Identification and functional binding analysis of GPR23/! LPA4 as a candidate G protein-coupled receptor for Guanosine.

Garozzo, R; Mudò, G; Caciagli, F; Condorelli, Df

Risultato della ricerca: Paper

Abstract

Several studies have shown that guanine-based purines exert biological effects on the central nervous system (CNS), possibly through membrane receptors, but at the present there are not reports related to the identification of such specific receptor(s). We have identified the first guanosine G protein-coupled receptor GPR23, also known as LPA4 receptor, involved in the modulation of guanosine-mediated antiproliferative effects in human glioma cell line (U87). We report that the silencing of GPR23 reduces significantly the antiproliferative effects of guanosine, while stably transfected cell clones over-expressing GPR23 increase sensitivity to guanosine. [3H] Guanosine radioligand binding assay reveals that [3H]-Guanosine binding to membrane fractions is greatly enhanced by GPR23 overexpression, and inhibited by GPR23 silencing. Furthermore, in [35S] GTPγS binding assay experiments, Guanosine causes a functional G protein coupled receptor activation in U87GPR23 overexp! ressing cells with an EC50= 8,067 nM. The binding site for [3H]-guanosine is highly specific and both lysophosphatidic acid (LPA) and guanine agonists are 10 times less effective than guanosine in displacing 50 nM [3H]-guanosine binding. In order to correlate the effects of guanosine in the CNS to a putative activation of GPR23, we performed, in different brain areas, the following investigations: by PCR, the expression levels of GPR23; by [3H]-Guanosine radioligand binding assay, the binding properties of Guanosine; by [35S] GTPγS binding assay, the receptor activation properties in response to Guanosine. Among the examined brain areas, the cerebral cortex shows the highest GPR23 expression levels which correlate with the highest Bmax values for [3H]-Guanosine as compared to other brain regions, with the following rank order: cerebral cortex>hippocampus>striatum>spinal cord. [35S] GTPγS binding assay experiments confirm an activation of a G protein-cou! pled receptor in response to guanosine in the cerebral cortex (EC50 10 0 nM). Although these observations do not exclude a possible involvement of other unidentified receptors, they can suggest an involvement of GPR23 in the functional response of cerebral cortex to Guanosine. Overall, together these data suggest that GPR23 may act as a functional membrane receptor for Guanosine.
Lingua originaleEnglish
Stato di pubblicazionePublished - 2013

Fingerprint

Guanosine
G-Protein-Coupled Receptors
Cerebral Cortex
Radioligand Assay
Guanine
Membranes
Brain
Central Nervous System
Purines

Cita questo

Identification and functional binding analysis of GPR23/! LPA4 as a candidate G protein-coupled receptor for Guanosine. / Garozzo, R; Mudò, G; Caciagli, F; Condorelli, Df.

2013.

Risultato della ricerca: Paper

@conference{960cb94bba324dd79a2bc9d8aa285a86,
title = "Identification and functional binding analysis of GPR23/! LPA4 as a candidate G protein-coupled receptor for Guanosine.",
abstract = "Several studies have shown that guanine-based purines exert biological effects on the central nervous system (CNS), possibly through membrane receptors, but at the present there are not reports related to the identification of such specific receptor(s). We have identified the first guanosine G protein-coupled receptor GPR23, also known as LPA4 receptor, involved in the modulation of guanosine-mediated antiproliferative effects in human glioma cell line (U87). We report that the silencing of GPR23 reduces significantly the antiproliferative effects of guanosine, while stably transfected cell clones over-expressing GPR23 increase sensitivity to guanosine. [3H] Guanosine radioligand binding assay reveals that [3H]-Guanosine binding to membrane fractions is greatly enhanced by GPR23 overexpression, and inhibited by GPR23 silencing. Furthermore, in [35S] GTPγS binding assay experiments, Guanosine causes a functional G protein coupled receptor activation in U87GPR23 overexp! ressing cells with an EC50= 8,067 nM. The binding site for [3H]-guanosine is highly specific and both lysophosphatidic acid (LPA) and guanine agonists are 10 times less effective than guanosine in displacing 50 nM [3H]-guanosine binding. In order to correlate the effects of guanosine in the CNS to a putative activation of GPR23, we performed, in different brain areas, the following investigations: by PCR, the expression levels of GPR23; by [3H]-Guanosine radioligand binding assay, the binding properties of Guanosine; by [35S] GTPγS binding assay, the receptor activation properties in response to Guanosine. Among the examined brain areas, the cerebral cortex shows the highest GPR23 expression levels which correlate with the highest Bmax values for [3H]-Guanosine as compared to other brain regions, with the following rank order: cerebral cortex>hippocampus>striatum>spinal cord. [35S] GTPγS binding assay experiments confirm an activation of a G protein-cou! pled receptor in response to guanosine in the cerebral cortex (EC50 10 0 nM). Although these observations do not exclude a possible involvement of other unidentified receptors, they can suggest an involvement of GPR23 in the functional response of cerebral cortex to Guanosine. Overall, together these data suggest that GPR23 may act as a functional membrane receptor for Guanosine.",
keywords = "Gpr23; Guanosine; Binding assay; Brain",
author = "{Garozzo, R; Mud{\`o}, G; Caciagli, F; Condorelli, Df} and Natale Belluardo and Giuseppa Mudo' and Maria Grillo and {Di Liberto}, Valentina",
year = "2013",
language = "English",

}

TY - CONF

T1 - Identification and functional binding analysis of GPR23/! LPA4 as a candidate G protein-coupled receptor for Guanosine.

AU - Garozzo, R; Mudò, G; Caciagli, F; Condorelli, Df

AU - Belluardo, Natale

AU - Mudo', Giuseppa

AU - Grillo, Maria

AU - Di Liberto, Valentina

PY - 2013

Y1 - 2013

N2 - Several studies have shown that guanine-based purines exert biological effects on the central nervous system (CNS), possibly through membrane receptors, but at the present there are not reports related to the identification of such specific receptor(s). We have identified the first guanosine G protein-coupled receptor GPR23, also known as LPA4 receptor, involved in the modulation of guanosine-mediated antiproliferative effects in human glioma cell line (U87). We report that the silencing of GPR23 reduces significantly the antiproliferative effects of guanosine, while stably transfected cell clones over-expressing GPR23 increase sensitivity to guanosine. [3H] Guanosine radioligand binding assay reveals that [3H]-Guanosine binding to membrane fractions is greatly enhanced by GPR23 overexpression, and inhibited by GPR23 silencing. Furthermore, in [35S] GTPγS binding assay experiments, Guanosine causes a functional G protein coupled receptor activation in U87GPR23 overexp! ressing cells with an EC50= 8,067 nM. The binding site for [3H]-guanosine is highly specific and both lysophosphatidic acid (LPA) and guanine agonists are 10 times less effective than guanosine in displacing 50 nM [3H]-guanosine binding. In order to correlate the effects of guanosine in the CNS to a putative activation of GPR23, we performed, in different brain areas, the following investigations: by PCR, the expression levels of GPR23; by [3H]-Guanosine radioligand binding assay, the binding properties of Guanosine; by [35S] GTPγS binding assay, the receptor activation properties in response to Guanosine. Among the examined brain areas, the cerebral cortex shows the highest GPR23 expression levels which correlate with the highest Bmax values for [3H]-Guanosine as compared to other brain regions, with the following rank order: cerebral cortex>hippocampus>striatum>spinal cord. [35S] GTPγS binding assay experiments confirm an activation of a G protein-cou! pled receptor in response to guanosine in the cerebral cortex (EC50 10 0 nM). Although these observations do not exclude a possible involvement of other unidentified receptors, they can suggest an involvement of GPR23 in the functional response of cerebral cortex to Guanosine. Overall, together these data suggest that GPR23 may act as a functional membrane receptor for Guanosine.

AB - Several studies have shown that guanine-based purines exert biological effects on the central nervous system (CNS), possibly through membrane receptors, but at the present there are not reports related to the identification of such specific receptor(s). We have identified the first guanosine G protein-coupled receptor GPR23, also known as LPA4 receptor, involved in the modulation of guanosine-mediated antiproliferative effects in human glioma cell line (U87). We report that the silencing of GPR23 reduces significantly the antiproliferative effects of guanosine, while stably transfected cell clones over-expressing GPR23 increase sensitivity to guanosine. [3H] Guanosine radioligand binding assay reveals that [3H]-Guanosine binding to membrane fractions is greatly enhanced by GPR23 overexpression, and inhibited by GPR23 silencing. Furthermore, in [35S] GTPγS binding assay experiments, Guanosine causes a functional G protein coupled receptor activation in U87GPR23 overexp! ressing cells with an EC50= 8,067 nM. The binding site for [3H]-guanosine is highly specific and both lysophosphatidic acid (LPA) and guanine agonists are 10 times less effective than guanosine in displacing 50 nM [3H]-guanosine binding. In order to correlate the effects of guanosine in the CNS to a putative activation of GPR23, we performed, in different brain areas, the following investigations: by PCR, the expression levels of GPR23; by [3H]-Guanosine radioligand binding assay, the binding properties of Guanosine; by [35S] GTPγS binding assay, the receptor activation properties in response to Guanosine. Among the examined brain areas, the cerebral cortex shows the highest GPR23 expression levels which correlate with the highest Bmax values for [3H]-Guanosine as compared to other brain regions, with the following rank order: cerebral cortex>hippocampus>striatum>spinal cord. [35S] GTPγS binding assay experiments confirm an activation of a G protein-cou! pled receptor in response to guanosine in the cerebral cortex (EC50 10 0 nM). Although these observations do not exclude a possible involvement of other unidentified receptors, they can suggest an involvement of GPR23 in the functional response of cerebral cortex to Guanosine. Overall, together these data suggest that GPR23 may act as a functional membrane receptor for Guanosine.

KW - Gpr23; Guanosine; Binding assay; Brain

UR - http://hdl.handle.net/10447/75955

M3 - Paper

ER -