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Cell Biology of G-protein-coupled receptors (GPCRs)


Schematic overview of G-protein-coupled
   receptor desensitisation and internalisation The effects of drugs often diminish over time. This "desensitization" phenomenon forms the basis of my research. Specifically, we are studying the desensitization of G-protein-coupled receptors. These proteins are usually localized within the plasma membrane where they sense a wide variety of extracellular signals and transmit them to the cell interior. They are targets for more than 30% of all prescription drugs. Therefore, understanding the mechanisms of their desensitization - and possibly preventing its occurrence - is of paramount interest.

As shown in the scheme, desensitisation, internalisation and recycling of G-protein-coupled receptors are intimately connected. The whole process is initiated by binding of an agonist to the receptor, which triggers a signal (usually via heterotrimeric G-proteins). However, agonist binding also converts the receptor into a substrate for a family of kinases, the G-protein-coupled receptor kinases (GRKs). These kinases phosphorylate only agonist-activated receptors. Subsequently, the phosphorylated receptor becomes a binding partner for arrestins. Arrestins are normally cytosolic proteins, but they recognise agonist-activated, phosphorylated receptors and bind them. This binding makes the receptor inacessible for G-proteins (i.e. the arrestin-bound receptor is desensitised), and it targets the receptor for internalisation. This is because arrestins do not only bind receptors, but they also bind components of clathrin-coated pits. Thus, arrestin-bound receptors move into clathrin-coated pits and are then internalised.

We are using a variety of methods to investigate these processes. We are able to purify most of the participating proteins from either bacteria or insect cells. Thereby we can investigate regulation of these proteins in vitro. Recently we have produced arrestin2 in a new crystal form (PDB entry 2WTR). We also look at binding between proteins in vitro. However, we also perform a range of experiments in living cells. For example, we can determine the internalisation of receptors by ligand binding. We also look at translocation of GFP-tagged proteins by live-cell confocal microscopy (see example below). Furthermore, we investigate protein-protein interactions in single living cells using fluorescence resonance energy transfer (FRET) between CFP- and YFP-tagged interaction partners. Finally, we determine the affinity of GPCR-binding proteins by two-color fluorescence recovery after photobleaching (FRAP).

The image on the right shows the translocation of arrestin3-YFP to the plasma membrane after stimulation of the β2-adrenergic receptor. The left picture was taken before stimulation; the right picture was taken 60 seconds after stimulation. Watch the whole 5-min sequence as Quicktime movie (3 MB).

At the moment, we are particularly interested in the interaction mechanism of arrestins with G-protein-coupled receptors. To address these questions, we use the methods mentioned above and complement them with more classical protein biochemistry. Recently, we could show that the co-internalization of arrestins with some GPCRs can be modulated by altering the affinity of GPCR-arrestin interaction. We could also demonstrate that receptor recycling to the plasma membrane after agonist removal is not related to co-internalization of arrestins with receptors, as previously postulated.

We also collaborate with other laboratories working on the interaction of G-protein-coupled receptors with GRKs and arrestins. These collaborations currently focus on the μ opioid receptor and the receptors for glucagon-like peptide 1 (GLP1) and glucose-dependent insulinotropic polypeptide (GIP).