A2A and A2B adenosine receptors: The extracellular loop 2 determines high (A2A) or low affinity (A2B) for adenosine

Elisabetta  De Filippo; Sonja  Hinz; Veronica  Pellizzari; Giuseppe Deganutti; Ali  El-Tayeb; Gemma  Navarro; Rafael  Franco; Stefano Moro; Anke C.  Schiedel; Christa E.  Müller

doi:10.1016/j.bcp.2019.113718

A2A and A2B adenosine receptors: The extracellular loop 2 determines high (A2A) or low affinity (A2B) for adenosine

Elisabetta De Filippo
, Sonja Hinz
, Veronica Pellizzari
, Giuseppe Deganutti
, Ali El-Tayeb
, Gemma Navarro
, Rafael Franco
, Stefano Moro
, Anke C. Schiedel
, Christa E. Müller

University of Bonn
Università di Padova
Universitat de Barcelona
Centro de Investigación en Red, Enfermedades Neurodegenerativas

Research output: Contribution to journal › Article › peer-review

35 Citations (Scopus)

Abstract

A2A and A2B adenosine receptors (ARs) are closely related G protein-coupled receptor subtypes, which represent important (potential) drug targets. Despite their almost identical binding sites for adenosine, A2AARs are activated by low (nanomolar) adenosine concentrations, while A2BARs require micromolar concentrations. In the present study, we exchanged the extracellular loop 2 (ECL2) of the human A2AAR for that of the A2BAR. The resulting chimeric A2A(ECL2-A2B)AR was investigated in radioligand binding and cAMP accumulation assays in comparison to the wildtype A2AAR. While the ribose-modified adenosine analog N-ethylcarboxamidoadenosine (NECA) and its 2-substituted derivative CGS-21680 did not exhibit significant changes, adenosine showed dramatically reduced potency and affinity for the A2A(ECL2-A2B)AR mutant displaying similarly low potency as for the wt A2BAR. Supervised molecular dynamics simulation studies predicted a meta-binding site with high affinity for adenosine, but not for NECA, which may contribute to the observed effects.

Original language	English
Article number	113718
Number of pages	14
Journal	Biochemical Pharmacology
Volume	172
Early online date	18 Nov 2019
DOIs	https://doi.org/10.1016/j.bcp.2019.113718
Publication status	Published - Feb 2020
Externally published	Yes

Funder

E.D.F., A.C.S., and C.E.M. were supported by the German Federal Ministery of Education and Research (BMBF project Bonn International Graduate School in Drug Sciences (BIGS DrugS)) and by the State of North-Rhine Westfalia (NRW International Research Graduate SchoolBIOTECH-PHARMA). [...] R.F. was supported by a grant from the Spanish Ministerio de Ciencia, Innovación y Universidades (# 2019_RTI2018-094204-B-I00; it may include EU FEDER funds). G.N. was supported by a grant from the Spanish Ministerio de Economía y Competitividad (MINECO #SAF2017-84117-R; it may include EU FEDER funds).

Funding

E.D.F., A.C.S., and C.E.M. were supported by the German Federal Ministery of Education and Research (BMBF project Bonn International Graduate School in Drug Sciences (BIGS DrugS)) and by the State of North-Rhine Westfalia (NRW International Research Graduate SchoolBIOTECH-PHARMA). [...] R.F. was supported by a grant from the Spanish Ministerio de Ciencia, Innovación y Universidades (# 2019_RTI2018-094204-B-I00; it may include EU FEDER funds). G.N. was supported by a grant from the Spanish Ministerio de Economía y Competitividad (MINECO #SAF2017-84117-R; it may include EU FEDER funds).

Funders	Funder number
Federal Ministry of Education and Research
State of North-Rhine Westfalia
Ministry of Science, Innovation and Universities	2019_RTI2018 -094204-B-I00
European Regional Development Fund
Ministerio de Economía y Competitividad	SAF2017-84117-R

Keywords

Adenosine
Chimeric receptor
Extracellular loop
GPCR
Radioligand binding
cAMP

ASJC Scopus subject areas

Biochemistry
Pharmacology

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10.1016/j.bcp.2019.113718

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title = "A2A and A2B adenosine receptors: The extracellular loop 2 determines high (A2A) or low affinity (A2B) for adenosine",

abstract = "A2A and A2B adenosine receptors (ARs) are closely related G protein-coupled receptor subtypes, which represent important (potential) drug targets. Despite their almost identical binding sites for adenosine, A2AARs are activated by low (nanomolar) adenosine concentrations, while A2BARs require micromolar concentrations. In the present study, we exchanged the extracellular loop 2 (ECL2) of the human A2AAR for that of the A2BAR. The resulting chimeric A2A(ECL2-A2B)AR was investigated in radioligand binding and cAMP accumulation assays in comparison to the wildtype A2AAR. While the ribose-modified adenosine analog N-ethylcarboxamidoadenosine (NECA) and its 2-substituted derivative CGS-21680 did not exhibit significant changes, adenosine showed dramatically reduced potency and affinity for the A2A(ECL2-A2B)AR mutant displaying similarly low potency as for the wt A2BAR. Supervised molecular dynamics simulation studies predicted a meta-binding site with high affinity for adenosine, but not for NECA, which may contribute to the observed effects.",

keywords = "Adenosine, Chimeric receptor, Extracellular loop, GPCR, Radioligand binding, cAMP",

author = "\{De Filippo\}, Elisabetta and Sonja Hinz and Veronica Pellizzari and Giuseppe Deganutti and Ali El-Tayeb and Gemma Navarro and Rafael Franco and Stefano Moro and Schiedel, \{Anke C.\} and M{\"u}ller, \{Christa E.\}",

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T2 - The extracellular loop 2 determines high (A2A) or low affinity (A2B) for adenosine

AU - De Filippo, Elisabetta

AU - Hinz, Sonja

AU - Pellizzari, Veronica

AU - Deganutti, Giuseppe

AU - El-Tayeb, Ali

AU - Navarro, Gemma

AU - Franco, Rafael

AU - Moro, Stefano

AU - Schiedel, Anke C.

AU - Müller, Christa E.

PY - 2020/2

Y1 - 2020/2

N2 - A2A and A2B adenosine receptors (ARs) are closely related G protein-coupled receptor subtypes, which represent important (potential) drug targets. Despite their almost identical binding sites for adenosine, A2AARs are activated by low (nanomolar) adenosine concentrations, while A2BARs require micromolar concentrations. In the present study, we exchanged the extracellular loop 2 (ECL2) of the human A2AAR for that of the A2BAR. The resulting chimeric A2A(ECL2-A2B)AR was investigated in radioligand binding and cAMP accumulation assays in comparison to the wildtype A2AAR. While the ribose-modified adenosine analog N-ethylcarboxamidoadenosine (NECA) and its 2-substituted derivative CGS-21680 did not exhibit significant changes, adenosine showed dramatically reduced potency and affinity for the A2A(ECL2-A2B)AR mutant displaying similarly low potency as for the wt A2BAR. Supervised molecular dynamics simulation studies predicted a meta-binding site with high affinity for adenosine, but not for NECA, which may contribute to the observed effects.

AB - A2A and A2B adenosine receptors (ARs) are closely related G protein-coupled receptor subtypes, which represent important (potential) drug targets. Despite their almost identical binding sites for adenosine, A2AARs are activated by low (nanomolar) adenosine concentrations, while A2BARs require micromolar concentrations. In the present study, we exchanged the extracellular loop 2 (ECL2) of the human A2AAR for that of the A2BAR. The resulting chimeric A2A(ECL2-A2B)AR was investigated in radioligand binding and cAMP accumulation assays in comparison to the wildtype A2AAR. While the ribose-modified adenosine analog N-ethylcarboxamidoadenosine (NECA) and its 2-substituted derivative CGS-21680 did not exhibit significant changes, adenosine showed dramatically reduced potency and affinity for the A2A(ECL2-A2B)AR mutant displaying similarly low potency as for the wt A2BAR. Supervised molecular dynamics simulation studies predicted a meta-binding site with high affinity for adenosine, but not for NECA, which may contribute to the observed effects.

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KW - Chimeric receptor

KW - Extracellular loop

KW - GPCR

KW - Radioligand binding

KW - cAMP

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ER -

A2A and A2B adenosine receptors: The extracellular loop 2 determines high (A2A) or low affinity (A2B) for adenosine

Abstract

Funder

Funding

Keywords

ASJC Scopus subject areas

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