Volume 11 Supplement 2
The folding interactome of GPCRs
© Bergmayr et al; licensee BioMed Central Ltd. 2011
Published: 5 September 2011
The A2A adenosine receptor is a prototypical G protein-coupled receptor. It is expressed in a wide variety of cells including as different types as neurons, platelets, cells of the immune system and muscle. The A2A receptor has an unusually long C-terminus (of >120 residues), which for the most part is dispensable for coupling to Gs. This C-terminus turned out to be the docking site for other proteins. Using a yeast-2-hybrid screen we have previously identified proteins interacting with the C-terminus including ARNO/cytohesin2, SAP102 and USP4.
To verify these interactions in vivo and to identify new interacting proteins of the A2A adenosine receptor we chose a two-step proteomics approach: we first expressed tagged receptors in HEK293 fibroblasts using various TAP (tandem affinity purification)-tag variants; the differently tagged receptors were analyzed for expression, localization and their pharmacological properties (ligand binding and cAMP accumulation) to identify tags suitable to further analyze the receptor’s interactome. These tagged receptors were then used to optimize the purification and to make the first initial screens using 2D-nano-LC-MS/MS approach. To prove the interaction of the A2A receptor with promising targets found in our screens, biochemical approaches, e.g. co-immunoprecipitation and whole-cell binding, were performed.
Results and conclusions
We could identify two tags suitable for further analysis of the A2A adenosine receptor interactome. Pharmacological properties of the tagged receptors were comparable to the native receptor. However, the tags seemed to retain the receptor to a large extent in the endoplasmic reticulum (ER) and hence we used this system to study the ER/folding interactome of the receptor. LC-MS/MS analysis of the purified ER-trapped version of the receptor revealed proteins putatively involved in the folding of the receptor, such as chaperones. We are currently generating a transgenic mouse-model expressing the TAP-tagged version of the A2A adenosine receptor under the control of its endogenous promotors (homologous knock-in). This will allow us to examine tissue- and development-specific interaction partners of the A2A adenosine receptor utilizing the optimized proteomics approach.
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