Interaction of ARNO/cytohesin2 with adenosine-2A receptors

G protein-coupled receptors are defined by their signaling mechanism, i.e. their ability to interact with and activate heterotrimeric G proteins. Besides 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 have used the intracellular carboxy terminal tail of the of the A 2A-adenosine receptor as bait in a yeast-two hybrid interaction screen for the following reasons. The A 2A-adenosine receptor has been proposed as a drug target in the treatment of Parkinson`s disease, psychotic disorder, Alzheimer`s disease etc. In addition, targeted deletion of this receptor in mice leads to hypertension, increased platelet aggregation, male aggressiveness and decreased susceptibility to ischaemic brain damage. Hence, the potential clinical relevance of this receptor is obvious. One of the proteins directly interacting with the A 2A-adenosine receptor was identified as ARNO/cytohesin-2. ARNO/cytohesin-2 was discovered as a nucleotide exchange factor for the small (monomeric) G proteins of the ADP-ribosylation factor (ARF) family, (while ARFs were originally identified as cofactors for cholera toxin-mediated ADP-ribosylation of Ga s ). In the working hypothesis underlying the current grant application, we postulate that (i) there is a direct interaction between the A2A-receptor and ARNO/cytohesin-2, (ii) that ARNO/cytohesin-2 affects the signaling properties and/or the cycle of desensitization and resensitization of the A2A-adenosine receptor and (iii) that the A2A- adenosine receptor regulates (i.e. stimulates or inhibits) the function of ARNO. The goals of the present project are to verify these assumptions; specifically we intend to investigate the structural requirements for the interaction and characterize pharmacological properties influencing the interaction and the function of both proteins. Thus, the project aims at generating insights that contribute to understanding the molecular mechanisms by which A 2A- adenosine receptors interact with ARNO/cytohesin-2. It is anticipated that the insights that will be obtained will also be relevant to other (related) receptors.

Adenosine receptors are involved in controlling a multitude of physiological and pathological processes. Thus, they are considered to be a very interesting target for the development of novel drugs. So far only adenosine itself is used in the acute treatment of tachycardia. It has been found that the A2A-subtype of the adenosine receptors has an important role in the pathogenesis of Parkinson`s disease, psychotic disorder, Alzheimer`s disease. Various antagonists of the A2A-adenosine receptor are currently tested in clinical trials. In addition, targeted deletion of this receptor subtype in mice leads to hypertension, increased platelet aggregation, male aggressiveness and decreased susceptibility to ischaemic brain damage. Hence, the potential clinical relevance of this receptor is obvious. G protein-coupled receptors like the A2A-adenosine receptor are defined by their ability to interact with and activate heterotrimeric G proteins. Besides 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 were able to discover at least eight novel interaction partners for the A 2A-adenosine receptor. One of the proteins directly interacting with the A 2A-adenosine receptor was identified as ARNO/cytohesin-2. ARNO/cytohesin-2 was discovered as a nucleotide exchange factor for the small (monomeric) G proteins of the ADP-ribosylation factor (ARF) family, (while ARFs were originally identified as cofactors for cholera toxin-mediated ADP-ribosylation of Ga s ). In the course of the project we proved the direct interaction between the two proteins using a variety of different methods. We also identified the parts of the proteins, which are necessary for this interaction. The most important finding of the project is that the presence of ARNO/cytohesin-2 is required to support the alternative signaling pathway of the A2A-adenosine receptor. This alternative signaling pathway leads to the activation of the mitogen-activated protein kinase and subsequently to the transcription of genes responsible for cell proliferation. The project generated novel insights into the molecular mechanisms of A2A-adenosine receptor signaling. In addition, we were able to shed new light on the diverse mechanisms that support cross-talk between G-protein coupled receptors and mitogen-activated protein kinase dependent signaling.