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Crystallization and structure determination of adrenergic G protein-coupled receptor subtypes

Final Report Summary - ADRENERGIC RECEPTORS (Crystallization and structure determination of adrenergic G protein-coupled receptor subtypes)

Adrenergic receptors (ARs) play central physiological roles by mediating the effects of the neurotransmitter noradrenaline and the hormone adrenaline. They are important drug targets, e.g. to treat hypertension and asthma. In humans, there are nine subtypes of ARs with different functions and pharmacology. In some ARs so called biased ligands can selectively activate specific downstream signaling pathways [1]. Structural studies of ARs are required to elucidate the molecular basis for their distinct pharmacological properties and to promote structure-based drug design, which will help to discover better drugs with fewer side effects. So far only the structure of the human beta2AR has been determined to high resolution [2]. In order to determine the crystal structures of additional subtypes of ARs we are engineering stabilized receptor constructs by introducing thermostabilizing point mutations and by fusing small and stable soluble proteins like T4 lysozyme to the receptor. To stabilize the human beta1AR we are applying a similar strategy as it was previously successfully developed for the homologous turkey beta1AR [3]. A construct of the human beta1AR with several thermostabilizing point mutations and truncations of flexible regions has been expressed in HEK293T and insect cells. Unlike the detergent-sensitive wild type human beta1AR, the stabilized receptor can be efficiently solubilized in an active form (measured by ligand binding) with a mild detergent (dodecyl maltoside).
We have successfully purified the stabilized human beta1AR by immobilized metal affinity and ligand affinity chromatography. With a thermal denaturation assay we demonstrate excellent stability characteristics ideal for crystallization. In addition, we are also exploring the possibility to stabilize all remaining AR subtypes by replacing the flexible third intracellular loop by T4 lysozyme. Crystallization experiments are ongoing with the human beta1AR. Meanwhile, we have determined the structure of the turkey beta1AR with a beta2-selective antagonist, which provides insight into the structural basis of ligand selectivity. In collaboration with internal and external partners, we are also using NMR spectroscopy to study the dynamical changes of the beta1ARs upon ligand binding.

[1] Galandrin, S. and Bouvier, M. (2006) Mol. Pharmacol. 70, 1575–1584.
[2] Rosenbaum, D.M. et al. (2007) Science 318, 1266–1273.
[3] Warne, T., Serrano-Vega, M.J. Tate, C.G. and Schertler, G.F.X. (2009) Protein Expr. Purif. 65, 204–213.