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Project area P

P1: Conformational control of GPCRs by functionally selective ligands

A wide variety of diseases is related to malfunction of transmembrane signalling events mediated by conformational changes of GPCRs. Thus, rational drug discovery requires a better understanding for both the structure of ligand-GPCR complexes and the ligand-induced conformational changes leading to individual signalling profiles. Upon activation, GPCRs can undergo a number of conformational changes. Functionally selective ligands are able to preferentially stabilize one or a subset of activated conformations. Using the β2-adrenergic receptor (β2AR) as a model system and employing 19F-NMR spectroscopy, R. Stevens, K. Wüthrich and coworkers recently observed a significant conformational impact of functionally selective ligands during receptor activation.[1]

To better understand the conformational changes upon receptor activation and the molecular origins for functional selectivity, dopamine D2 receptors and site-specifically mutated variants thereof will be purified and solubilized. After treatment with the ligands of interest and site specific labeling with 2,2,2-trifluoroethanethiol, relative populations of active and inactive states will be determined from the 1D 19F-NMR spectra as described before for the β2-adrenergic receptor. A plot of relative peak volumes for diagnostic Cys mutants will represent the populations of the active states. The changes observed by 19F-NMR will provide novel insight into correlations between the chemical structure of the ligands and their interactions with the dopamine D2 receptor. This may lead to a general principle between ligand structure and the specific conformational changes that are necessary for G-protein binding or -arrestin recruitment. Applications to other GPCRs that are relevant for the aims of the RTG are envisioned.

R. Lefkowitz and coworkers developed a quantitative mass spectrometry strategy measuring the reactivity of particular side-chains to undergo structural changes induced by ligands with functionally distinct ligands. Their findings demonstrate significant conformational variability with significant implications for the design of functionally selective adrenergic drugs.

To better understand the conformational changes upon receptor activation of the dopamine receptor subtypes and the molecular origins for functional selectivity, the above mentioned quantitative mass spectrometry strategy measuring the reactivity of particular side-chains to undergo structural changes induced by ligands with functionally distinct ligands. This will provide us with further insight into ligand-specific conformational rearrangements of the dopamine D2-like receptors involving D2long, D2short, D3 and D4.

 

Research stay abroad

The ‘research stay abroad’ will facilitate that the postdoctoral researcher, who will be recruited for this project, can learn how to purify and reconstitute GPCRs in the laboratory of our collaborator Prof. Kobilka (Stanford University).

References:

  1. Liu J., Horst R., Katritch V., Stevens R., Wüthrich K, Biased Signaling Pathways in β2-Adrenergic Receptor Characterized by 19F-NMRScience, 335:1106-1110 (2012), DOI: 10.1126/science.1215802

P2: Mapping GPCR-activation in adherent cell monolayers using imaging SPR

Surface plasmon resonance spectroscopy (SPR) is a surface sensitive experimental technique capable of monitoring changes of the refractive index (RI) within a distance of typically 100 - 200 nm from a gold surface. When cells are grown on such gold surfaces the SPR readout returns very sensitively the integral changes of the RI inside the cells as response to external stimuli like toxins or drugs. The RI-changes have been assigned to dynamic mass redistributions (DMR) inside the cell caused by the stimulant directly or indirectly by the associated signal transduction pathway. It has been shown recently that DMR induced by stimulation of GPCRs can be measured and used as a label-free tool to identify the associated signal transduction pathway by pattern recognition.

A more recent technical development provides the ability to use SPR spectroscopy with microscopic resolution (imaging SPR = iSPR). It is the major objective of this project to develop an imaging SPR setup capable of studying adherent cell monolayers during GPCR-activation with microscopic resolution. The image contrast will rely on DMR associated with GPCR-mediated signal transduction. Thus, iSPR will not integrate over the entire population but reveal the spatial and temporal heterogeneity of receptor activation and downstream cell response without using labels for analysis. From a methodological viewpoint this project is located at the the interface between medicinal, analytical chemistry and optical engineering. It comprises establishment of an imgaing SPR setup, cell culture work and confocal microscopy.