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Kinetic analysis of antagonist-occupied adenosine-A(3) receptors within membrane microdomains of individual cells provides evidence of receptor dimerization and allosterism
In our previous work, using a fluorescent adenosine-A(3) receptor (A(3)AR) agonist and fluorescence correlation spectroscopy (FCS), we demonstrated high-affinity labeling of the active receptor (R*) conformation. In the current study, we used a fluorescent A(3)AR antagonist (CA200645) to study the b...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Federation of American Societies for Experimental Biology
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4202110/ https://www.ncbi.nlm.nih.gov/pubmed/24970394 http://dx.doi.org/10.1096/fj.13-247270 |
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author | Corriden, Ross Kilpatrick, Laura E. Kellam, Barrie Briddon, Stephen J. Hill, Stephen J. |
author_facet | Corriden, Ross Kilpatrick, Laura E. Kellam, Barrie Briddon, Stephen J. Hill, Stephen J. |
author_sort | Corriden, Ross |
collection | PubMed |
description | In our previous work, using a fluorescent adenosine-A(3) receptor (A(3)AR) agonist and fluorescence correlation spectroscopy (FCS), we demonstrated high-affinity labeling of the active receptor (R*) conformation. In the current study, we used a fluorescent A(3)AR antagonist (CA200645) to study the binding characteristics of antagonist-occupied inactive receptor (R) conformations in membrane microdomains of individual cells. FCS analysis of CA200645-occupied A(3)ARs revealed 2 species, τ(D2) and τ(D3), that diffused at 2.29 ± 0.35 and 0.09 ± 0.03 μm(2)/s, respectively. FCS analysis of a green fluorescent protein (GFP)-tagged A(3)AR exhibited a single diffusing species (0.105 μm(2)/s). The binding of CA200645 to τ(D3) was antagonized by nanomolar concentrations of the A(3) antagonist MRS 1220, but not by the agonist NECA (up to 300 nM), consistent with labeling of R. CA200645 normally dissociated slowly from the A(3)AR, but inclusion of xanthine amine congener (XAC) or VUF 5455 during washout markedly accelerated the reduction in the number of particles exhibiting τ(D3) characteristics. It is notable that this effect was accompanied by a significant increase in the number of particles with τ(D2) diffusion. These data show that FCS analysis of ligand-occupied receptors provides a unique means of monitoring ligand A(3)AR residence times that are significantly reduced as a consequence of allosteric interaction across the dimer interface.—Corriden, R., Kilpatrick, L. E., Kellam, B., Briddon, S. J., Hill, S. J. Kinetic analysis of antagonist-occupied adenosine-A(3) receptors within membrane microdomains of individual cells provides evidence for receptor dimerization and allosterism. |
format | Online Article Text |
id | pubmed-4202110 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Federation of American Societies for Experimental Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-42021102014-11-03 Kinetic analysis of antagonist-occupied adenosine-A(3) receptors within membrane microdomains of individual cells provides evidence of receptor dimerization and allosterism Corriden, Ross Kilpatrick, Laura E. Kellam, Barrie Briddon, Stephen J. Hill, Stephen J. FASEB J Research Communications In our previous work, using a fluorescent adenosine-A(3) receptor (A(3)AR) agonist and fluorescence correlation spectroscopy (FCS), we demonstrated high-affinity labeling of the active receptor (R*) conformation. In the current study, we used a fluorescent A(3)AR antagonist (CA200645) to study the binding characteristics of antagonist-occupied inactive receptor (R) conformations in membrane microdomains of individual cells. FCS analysis of CA200645-occupied A(3)ARs revealed 2 species, τ(D2) and τ(D3), that diffused at 2.29 ± 0.35 and 0.09 ± 0.03 μm(2)/s, respectively. FCS analysis of a green fluorescent protein (GFP)-tagged A(3)AR exhibited a single diffusing species (0.105 μm(2)/s). The binding of CA200645 to τ(D3) was antagonized by nanomolar concentrations of the A(3) antagonist MRS 1220, but not by the agonist NECA (up to 300 nM), consistent with labeling of R. CA200645 normally dissociated slowly from the A(3)AR, but inclusion of xanthine amine congener (XAC) or VUF 5455 during washout markedly accelerated the reduction in the number of particles exhibiting τ(D3) characteristics. It is notable that this effect was accompanied by a significant increase in the number of particles with τ(D2) diffusion. These data show that FCS analysis of ligand-occupied receptors provides a unique means of monitoring ligand A(3)AR residence times that are significantly reduced as a consequence of allosteric interaction across the dimer interface.—Corriden, R., Kilpatrick, L. E., Kellam, B., Briddon, S. J., Hill, S. J. Kinetic analysis of antagonist-occupied adenosine-A(3) receptors within membrane microdomains of individual cells provides evidence for receptor dimerization and allosterism. Federation of American Societies for Experimental Biology 2014-10 /pmc/articles/PMC4202110/ /pubmed/24970394 http://dx.doi.org/10.1096/fj.13-247270 Text en © FASEB This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 international (CC BY 4.0) (http://creativecommons.org/licenses/by/4.0/) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Communications Corriden, Ross Kilpatrick, Laura E. Kellam, Barrie Briddon, Stephen J. Hill, Stephen J. Kinetic analysis of antagonist-occupied adenosine-A(3) receptors within membrane microdomains of individual cells provides evidence of receptor dimerization and allosterism |
title | Kinetic analysis of antagonist-occupied adenosine-A(3) receptors within membrane microdomains of individual cells provides evidence of receptor dimerization and allosterism |
title_full | Kinetic analysis of antagonist-occupied adenosine-A(3) receptors within membrane microdomains of individual cells provides evidence of receptor dimerization and allosterism |
title_fullStr | Kinetic analysis of antagonist-occupied adenosine-A(3) receptors within membrane microdomains of individual cells provides evidence of receptor dimerization and allosterism |
title_full_unstemmed | Kinetic analysis of antagonist-occupied adenosine-A(3) receptors within membrane microdomains of individual cells provides evidence of receptor dimerization and allosterism |
title_short | Kinetic analysis of antagonist-occupied adenosine-A(3) receptors within membrane microdomains of individual cells provides evidence of receptor dimerization and allosterism |
title_sort | kinetic analysis of antagonist-occupied adenosine-a(3) receptors within membrane microdomains of individual cells provides evidence of receptor dimerization and allosterism |
topic | Research Communications |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4202110/ https://www.ncbi.nlm.nih.gov/pubmed/24970394 http://dx.doi.org/10.1096/fj.13-247270 |
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