and serving as scaffolds for ERK1/2 activation. Studies using CRISPR/Cas9 to delete βArr1/2 and G proteins have cast doubt on the role of β-arrestins in activating specific pools of ERK1/2. We compared the effects of siRNA-mediated knockdown of βArr1/2 and reconstitution with βArr1/2 in three different parental and CRISPR-derived βArr1/2 knockout HEK293 cell pairs to assess the effect of βArr1/2 deletion on ERK1/2 activation by four Gs-coupled GPCRs. In all parental lines with all receptors, ERK1/2 stimulation was reduced by siRNAs specific for βArr2 or βArr1/2. In contrast, variable effects were observed with CRISPR-derived cell lines both between different lines and with activation of different receptors. For β2adrenergic receptors (β2ARs) and β1ARs, βArr1/2 deletion increased, decreased, or had no effect on isoproterenol-stimulated ERK1/2 activation in different CRISPR clones. ERK1/2 activation by the vasopressin V2and follicle-stimulating hormone receptors was reduced in these cells but was enhanced by reconstitution with βArr1/2. Loss of desensitization and receptor internalization in CRISPR βArr1/2 knockout cells caused β2AR-mediated stimulation of ERK1/2 to become more dependent on G proteins, which was reversed by reintroducing βArr1/2. These data suggest that βArr1/2 function as a regulatory hub, determining the balance between mechanistically different pathways that result in activation of ERK1/2, and caution against extrapolating results obtained from βArr1/2- or G protein–deleted cells to GPCR behavior in native systems.

Role of receptor kinase in long-term desensitization of the cardiac muscarinic receptor-K+ channel system

2002 ◽  
Vol 283 (2) ◽  
pp. H819-H828 ◽  
Author(s):  
Z. Shui ◽  
I. A. Khan ◽  
H. Tsuga ◽  
H. Dobrzynski ◽  
T. Haga ◽  
...  
Keyword(s):  
G Protein ◽  
Muscarinic Receptor ◽  
Cho Cells ◽  
Receptor Kinase ◽  
Wild Type ◽  
Channel System ◽  
Atrial Cells ◽  
G Protein Coupled ◽  
Type Receptor

Desensitization of the cardiac muscarinic K+ channel was studied in cultured neonatal rat atrial cells and in Chinese hamster ovary (CHO) cells transfected with muscarinic receptor (HM2), G protein-coupled inward rectifying K+ channels 1 and 4, and G protein-coupled receptor kinase 2. In atrial cells incubated in 10 μM carbachol for 24 h, channel activity in cell-attached patches was substantially reduced as a result of long-term desensitization. The long-term desensitization was also observed in CHO cells transfected with the wild-type receptor and receptor kinase (as well as the channel). However, long-term desensitization was greatly reduced or abolished if the cells were 1) not transfected with the receptor kinase, 2) transfected with a mutant receptor lacking phosphorylation sites (rather than the wild-type receptor), or 3) transfected with a mutant receptor kinase lacking kinase activity (rather than the wild-type receptor kinase). We suggest that long-term desensitization of the cardiac muscarinic receptor-K+ channel system to muscarinic agonist may involve phosphorylation of the receptor by receptor kinase.

scholarly journals Mechanisms Governing the Activation and Trafficking of Yeast G Protein-coupled Receptors

1998 ◽  
Vol 9 (4) ◽  
pp. 885-899 ◽  
Author(s):  
Christopher J. Stefan ◽  
Mark C. Overton ◽  
Kendall J. Blumer
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
G Protein ◽  
G Proteins ◽  
Structural Changes ◽  
G Protein Coupled Receptors ◽  
Wild Type ◽  
Proline Residue ◽  
G Protein Coupled ◽  
Constitutively Active

We have addressed the mechanisms governing the activation and trafficking of G protein-coupled receptors (GPCRs) by analyzing constitutively active mating pheromone receptors (Ste2p and Ste3p) of the yeast Saccharomyces cerevisiae. Substitution of the highly conserved proline residue in transmembrane segment VI of these receptors causes constitutive signaling. This proline residue may facilitate folding of GPCRs into native, inactive conformations, and/or mediate agonist-induced structural changes leading to G protein activation. Constitutive signaling by mutant receptors is suppressed upon coexpression with wild-type, but not G protein coupling-defective, receptors. Wild-type receptors may therefore sequester a limiting pool of G proteins; this apparent “precoupling” of receptors and G proteins could facilitate signal production at sites where cell surface projections form during mating partner discrimination. Finally, rather than being expressed mainly at the cell surface, constitutively active pheromone receptors accumulate in post-endoplasmic reticulum compartments. This is in contrast to other defective membrane proteins, which apparently are targeted by default to the vacuole. We suggest that the quality-control mechanism that retains receptors in post-endoplasmic reticulum compartments may normally allow wild-type receptors to fold into their native, fully inactive conformations before reaching the cell surface. This may ensure that receptors do not trigger a response in the absence of agonist.

scholarly journals GRKs as Modulators of Neurotransmitter Receptors

Cells ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 52
Author(s):  
Eugenia V. Gurevich ◽  
Vsevolod V. Gurevich
Keyword(s):  
G Protein ◽  
General Model ◽  
G Protein Coupled Receptors ◽  
Receptor Internalization ◽  
Neurotransmitter Receptors ◽  
Receptor Subtype ◽  
Receptor Subtypes ◽  
Receptor Kinase ◽  
G Protein Coupled ◽  
Homologous Desensitization

Many receptors for neurotransmitters, such as dopamine, norepinephrine, acetylcholine, and neuropeptides, belong to the superfamily of G protein-coupled receptors (GPCRs). A general model posits that GPCRs undergo two-step homologous desensitization: the active receptor is phosphorylated by kinases of the G protein-coupled receptor kinase (GRK) family, whereupon arrestin proteins specifically bind active phosphorylated receptors, shutting down G protein-mediated signaling, facilitating receptor internalization, and initiating distinct signaling pathways via arrestin-based scaffolding. Here, we review the mechanisms of GRK-dependent regulation of neurotransmitter receptors, focusing on the diverse modes of GRK-mediated phosphorylation of receptor subtypes. The immediate signaling consequences of GRK-mediated receptor phosphorylation, such as arrestin recruitment, desensitization, and internalization/resensitization, are equally diverse, depending not only on the receptor subtype but also on phosphorylation by GRKs of select receptor residues. We discuss the signaling outcome as well as the biological and behavioral consequences of the GRK-dependent phosphorylation of neurotransmitter receptors where known.

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