SAP102 organizes adenosine(2A)-receptor signaling

The A2A-adenosine receptor has become a drug target in the treatment of Parkinson`s disease, psychotic behavior and dementia. In addition, targeted deletion of this receptor in mice leads to hypertension, increased platelet aggregation, male aggressiveness and decreased susceptibility to ischemic brain damage. Hence, the potential clinical relevance of this receptor is obvious. Besides the activation of heterotrimeric G proteins, G protein-coupled receptors interact with additional components that impinge on the signaling cascade. In addition to G protein- coupled kinases and arrestins, there are several examples where known adapter proteins as well as signaling proteins other than G proteins have been shown to bind directly to a G protein-coupled receptor. In a yeast-two hybrid interaction hunt, we previously identified SAP102 ("synapse associated protein of 102 kD")as a novel interaction partner, which also bound to the carboxyl terminal of the A2A-adenosine receptor. These results were already confirmed by direct "pull-down" assays of purified proteins and co-immunoprecipitation. SAP102 belongs to the MAGUK ("membrane associated guanylate kinase-like domain") protein family and was identified as important component of the postsynaptic density. A generally accepted function of MAGUK proteins is the clustering receptors and proteins of the signaling cascade at their postsynaptic sites. In some instances timely expression of different MAGUK proteins regulates synapse development. In the working hypothesis underlying the current grant application, I postulate that (i) the adenosine A2A-receptor interacts specifically with SAP102, (ii) this interaction is crucial for proper targeting of the A 2A-receptor, and (iii) SAP102 influences the mobility of the A2A-receptor. The goals of the present project are to verify these assumptions; specifically, I intend to investigate the basic structural requirements for the interaction and characterize consequences of the interaction on the function of both proteins. Thus, the project aims at generating insights that contribute to understanding the biological relevance of the interaction of MAGUK proteins with a G protein-coupled receptor.

Adenosine A2A-receptors have become drug targets for the treatment of Parkinsons disease, psychotic behavior and dementia. Topical preparations of A2A-selective agonists are tested in clinical trials for the promotion of dermal wound healing and for suppression of asthma, because receptor activation stimulates endothelial cell proliferation and suppresses proinflammatory signals in macrophages and T-cells. The potential clinical relevance of this receptor in various diseases is obvious, which makes it important to fully understand the signaling properties of these receptors. Besides the activation of heterotrimeric G proteins, these receptors interact with additional components that impinge on the signaling cascade. In addition to G protein-coupled kinases and arrestins, there are several examples where known adapter proteins as well as signaling proteins other than G proteins have been shown to bind directly to a G protein-coupled receptor. We found SAP102 (synapse associated protein of 102 kD) as a novel interaction partner for the A2A-adenosine receptor, which binds to its intracellular carboxyl terminal. SAP102 belongs to the MAGUK (membrane associated guanylate kinase-like domain) protein family and was previously identified as an important component of the postsynaptic density. A generally accepted function of MAGUK proteins is the clustering of receptors and proteins of the signaling cascade at their postsynaptic sites. In our working hypothesis we postulated that SAP102 influences the mobility of the A2A-receptor. We established the method of single particle tracking of quantum dot-labeled receptors and using this method we were able to demonstrate that in primary neuronal cells the A2A-receptor shuttles between two diffusion states. Agonist activation reduced the transition between the two states and promoted the accumulation of receptors in a compartment with slow mobility, an area consistent with the dimensions of a lipid raft. However, overexpression of SAP102 prevented access of the receptors to this compartment with restricted mobility. We also identified the basic structural requirements for the interaction on both proteins. Our observations revealed that the hydrophobic core per se does not fully account for the agonist-promoted change in mobility of the A2A-receptor. In addition, the extended carboxyl terminus allows for regulatory input by scaffolding molecules such as SAP102 affecting receptor localization. Especially in nerve cells the regulation of receptor localization has major effects on its signaling properties.