Negative cooperativity across β1-adrenoceptor homodimers provides insights into the nature of the secondary low-affinity CGP 12177 β1-adrenoceptor binding conformation

Karolina Gherbi, Lauren T. May, Jillian G Baker, Stephen J Briddon, Stephen J Hill

Research output: Contribution to journalArticleResearchpeer-review

Abstract

At the β1-adrenoceptor, CGP 12177 potently antagonizes agonist responses at the primary highaffinity catecholamine conformation while also exerting agonist effects of its own through a secondary low-affinity conformation. A recent mutagenesis study identified transmembrane region (TM)4 of the β1-adrenoceptor as key for this low-affinity conformation. Others suggested that TM4 has a role in β1-adrenoceptor oligomerization. Here, assessment of the dissociation rate of a fluorescent analog of CGP 12177 [bordifluoropyrromethane-tetramethylrhodamine-(±)CGP 12177 (BODIPY-TMR-CGP)] at the human β1-adrenoceptor expressed in Chinese hamster ovary cells revealed negative cooperative interactions between 2 distinct β1-adrenoceptor conformations. The dissociation rate of 3 nM BODIPY-TMR-CGP was 0.09 ± 0.01 min-1 in the absence of competitor ligands, and this was enhanced 2.2- and 2.1-fold in the presence of 1 μM CGP 12177 and 1 μM propranolol, respectively. These effects on the BODIPY-TMR-CGP dissociation rate were markedly enhanced in β1-adrenoceptor homodimers constrainedbybimolecular fluorescencecomplementation(9.8-and9.9-foldfor1μM CGP 12177 and 1 μM propranolol, respectively) and abolished in β1-adrenoceptors containing TM4 mutations vital for the second conformation pharmacology. This study suggests that negative cooperativity across a β1-adrenoceptor homodimer may be responsible for generating the low-affinity pharmacology of the secondary β1-adrenoceptor conformation.

Original languageEnglish
Pages (from-to)2859-2871
Number of pages13
JournalFASEB Journal
Volume29
Issue number7
DOIs
Publication statusPublished - 1 Jul 2015
Externally publishedYes

Keywords

  • Allosterism
  • Dissociation
  • GPCR
  • Receptor dimerization

Cite this

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title = "Negative cooperativity across β1-adrenoceptor homodimers provides insights into the nature of the secondary low-affinity CGP 12177 β1-adrenoceptor binding conformation",
abstract = "At the β1-adrenoceptor, CGP 12177 potently antagonizes agonist responses at the primary highaffinity catecholamine conformation while also exerting agonist effects of its own through a secondary low-affinity conformation. A recent mutagenesis study identified transmembrane region (TM)4 of the β1-adrenoceptor as key for this low-affinity conformation. Others suggested that TM4 has a role in β1-adrenoceptor oligomerization. Here, assessment of the dissociation rate of a fluorescent analog of CGP 12177 [bordifluoropyrromethane-tetramethylrhodamine-(±)CGP 12177 (BODIPY-TMR-CGP)] at the human β1-adrenoceptor expressed in Chinese hamster ovary cells revealed negative cooperative interactions between 2 distinct β1-adrenoceptor conformations. The dissociation rate of 3 nM BODIPY-TMR-CGP was 0.09 ± 0.01 min-1 in the absence of competitor ligands, and this was enhanced 2.2- and 2.1-fold in the presence of 1 μM CGP 12177 and 1 μM propranolol, respectively. These effects on the BODIPY-TMR-CGP dissociation rate were markedly enhanced in β1-adrenoceptor homodimers constrainedbybimolecular fluorescencecomplementation(9.8-and9.9-foldfor1μM CGP 12177 and 1 μM propranolol, respectively) and abolished in β1-adrenoceptors containing TM4 mutations vital for the second conformation pharmacology. This study suggests that negative cooperativity across a β1-adrenoceptor homodimer may be responsible for generating the low-affinity pharmacology of the secondary β1-adrenoceptor conformation.",
keywords = "Allosterism, Dissociation, GPCR, Receptor dimerization",
author = "Karolina Gherbi and May, {Lauren T.} and Baker, {Jillian G} and Briddon, {Stephen J} and Hill, {Stephen J}",
year = "2015",
month = "7",
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doi = "10.1096/fj.14-265199",
language = "English",
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Negative cooperativity across β1-adrenoceptor homodimers provides insights into the nature of the secondary low-affinity CGP 12177 β1-adrenoceptor binding conformation. / Gherbi, Karolina; May, Lauren T.; Baker, Jillian G; Briddon, Stephen J; Hill, Stephen J.

In: FASEB Journal, Vol. 29, No. 7, 01.07.2015, p. 2859-2871.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Negative cooperativity across β1-adrenoceptor homodimers provides insights into the nature of the secondary low-affinity CGP 12177 β1-adrenoceptor binding conformation

AU - Gherbi, Karolina

AU - May, Lauren T.

AU - Baker, Jillian G

AU - Briddon, Stephen J

AU - Hill, Stephen J

PY - 2015/7/1

Y1 - 2015/7/1

N2 - At the β1-adrenoceptor, CGP 12177 potently antagonizes agonist responses at the primary highaffinity catecholamine conformation while also exerting agonist effects of its own through a secondary low-affinity conformation. A recent mutagenesis study identified transmembrane region (TM)4 of the β1-adrenoceptor as key for this low-affinity conformation. Others suggested that TM4 has a role in β1-adrenoceptor oligomerization. Here, assessment of the dissociation rate of a fluorescent analog of CGP 12177 [bordifluoropyrromethane-tetramethylrhodamine-(±)CGP 12177 (BODIPY-TMR-CGP)] at the human β1-adrenoceptor expressed in Chinese hamster ovary cells revealed negative cooperative interactions between 2 distinct β1-adrenoceptor conformations. The dissociation rate of 3 nM BODIPY-TMR-CGP was 0.09 ± 0.01 min-1 in the absence of competitor ligands, and this was enhanced 2.2- and 2.1-fold in the presence of 1 μM CGP 12177 and 1 μM propranolol, respectively. These effects on the BODIPY-TMR-CGP dissociation rate were markedly enhanced in β1-adrenoceptor homodimers constrainedbybimolecular fluorescencecomplementation(9.8-and9.9-foldfor1μM CGP 12177 and 1 μM propranolol, respectively) and abolished in β1-adrenoceptors containing TM4 mutations vital for the second conformation pharmacology. This study suggests that negative cooperativity across a β1-adrenoceptor homodimer may be responsible for generating the low-affinity pharmacology of the secondary β1-adrenoceptor conformation.

AB - At the β1-adrenoceptor, CGP 12177 potently antagonizes agonist responses at the primary highaffinity catecholamine conformation while also exerting agonist effects of its own through a secondary low-affinity conformation. A recent mutagenesis study identified transmembrane region (TM)4 of the β1-adrenoceptor as key for this low-affinity conformation. Others suggested that TM4 has a role in β1-adrenoceptor oligomerization. Here, assessment of the dissociation rate of a fluorescent analog of CGP 12177 [bordifluoropyrromethane-tetramethylrhodamine-(±)CGP 12177 (BODIPY-TMR-CGP)] at the human β1-adrenoceptor expressed in Chinese hamster ovary cells revealed negative cooperative interactions between 2 distinct β1-adrenoceptor conformations. The dissociation rate of 3 nM BODIPY-TMR-CGP was 0.09 ± 0.01 min-1 in the absence of competitor ligands, and this was enhanced 2.2- and 2.1-fold in the presence of 1 μM CGP 12177 and 1 μM propranolol, respectively. These effects on the BODIPY-TMR-CGP dissociation rate were markedly enhanced in β1-adrenoceptor homodimers constrainedbybimolecular fluorescencecomplementation(9.8-and9.9-foldfor1μM CGP 12177 and 1 μM propranolol, respectively) and abolished in β1-adrenoceptors containing TM4 mutations vital for the second conformation pharmacology. This study suggests that negative cooperativity across a β1-adrenoceptor homodimer may be responsible for generating the low-affinity pharmacology of the secondary β1-adrenoceptor conformation.

KW - Allosterism

KW - Dissociation

KW - GPCR

KW - Receptor dimerization

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U2 - 10.1096/fj.14-265199

DO - 10.1096/fj.14-265199

M3 - Article

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