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

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

scholarly journals Rescue of defective G protein–coupled receptor function in vivo by intermolecular cooperation

2010 ◽  
Vol 107 (5) ◽  
pp. 2319-2324 ◽  
Author(s):  
Adolfo Rivero-Müller ◽  
Yen-Yin Chou ◽  
Inhae Ji ◽  
Svetlana Lajic ◽  
Aylin C. Hanyaloglu ◽  
...  
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptors ◽  
Luteinizing Hormone Receptor ◽  
Wild Type ◽  
Gpcr Signaling ◽  
Physiological Relevance ◽  
Biosynthesis Activation ◽  
Mutant Receptors ◽  
G Protein Coupled

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.

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

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.

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

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

Blood ◽  
2004 ◽  
Vol 104 (5) ◽  
pp. 1327-1334 ◽  
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.

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

1995 ◽  
Vol 310 (2) ◽  
pp. 553-558 ◽  
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).

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

eLife ◽  
2019 ◽  
Vol 8 ◽  
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.

scholarly journals Substitution of the cysteine 438 residue in the cytoplasmic tail of the glucagon-like peptide-1 receptor alters signal transduction activity

2005 ◽  
Vol 185 (1) ◽  
pp. 35-44 ◽  
Author(s):  
Patricia Vázquez ◽  
Isabel Roncero ◽  
Enrique Blázquez ◽  
Elvira Alvarez
Keyword(s):  
Adenylate Cyclase ◽  
G Protein ◽  
Cytoplasmic Tail ◽  
G Protein Coupled Receptors ◽  
Cysteine Residues ◽  
Wild Type ◽  
Coated Pits ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1 ◽  
G Protein Coupled

Several G-protein-coupled receptors contain cysteine residues in the C-terminal tail that may modulate receptor function. In this work we analysed the substitution of Cys438 by alanine in the glucagon-like peptide-1 (GLP-1) receptor (GLPR), which led to a threefold decrease in cAMP production, although endocytosis and cellular redistribution of GLP-1 receptor agonist-induced processes were unaffected. Additionally, cysteine residues in the C-terminal tail of several G-protein-coupled receptors were found to act as substrates for palmitoylation, which might modify the access of protein kinases to this region. His-tagged GLP-1 receptors incorporated 3H-palmitate. Nevertheless, substitution of Cys438 prevented the incorporation of palmitate. Accordingly, we also investigated the effect of substitution of the consensus sequence by protein kinase C (PKC) Ser431/432 in both wild-type and Ala438 GLP-1 receptors. Substitution of Ser431/432 by alanine did not modify the ability of wild-type receptors to stimulate adenylate cyclase or endocytosis and recycling processes. By contrast, the substitution of Ser431/432 by alanine in the receptor containing Ala438 increased the ability to stimulate adenylate cyclase. All types of receptors were mainly internalised through coated pits. Thus, cysteine 438 in the cytoplasmic tail of the GLP-1 receptor would regulate its interaction with G-proteins and the stimulation of adenylyl cyclase. Palmitoylation of this residue might control the access of PKC to Ser431/432.

scholarly journals Dominant-Negative Mutations in the G-Protein-Coupled α-Factor Receptor Map to the Extracellular Ends of the Transmembrane Segments

1998 ◽  
Vol 18 (10) ◽  
pp. 5981-5991 ◽  
Author(s):  
Mercedes Dosil ◽  
Loïc Giot ◽  
Colleen Davis ◽  
James B. Konopka
Keyword(s):  
G Protein ◽  
Receptor Activation ◽  
G Protein Coupled Receptors ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Wild Type ◽  
Sensory Stimuli ◽  
Transmembrane Segments ◽  
Dominant Negative Mutations ◽  
G Protein Coupled

ABSTRACT G-protein-coupled receptors (GPCRs) transduce the signals for a wide range of hormonal and sensory stimuli by activating a heterotrimeric guanine nucleotide-binding protein (G protein). The analysis of loss-of-function and constitutively active receptor mutants has helped to reveal the functional properties of GPCRs and their role in human diseases. Here we describe the identification of a new class of mutants, dominant-negative mutants, for the yeast G-protein-coupled α-factor receptor (Ste2p). Sixteen dominant-negative receptor mutants were isolated based on their ability to inhibit the response to mating pheromone in cells that also express wild-type receptors. Detailed analysis of two of the strongest mutant receptors showed that, unlike other GPCR interfering mutants, they were properly localized at the plasma membrane and did not alter the stability or localization of wild-type receptors. Furthermore, their dominant-negative effect was inversely proportional to the relative amount of wild-type receptors and was reversed by overexpressing the G-protein subunits, suggesting that these mutants compete with the wild-type receptors for the G protein. Interestingly, the dominant-negative mutations are all located at the extracellular ends of the transmembrane segments, defining a novel region of the receptor that is important for receptor signaling. Altogether, our results identify residues of the α-factor receptor specifically involved in ligand binding and receptor activation and define a new mechanism by which GPCRs can be inactivated that has important implications for the evaluation of receptor mutations in other G-protein-coupled receptors.

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