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

G protein–coupled receptors (GPCRs) are ubiquitous mediators of signaling of hormones, neurotransmitters, and sensing. The old dogma is that a one ligand/one receptor complex constitutes the functional unit of GPCR signaling. However, there is mounting evidence that some GPCRs form dimers or oligomers during their biosynthesis, activation, inactivation, and/or internalization. This evidence has been obtained exclusively from cell culture experiments, and proof for the physiological significance of GPCR di/oligomerization in vivo is still missing. Using the mouse luteinizing hormone receptor (LHR) as a model GPCR, we demonstrate that transgenic mice coexpressing binding-deficient and signaling-deficient forms of LHR can reestablish normal LH actions through intermolecular functional complementation of the mutant receptors in the absence of functional wild-type receptors. These results provide compelling in vivo evidence for the physiological relevance of intermolecular cooperation in GPCR signaling.

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Melatonin promotes sleep by activating the BK channel in C. elegans

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2010928117 ◽

2020 ◽

Vol 117 (40) ◽

pp. 25128-25137

Author(s):

Longgang Niu ◽

Yan Li ◽

Pengyu Zong ◽

Ping Liu ◽

Yuan Shui ◽

...

Keyword(s):

G Protein ◽

Neurotransmitter Release ◽

Expression System ◽

Molecular Target ◽

Bk Channel ◽

G Protein Coupled Receptors ◽

Wild Type ◽

Heterologous Expression System ◽

C Elegans ◽

G Protein Coupled

Melatonin (Mel) promotes sleep through G protein-coupled receptors. However, the downstream molecular target(s) is unknown. We identified the Caenorhabditis elegans BK channel SLO-1 as a molecular target of the Mel receptor PCDR-1-. Knockout of pcdr-1, slo-1, or homt-1 (a gene required for Mel synthesis) causes substantially increased neurotransmitter release and shortened sleep duration, and these effects are nonadditive in double knockouts. Exogenous Mel inhibits neurotransmitter release and promotes sleep in wild-type (WT) but not pcdr-1 and slo-1 mutants. In a heterologous expression system, Mel activates the human BK channel (hSlo1) in a membrane-delimited manner in the presence of the Mel receptor MT1 but not MT2. A peptide acting to release free Gβγ also activates hSlo1 in a MT1-dependent and membrane-delimited manner, whereas a Gβλ inhibitor abolishes the stimulating effect of Mel. Our results suggest that Mel promotes sleep by activating the BK channel through a specific Mel receptor and Gβλ.

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

The Journal of Cell Biology ◽

10.1083/jcb.200610154 ◽

2007 ◽

Vol 177 (5) ◽

pp. 905-916 ◽

Cited By ~ 76

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.

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Defining the Functional Equivalence of Wild-Type and Chemically Engineered G Protein-Coupled Receptors

Neuromethods - Designer Receptors Exclusively Activated by Designer Drugs ◽

10.1007/978-1-4939-2944-3_1 ◽

2015 ◽

pp. 1-28

Author(s):

Elisa Alvarez-Curto ◽

Graeme Milligan

Keyword(s):

G Protein ◽

G Protein Coupled Receptors ◽

Functional Equivalence ◽

Wild Type ◽

G Protein Coupled

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Caspase-12: a developmental link between G-protein–coupled receptors and integrin αIIbβ3 activation

Blood ◽

10.1182/blood-2003-10-3633 ◽

2004 ◽

Vol 104 (5) ◽

pp. 1327-1334 ◽

Cited By ~ 34

Author(s):

Steven W. Kerrigan ◽

Meenakshi Gaur ◽

Ronan P. Murphy ◽

Sanford J. Shattil ◽

Andrew D. Leavitt

Keyword(s):

G Protein ◽

Adenosine Diphosphate ◽

G Protein Coupled Receptors ◽

Free Calcium ◽

Wild Type ◽

Integrin Αiibβ3 ◽

Caspase 12 ◽

Fibrinogen Binding ◽

G Protein Coupled ◽

Inside Out

Abstract Fibrinogen binding by integrin αIIbβ3 is promoted by platelet agonists that increase the affinity and avidity of αIIbβ3 for fibrinogen through a process called “inside-out” signaling. Having previously demonstrated that inside-out activation of αIIbβ3 is defective in murine megakaryocytes that lack the transcription factor NF-E2, we screened for NF-E2–regulated genes that affect αIIbβ3 activation. Caspase-12 is the most down-regulated gene we identified in NF-E2–/– megakaryocytes. Therefore, the role of this protein in αIIbβ3 activation was determined using platelets from caspase-12–/– mice. Despite wild-type levels of αIIbβ3, caspase-12–/– platelets exhibit reduced fibrinogen binding to αIIbβ3 following stimulation by adenosine diphosphate (ADP) or protease-activated receptor 4 (PAR4) receptor-activating peptide. The defect in αIIbβ3 activation is associated with decreased cytosolic free calcium and inositol triphosphate levels, and with reduced aggregation, despite wild-type phospholipase Cβ expression levels. In contrast, agonist-induced surface expression of P-selectin, suppression of cAMP levels following ADP stimulation, and spreading on immobilized fibrinogen are unimpaired. Moreover, although caspase-12 is highly expressed in mature megakaryocytes, it is undetectable in platelets. Taken together, these studies establish that caspase-12 expression in murine megakaryocytes is regulated, directly or indirectly, by NF-E2, and suggest that caspase-12 participates in the development of fully functional signaling pathways linking some G-protein–coupled receptors to αIIbβ3 activation.

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Structural and functional analysis of the canine histamine H2 receptor by site-directed mutagenesis: N-glycosylation is not vital for its action

Biochemical Journal ◽

10.1042/bj3100553 ◽

1995 ◽

Vol 310 (2) ◽

pp. 553-558 ◽

Cited By ~ 39

Author(s):

Y Fukushima ◽

Y Oka ◽

T Saitoh ◽

H Katagiri ◽

T Asano ◽

...

Keyword(s):

Molecular Mass ◽

G Protein ◽

G Protein Coupled Receptors ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

Histamine H2 Receptor ◽

H2 Receptor ◽

H2 Receptors ◽

G Protein Coupled

G-protein-coupled receptors generally share a similar structure containing seven membrane-spanning domains and extracellular site(s) for N-glycosylation. The histamine H2 receptor is a member of the family of G-protein-coupled receptors, and has three extracellular potential sites for N-glycosylation (Asn-4, Asn-162 and Asn-168). To date, however, no information has been presented regarding N-glycosylation of the H2 receptor. To investigate the presence, location and functional roles of N-glycosylation of the H2 receptor, site-directed mutagenesis was performed to eliminate the potential site(s) for N-glycosylation singly and collectively. The wild-type and mutated H2 receptors were expressed stably in Chinese hamster ovary (CHO) cells or transiently in COS7 cells. Immunoblotting of the wild-type and mutated H2 receptors with an antiserum directed against the C-terminus of the H2 receptor showed that mutation at Asn-162, but not at Asn-168, resulted in a substantial decrease in the molecular mass. A mutation at Asn-4 led to a further decrease in the molecular mass. Tunicamycin treatment of the transfected cells yielded a sharp band with a molecular mass identical to that of the mutant devoid of all three potential sites for N-glycosylation. These findings indicate that the H2 receptor is N-glycosylated, and that N-glycosylation takes place mainly at two sites, Asn-4 and Asn-162. Neither the affinity for tiotidine nor that for histamine was affected by the mutagenesis. Immunocytochemistry and tiotidine binding showed that the mutated receptors were exclusively distributed on the cell surface in a fashion similar to that of the wild-type. In addition, the glycosylation-defective receptor was capable of activating adenylate cyclase and elevating the intracellular Ca2+ concentration in response to histamine in stable CHO cell lines. Thus N-glycosylation of the H2 receptor is not required for cell surface localization, ligand binding or functional coupling to G-protein(s).

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

International Journal of Molecular Sciences ◽

10.3390/ijms22105318 ◽

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.

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Identification of a bilirubin receptor that may mediate a component of cholestatic itch

eLife ◽

10.7554/elife.44116 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 28

Author(s):

James Meixiong ◽

Chirag Vasavda ◽

Dustin Green ◽

Qin Zheng ◽

Lijun Qi ◽

...

Keyword(s):

G Protein ◽

Mouse Models ◽

Sensory Neurons ◽

G Protein Coupled Receptors ◽

Primary Sensory Neurons ◽

Wild Type ◽

Biliverdin Reductase ◽

Promising Strategy ◽

Genetic Deletion ◽

G Protein Coupled

Various pathologic conditions result in jaundice, a yellowing of the skin due to a buildup of bilirubin. Patients with jaundice commonly report experiencing an intense non-histaminergic itch. Despite this association, the pruritogenic capacity of bilirubin itself has not been described, and no bilirubin receptor has been identified. Here, we demonstrate that pathophysiologic levels of bilirubin excite peripheral itch sensory neurons and elicit pruritus through MRGPRs, a family of G-protein coupled receptors expressed in primary sensory neurons. Bilirubin binds and activates two MRGPRs, mouse MRGPRA1 and human MRGPRX4. In two mouse models of pathologic hyperbilirubinemia, we show that genetic deletion of either Mrgpra1 or Blvra, the gene that encodes the bilirubin-producing enzyme biliverdin reductase, attenuates itch. Similarly, plasma isolated from hyperbilirubinemic patients evoked itch in wild-type animals but not Mrgpra1-/- animals. Removing bilirubin decreased the pruritogenic capacity of patient plasma. Based on these data, targeting MRGPRs is a promising strategy for alleviating jaundice-associated itch.

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Faculty Opinions recommendation of Constitutive activity of G-protein-coupled receptors: cause of disease and common property of wild-type receptors.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.12818978.14100082 ◽

2011 ◽

Author(s):

Jean-Pierre Changeux

Keyword(s):

G Protein ◽

Common Property ◽

G Protein Coupled Receptors ◽

Constitutive Activity ◽

Wild Type ◽

G Protein Coupled

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

Molecular Endocrinology ◽

10.1210/mend.11.9.9966 ◽

1997 ◽

Vol 11 (9) ◽

pp. 1305-1318 ◽

Cited By ~ 47

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.

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