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ERC

EMPSI Report Summary

Project ID: 339995
Funded under: FP7-IDEAS-ERC
Country: United Kingdom

Mid-Term Report Summary - EMPSI (Receptors, Channels and Transporters:Development and Application of Novel Technologies for Structure Determination)

The overall aim of the project is a deeper understanding of the structure and function of G protein-coupled receptors through a combination of structural biology and high-throughput screening of receptor mutants for all functionalities (folding, stability, antagonist binding, agonist binding, G protein-coupling, arrestin coupling, basal activity). In the first two years of the project we have achieved a number of milestones towards these goals.

The structure of the adenosine A2A receptor bound to an agonist and an engineered G protein (mini-Gs) was determined by x-ray crystallography. The structure showed that the binding of the G protein resulted in only the rotamer change of 3 side chains of amino acid residues in the receptor and the bending of helix 6, which also resulted in the inward displacement of helix 5. This is in contrast to the large conformation changes throughout the receptor, characterised by helix rotations of helices 5,6 and 7, which result from agonist binding. Binding of the mini-Gs had little effect on the agonist binding site, suggesting that the structure of the receptor with only agonist bound represents the high affinity state.

A comparison of the structural changes in a peptide receptor is highly desirable, so we are working towards the structure of the angiotensin receptor bound to agonists, either with or without G protein bound. We have thermostabilised the receptor in the agonist bound state, which we anticipate will represent the intermediate active state of the adenosine A2A receptor. In addition, binding of mini-Gq significantly thermostabilises the fully active conformation of the receptor. Both conformations of the receptor are being purified for crystallisation.

A cell sorting strategy has been developed for identifying mammalian cells expressing higher levels of expression of specific mutants of a membrane protein from a library of mutants. A number of technical hurdles had to be overcome before this methodology worked robustly.

Contact

Lisa Fields, (External Funding Officer)
Tel.: +44 1223 267201
E-mail
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