A method for evaluating the effects of ligands upon Gs protein-coupled receptors using a recombinant melanophore-based bioassay

Potenza, Marc N.; Graminski, Gerard F.; Lerner, Michael R.

doi:10.1016/0003-2697(92)90372-e

Cited by 54 publications

(13 citation statements)

References 11 publications

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“…Under voltage clamp conditions, oocytes injected with mRNAs for GPR 51, GIRK1, and GIRK2 or combinations of GPR 51, GIRK1, and GIRK4 showed no response to 100 M concentrations of (Ϫ)baclofen, GABA, or (L)-glutamic acid (data not shown). The Xenopus melanophore pigment aggregation/dispersion assay has been shown to be highly suitable for monitoring agonist activation of Gi-, Gq-, and Gs-coupled GPCRs and potentially orphan GPCRs as well (Potenza et al, 1992;Lerner, 1994). Agonist activation of Gi-coupled receptors expressed in melanophores results in pigment aggregation via a reduction in intracellular cAMP levels, whereas activation of Gs-and Gq-coupled receptors results in pigment dispersion via elevations in intracellular cAMP and calcium levels, respectively.…”

Section: Recombinant Expression Of Gpr 51mentioning

confidence: 99%

Cloning of a Novel G-Protein-Coupled Receptor GPR 51 Resembling GABABReceptors Expressed Predominantly in Nervous Tissues and Mapped Proximal to the Hereditary Sensory Neuropathy Type 1 Locus on Chromosome 9

McDonald

Bonnert

et al. 1999

Genomics

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Section: Recombinant Expression Of Gpr 51mentioning

confidence: 99%

Cloning of a Novel G-Protein-Coupled Receptor GPR 51 Resembling GABABReceptors Expressed Predominantly in Nervous Tissues and Mapped Proximal to the Hereditary Sensory Neuropathy Type 1 Locus on Chromosome 9

McDonald

Bonnert

et al. 1999

Genomics

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“…Because rat GALR2 would appear to couple to both G 1-and Gqc oupled signaling pathways, we chose to investigate the signaling mechanism of human GALR2 in Xenopus melanophores (Daniolos et a!., 1990;Potenza et al, 1992;Lerner, 1994). The melanophore assay system is based on the dispersion and aggregation of intracellular pigment granules in response to changes in intracellular second messenger molecules.…”

Section: Signal Transduction Pathway Of Human Galr2 and Galr3mentioning

confidence: 99%

“…Conversely, agonist activation of a recombinant G~-coupledGPCR expressed heterologously in melanophores will lead to pigment aggregation. Changes in melanophore pigmentation show a dosedependent correlation with the level of specific receptor activation and can be quantified by the change in absorbance at 600 nm between the nonactivated and agonist-activated cells (Daniolos et al, 1990;Potenza et a!., 1992;Lerner, 1994).…”

Section: Signal Transduction Pathway Of Human Galr2 and Galr3mentioning

confidence: 99%

Molecular Characterization and Expression of Cloned Human Galanin Receptors GALR2 and GALR3

Kolakowski

O’Neill

Howard

et al. 1998

Journal of Neurochemistry

168

129

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Abstract:Galanin is a 29-or 30-amino acid peptide with wide-ranging effects on hormone release, feeding behavior, smooth muscle contractility, and somatosensory neuronal function. Three distinct galanin receptor (GALR) subtypes, designated GALR1, 2, and 3, have been cloned from the rat. We report here the cloning of the human GALR2 and GALR3 genes, an initial characterization of their pharmacology with respect to radioligand binding and signal transduction pathways, and a profile of their expression in brain and peripheral tissues. Human GALR2 and GALR3 show, respectively, 92 and 89% amino acid sequence identity with their rat homologues. Radioligand binding studies with 125l-galanin show that recombinant human GALR2 binds with high affinity to human galanin (K 0 = 0.3 nM). Human GALR3 binds galanin with less affinity (IC50 of 12 nM for porcine galanin and 75 nM for human galanin). Human GALR2 was shown to couple to phospholipase C and elevation of intracellular calcium levels as assessed by aequorin luminescence in HEK-293 cells and by Xenopus melanophore pigment aggregation and dispersion assays, in contrast to human GALR1 and human GALR3, which signal predominantly through inhibition of adenylate cyclase. GALR2 mRNA shows a wide distribution in the brain (mammillary nuclei, dentate gyrus, cingulate gyrus, and posterior hypothalamic, supraoptic, and arcuate nuclei), and restricted peripheral tissue distritution with highest mRNA levels detected in human small intestine. In comparison, whereas GALR3 mRNA was expressed in many areas of the rat brain, there was abundant expression in the primary 01-factory cortex, olfactory tubercie, the islands of Calleja, the hippocampal CA regions of Ammon's horn, and the dentate gyrus. GALR3 mRNA was highly expressed in human testis and was detectable in adrenal gland and pancreas. The genes for human GALR2 and 3 were localized to chromosomes 17q25 and 22q12.2-13.1, respectively. Key Words: Galanin receptor-G protein-coupled receptor-Cloning.

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“…In these cells, melanosome dispersion can be affected via activation of adenylyl cyclase (Potenza et al, 1992;McClintock et al, 1993) or phospholipase C , whereas melanosome aggregation results from the inhibition of adenylyl cyclase (Potenza et al, 1992;McClintock et al, 1993). Melanophore cells contain a wide range of G ␣ -proteins (Jayawickreme et al, 1994); therefore, the expression of numerous foreign G protein-coupled receptors can be facilitated (Potenza et al, 1992(Potenza et al, , 1994Karne et al, 1993;McClintock et al, 1993;Graminski et al, 1993 and1994;Jayawickreme et al, 1994aJayawickreme et al, , 1994bLerner, 1994). Because both states of in-ABBREVIATIONS: HIV-1, human immunodeficiency virus type 1; CXCRx, CXC chemokine receptor, where x is the receptor number; SDF, stromal cell-derived factor; BSA, bovine serum albumin; PCR, polymerase chain reaction; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; SDS, sodium dodecyl sulfate; ED 50 , dose effective on 50% of the population.…”

Section: Introductionmentioning

confidence: 99%

“…In these cells, melanosome dispersion can be affected via activation of adenylyl cyclase (Potenza et al, 1992;McClintock et al, 1993) or phospholipase C , whereas melanosome aggregation results from the inhibition of adenylyl cyclase (Potenza et al, 1992;McClintock et al, 1993). Melanophore cells contain a wide range of G ␣ -proteins (Jayawickreme et al, 1994); therefore, the expression of numerous foreign G protein-coupled receptors can be facilitated (Potenza et al, 1992(Potenza et al, , 1994Karne et al, 1993;McClintock et al, 1993;Graminski et al, 1993 and1994;Jayawickreme et al, 1994aJayawickreme et al, , 1994bLerner, 1994). Because both states of in-tracellular melanosome distribution (dispersion or aggregation) are easily detectable, various G protein-coupled receptors have been studied by monitoring ligand-mediated melanosome translocation, either by measuring the change in light transmittance through the cells or by imaging the cell response (Potenza et al, 1992(Potenza et al, , 1994Karne et al, 1993;McClintock et al, 1993;Graminski et al, 1993 and1994;Jayawickreme et al, 1994aJayawickreme et al, , 1994bLerner, 1994).…”

mentioning

confidence: 99%

Recombinant Human CXC-Chemokine Receptor-4 in Melanophores Are Linked to G_iProtein: Seven Transmembrane Coreceptors for Human Immunodeficiency Virus Entry into Cells

et al. 1998

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This article describes the transient expression of the CXC chemokine receptor-4 in Xenopus laevis melanophores and the resulting functional assay for the endogenous ligand for this receptor stromal cell-derived factor (SDF)-1␣. Specifically, it will be shown that SDF-1␣ produces increased light transmittance in transfected cells that is consistent with the activation of G i protein. This stimulus pathway is further implicated by the abolition of this response after pretreatment of the cells with pertussis toxin, a known method for the inactivation of G i protein. The fact that SDF-1␣ does not produce responses in nontransfected cells and that treatment of the cells with 12G5, an antibody specific for the CXC chemokine receptor-4, eliminates this response indicates that this ligand produces responses by activation of this receptor in these cells. The possible relevance to human immunodeficiency virus (HIV) entry into cells was explored by observing the effects of SDF-1␣ on HIV-mediated cell fusion. It was found that SDF-1␣ blocked cell-to-cell fusion (as has been previously reported) at concentrations 1200-fold greater than those required to produce G i protein mediated responses. The implications of the functional assay to screening for new drugs to block HIV-mediated fusion is discussed.The discovery that HIV-1 utilizes seven transmembrane receptors as coreceptors for viral fusion has introduced a new target into the therapeutic field of view. Studies on HIV-1 envelope-mediated cell fusion have identified a 46-kDa, integral membrane protein named "fusin" that serves as a cofactor for HIV fusion and entry (Feng et al., 1996). Fusin is a 352-amino-acid, seven-transmembrane receptor, the sequence of which is identical to a previously cloned orphan receptor denoted LESTR (leukocyte-derived seven-transmembrane domain receptor). Sequence comparison has shown that fusin (or LESTR) is a member of the CXC chemokine receptor family with a sequence homology to the CXCR2 receptor (Loetscher et al., 1994;Raport et al., 1996;Wells et al., 1996). The identification of SDF-1␣ (a CXC chemokine) as a ligand for fusin has led to the suggested classification of this receptor as CXCR4 in the chemokine receptor nomenclature system (Bleul et al., 1996;Oberlin et al., 1996). This article describes the construction of a receptor assay in which the human CXCR4 couples to G i -protein in recombinant Xenopus laevis melanophores. In these cells, melanosome dispersion can be affected via activation of adenylyl cyclase (Potenza et al., 1992;McClintock et al., 1993) or phospholipase C , whereas melanosome aggregation results from the inhibition of adenylyl cyclase (Potenza et al., 1992;McClintock et al., 1993). Melanophore cells contain a wide range of G ␣ -proteins (Jayawickreme et al., 1994); therefore, the expression of numerous foreign G protein-coupled receptors can be facilitated (Potenza et al., 1992(Potenza et al., , 1994Karne et al., 1993;McClintock et al., 1993;Graminski et al., 1993 and1994;Jayawickreme et al., 1994aJayawic...

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A method for evaluating the effects of ligands upon Gs protein-coupled receptors using a recombinant melanophore-based bioassay

Cited by 54 publications

References 11 publications

Cloning of a Novel G-Protein-Coupled Receptor GPR 51 Resembling GABABReceptors Expressed Predominantly in Nervous Tissues and Mapped Proximal to the Hereditary Sensory Neuropathy Type 1 Locus on Chromosome 9

Cloning of a Novel G-Protein-Coupled Receptor GPR 51 Resembling GABABReceptors Expressed Predominantly in Nervous Tissues and Mapped Proximal to the Hereditary Sensory Neuropathy Type 1 Locus on Chromosome 9

Molecular Characterization and Expression of Cloned Human Galanin Receptors GALR2 and GALR3

Recombinant Human CXC-Chemokine Receptor-4 in Melanophores Are Linked to G_iProtein: Seven Transmembrane Coreceptors for Human Immunodeficiency Virus Entry into Cells

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A method for evaluating the effects of ligands upon Gs protein-coupled receptors using a recombinant melanophore-based bioassay

Cited by 54 publications

References 11 publications

Cloning of a Novel G-Protein-Coupled Receptor GPR 51 Resembling GABABReceptors Expressed Predominantly in Nervous Tissues and Mapped Proximal to the Hereditary Sensory Neuropathy Type 1 Locus on Chromosome 9

Cloning of a Novel G-Protein-Coupled Receptor GPR 51 Resembling GABABReceptors Expressed Predominantly in Nervous Tissues and Mapped Proximal to the Hereditary Sensory Neuropathy Type 1 Locus on Chromosome 9

Molecular Characterization and Expression of Cloned Human Galanin Receptors GALR2 and GALR3

Recombinant Human CXC-Chemokine Receptor-4 in Melanophores Are Linked to GiProtein: Seven Transmembrane Coreceptors for Human Immunodeficiency Virus Entry into Cells

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Recombinant Human CXC-Chemokine Receptor-4 in Melanophores Are Linked to G_iProtein: Seven Transmembrane Coreceptors for Human Immunodeficiency Virus Entry into Cells