The protein interactome of the A2A-adenosine receptor

The A2A-adenosine receptor is a prototypical Gs -coupled receptor. However, the A2A-receptor has several structural and functional characteristics that make it unique. In contrast to the classical model of collision coupling described for the ß-adrenergic receptors, the A2A-receptor couples to adenylyl cyclase by restricted collision coupling and forms a tight complex with Gs . The mechanistic basis for this is not clear. The A 2A-receptor has a long C-terminus (of > 120 residues), which is for the most part dispensable for coupling to G s . It was originally viewed as the docking site for kinases and the ß-arrestins, which initiate receptor desensitization and endocytosis. The A2A-receptor is, however, fairly resistant to agonist-induced internalization. Recently, the C-terminus has also been appreciated as a binding site for several additional "accessory" proteins. Established interaction partners include a-actinin, ARNO, USP4 and translin-associated protein-X. In addition, the A2A-receptor has also been reported to form a heteromeric complex with the D2 -dopamine receptor and the metabotropic glutamate receptor- 5. It is clear that (i) this list cannot be exhaustive and (ii) that all these proteins cannot bind simultaneously to the receptor. The challenging, but very important task is to document which of all these interactions do occur in the living organisms. Here we propose to meet this challenge by characterizing the interactome of the A 2A-receptor by using a two-step proteomics approach; i.e., the receptor will first be expressed in tagged versions in cells, which can be differentiated into neurons. This will allow for optimization of the conditions and for an initial survey of interaction partners; subsequently, we intend to generate mice, in which the receptor has been engineered to afford the isolation of receptor-complexes from native tissues, in particular the striatum. It is anticipated that this approach will allow for testing which complexes are preferentially formed under specific conditions: agonist treatment may, for instance, result in the sequential interaction with different signaling molecules and cause the assembly of large complexes that have been referred to as signalosomes. The nature of this elusive signalosomes may be explored by our approach and their composition defined. Similarly, antagonists are likely to facilitate folding and ER export of the A 2A-adenosine receptor. Accordingly, a differential analysis of interacting proteins, will also allow identifying ER chaperones, which assist in folding and ER export of the protein. To the best of our knowledge, the proposed strategy has not yet been pursued with other G protein-coupled receptors. Thus, if successful, it may set a precedent and provide the proof-of-principle for the entire family of GPCRs. At the very least, the current approach provides the `acid test` for all purported interactors of the A 2A- receptor.