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A pocket guide to GPCRs

PSI-SGKB [doi:10.1038/fa_psisgkb.2008.16]
Featured Article - December 2008
Short description: The structure of the G-protein-coupled receptor adenosine receptor A2A reveals differences between its ligand-binding site and those of other family members, and begins to explain this family's diversity.Science, doi:10.1126/science.1164772

Structure of the human adenosine A2a receptor bound with the potential Parkinson's drug ZM241385 (blue). Four disulfide bridges (yellow) form a complex network to shape the extracellular loops (green) and binding site region. Image courtesy of the Stevens Laboratory, The Scripps Research Institute. (PDB 3EML)

G-protein-coupled receptors (GPCRs) are implicated in numerous diseases and are the target of many drug treatments. Obtaining structures of these receptors has been a challenge, and is still proving to be difficult. Until recently, of the almost 1,000 proteins in this family, only two crystallographic structures had been solved.

Now, a third family member has been added. Jaakola et al., part of the NIH Roadmap center JCIMPT and PSI center ATCG3D, in collaboration with colleagues from the Leiden/Amsterdam Center for Drug Research, have elucidated the structure of the human adenosine A2A receptor.

The adenosine A2A receptor is a class A GPCR, and is important for neurotransmission, coronary blood flow and respiration. It is blocked by caffeine, and is the subject of much research after epidemiological evidence suggested that coffee drinkers have a lower risk of Parkinson's disease. Selective compounds are likely to be useful for the treatment of pain, Parkinson's disease, Huntington's disease and asthma.

Crystals of GPCRs, in general, are difficult to obtain because of the receptors' flexibility and because of their structural heterogeneity. The structures previously solved, rhodopsin and the human β2 adrenergic receptors, are among the most studied GPCRs. The adenosine A2A receptor, similar to the β2-adrenergic receptor, is rapidly denatured upon concentration without the presence of an inverse agonist or antagonist.

The strategy

To solve the problem of the adenosine A2A receptor's instability and to increase the chances of obtaining crystals, Jaakola et al. used a T4L fusion strategy. For this, they replaced the third cytoplasmic loop (Leu209–Ala221) of the receptor with lysozyme from T4 bacteriophage, deleted the C-terminal tail, and conducted all structural studies in a cholesterol-enriched lipidic cubic phase (LCP). Multiple technologies had to be developed to successfully observe, extract and collect X-ray diffraction data on the delicate and tiny crystalline samples.

The resulting receptor was also stabilized during purification by adding sodium chloride and a saturating concentration of a non-specific adenosine receptor antagonist, which was swapped for its specific antagonist ZM241385 at the last step. In addition, cholesteryl hemisuccinate was present throughout purification. Comparison of the binding properties of the fusion construct with the wild-type receptor confirmed that the antagonist binds with similar affinity to both receptors.

Using the T4L fusion protein, the team obtained the crystal structure of the human adenosine A2A receptor in complex with a high-affinity A2A-selective antagonist ZM241385 at 2.6 Å resolution. It reveals three features that are distinct from those of previously reported GPCR structures and that explain the selectivity of this receptor.

Family differences

The first discrepancy is that the antagonist ZM241385 binds to A2A in an extended conformation perpendicular to the plane of the membrane. This is different from predictions from earlier modeling studies, based on the structures of known GPCR structures, that docked ZM241835 in a binding site that was like that of β2-adrenergic receptors and rhodopsin.

The subtle differences in helical positions and orientations relative to rhodopsin and the β2-adrenergic receptors created this new antagonist-binding cavity, and show an important role for extracellular loops and helical core in ligand recognition.

The second difference is in the extracellular loop organization. That of the adenosine A2A receptor is mainly a spatially constrained random coil, whereas rhodopsin and β-adrenergic receptors have some secondary structure elements such as a β-sheet and α-helix.

The third discrepancy is that the antagonist for adenosine A2A restricts the movement of a tryptophan residue important for activating all three GPCR receptors. This tryptophan is thought to act as a 'toggle switch', and Jaakola et al. suggest that ZM241385 prevents structural rearrangements needed for activation of adenosine A2A, thus locking it in the inactive/resting state.

Overall, this structure suggests that there is no general, family-conserved receptor-binding pocket for GPCRs. Instead, the pocket varies in position and orientation, which would give rise to diverse receptors and enhance ligand selectivity.

Maria Hodges


  1. Veli-Pekka Jaakola, Mark T. Griffith, Michael A. Hanson, Vadim Cherezov, Ellen Y. T. Chien et al. The 2.6 Å crystal structure of a human A2A adenosine receptor bound to an antagonist.
    Science (2 October 2008). doi:10.1126/science.1164772

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