Accessory Proteins in A2A-Receptor Signalling

G protein-coupled receptors are defined by their signalling mechanism, i.e. their ability to interact with and activate heterotrimeric G proteins. However, in many cases the functional properties of these receptors are inconsistent with the current model of G protein-dependent signalling. This indicates that there are must be additional components that impinge on the signalling cascade. In fact, there are several examples where adapter proteins as well as signalling proteins other than G proteins have been shown to bind directly to a receptor. In the working hypothesis underlying the current grant application, we postulate that the complexity of cellular signal transduction can only be understood if the factors that lead to signal integration at the level of the receptor are known. We propose to focus on the A2A-adenosine receptor for the following reasons: (i) targeted deletion of this receptor in mice leads to hypertension, increased platelet aggregation, male aggressiveness and decreased susceptibility to ischaemic brain damage. In addition, the A2A-adenosine receptor has been proposed as a drug target in the treatment of Parkinson`s disease. Hence, the potential clinical relevance of this receptor is obvious. (ii) The A2A-adenosine receptor has an extended carboxy terminus that is enriched in proline residues and that is subject to extensive phosphorylation. (iii) Preliminary experiments indicate that truncation of the carboxy terminus alters the signalling properties of the A2A-adenosine receptor. Our assumptions are as follows: -the extended carboxy terminus of the A2A-adenosine receptor plays an important role in determining the output of the receptor, i.e. both the type of signal (signalling via Gs to cAMP/pro-tein kinase A or via p21ras to MAP kinase) and the level of constitutive (agonist-independent) activity. -This regulatory role of the carboxy terminus results from its interaction with accessory proteins. - The interaction between the A2A-adenosine receptor and the accessory proteins is subject to regulation. There must be mechanisms that allow for modulation of this in this interaction; phosphorylation of the carboxy terminus is one obvious candidate mechanism. The goals of the current project are to verify these conjectures; specifically, we intend (i) to verify the role of the carboxy terminus in specifying the nature of the signal generated by the receptor, (ii) to identify accessory proteins that bind to the carboxy terminus of the A2A-adenosine receptor (iii) and to prove that these proteins participate in regulating the signalling properties of the A2A-adenosine receptor. Thus, the project aims at the generating insights that contribute to understanding the mecha-nisms by which receptors act as signal integrators and coincidence detectors. We hope to develop a model that may also serve as a framework to understand the role of A2A-adenosine receptors and of the accessory proteins in diseases. Finally, it is anticipated that the insights that will be obtained will also be relevant to other (related) receptor.