Ligand-induced conformational changes in a SMALP-encapsulated GPCR.

https://doi.org/10.1016/j.bbamem.2020.183235Get rights and content
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Highlights

  • Conformational changes in the A2AR monitored in a nano-scale membrane disc (SMALP).

  • Profile of phospholipids in A2AR-SMALP similar to the plasma membrane.

  • A partially-active conformation of A2AR is supported in a SMALP.

  • Inverse agonist induced dose-dependent conformational transitions in A2AR-SMALP.

  • In contrast to inverse agonist, agonist induced only small conformational changes.

Abstract

The adenosine 2A receptor (A2AR), a G-protein-coupled receptor (GPCR), was solubilised and purified encapsulated in styrene maleic acid lipid particles (SMALPs). The purified A2AR-SMALP was associated with phospholipids characteristic of the plasma membrane of Pichia pastoris, the host used for its expression, confirming that the A2AR-SMALP encapsulated native lipids. The fluorescence spectrum of the A2AR-SMALP showed a characteristic broad emission peak at 330 nm, produced by endogenous Trp residues. The inverse agonist ZM241385 caused 30% increase in fluorescence emission, unusually accompanied by a red-shift in the emission wavelength. The emission spectrum also showed sub-peaks at 321 nm, 335 nm and 350 nm, indicating that individual Trp inhabited different environments following ZM241385 addition. There was no effect of the agonist NECA on the A2AR-SMALP fluorescence spectrum. Substitution of two Trp residues by Tyr suggested that ZM241385 affected the environment and mobility of Trp2466.48 in TM6 and Trp2687.33 at the extracellular face of TM7, causing transition to a more hydrophobic environment. The fluorescent moiety IAEDANS was site-specifically introduced at the intracellular end of TM6 (residue 2316.33) to report on the dynamic cytoplasmic face of the A2AR. The inverse agonist ZM241385 caused a concentration-dependent increase in fluorescence emission as the IAEDANS moved to a more hydrophobic environment, consistent with closing the G-protein binding crevice. NECA generated only 30% of the effect of ZM241385. This study provides insight into the SMALP environment; encapsulation supported constitutive activity of the A2AR and ZM241385-induced conformational transitions but the agonist NECA generated only small effects.

Abbreviations

A2AR
adenosine A2A receptor
DMPC
1,2-dimyristoyl-sn-glycero-3-phosphocholine
ECL
extracellular loop
GPCR
G-protein-coupled receptor
IAEDANS
5-({2-[(iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid
ICL
intracellular loop
LC-MS/MS
liquid chromatography with tandem mass spectrometry
m/z
mass to charge ratio
PC
phosphatidycholine
PE
phosphatidylethanolamine
PG
phosphatidyglycerol
PS
phosphatidylserine
SMA
styrene maleic acid
SMALP
styrene maleic acid lipid particle
ZM241385
{4-(2-[7-amino-2-(furan-2-yl)[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-yl amino]ethyl)phenol}

Keywords

GPCR
SMALP
Adenosine receptor
Fluorescence

Cited by (0)

1

Co-first authors.