title: A purified C-terminally truncated human adenosine A(2A) receptor construct is functionally stable and degradation resistant.
abstract: Recent high resolution structures of modified G-protein coupled receptors (GPCRs) have provided major insight into the mechanisms of receptor-ligand binding. However understanding of the complete mechanism of GPCR function remains limited. This study characterised C-terminally truncated versions of the human adenosine A(2A) receptor (A(2A)R) with a view to producing protein suitable for structural studies. The constructs terminated at residue A316, removing the intracellular C-terminal tail, or V334, producing a C-terminal tail equivalent in length to that of rhodopsin. Higher levels of functional receptor before and after solubilisation were obtained for both C-terminally truncated constructs compared to the wild-type receptor (WT) as assessed by radioligand binding analysis using [(3)H]ZM241385. The construct which yielded the highest level of functional receptor, V334 A(2A)R, was purified in DDM to high homogeneity with a final yield of 2 mg/L. Binding analysis revealed that the purified receptor had a specific activity of 20.2+/-1.2 nmol/mg, close to the theoretical maximum. Pure V334 A(2A)R was resistant to degradation over 15 days when stored at 4 degrees C or 20 degrees C and showed remarkable functional stability when stored at 4 degrees C, retaining 84% of initial functionality after 30 days. This construct is an excellent candidate for structural studies.
authors.affiliation: Membrane Protein Crystallography Group, Division of Molecular Biosciences, Imperial College London, South Kensington, London, SW7 2AZ, UK.
authors.forename: Shweta
authors.initials: S
authors.lastname: Singh
authors.forename: Diana
authors.initials: D
authors.lastname: Hedley
authors.forename: Elodie
authors.initials: E
authors.lastname: Kara
authors.forename: Adrien
authors.initials: A
authors.lastname: Gras
authors.forename: So
authors.initials: S
authors.lastname: Iwata
authors.forename: Jonathan
authors.initials: J
authors.lastname: Ruprecht
authors.forename: Philip G
authors.initials: PG
authors.lastname: Strange
authors.forename: Bernadette
authors.initials: B
authors.lastname: Byrne
mesh_terms: D002460:Cell Line; D002788:Cholesterol Esters / Q000378:metabolism; D003902:Detergents; D015870:Gene Expression; D006801:Humans; D055550:Protein Stability; D043705:Receptor, Adenosine A2A / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D012995:Solubility
chemical_list: D002788:Cholesterol Esters; D003902:Detergents; D043705:Receptor, Adenosine A2A; C013440:cholesteryl succinate