Regulatin of the A2A - Adenosinreceptor by the Ubiquitin Specific ProteaseUSP4 and Astrin: Two Novel Proteins with Potential Importance in the Biology ofr the A2A receptor

The A2A-adenosine receptor is a pharmacological target in the central nervous system and the vasculature. Targeted deletion of the receptor gene causes behavioral defects in mice including anxiety and psychomotor hyperactivity, increased blood pressure and platelet aggregation. We found that truncation of the A2A-receptor carboxyl terminus leads to altered receptor regulation in cell culture. (i) Degradation of the receptor by the proteasome is abolished and (ii) subcellular localization in nerve cells is different from that of the full-length receptor. Our working hypothesis is that the truncated receptor fails to interact with USP4, a ubiquitin-specific protease, and with the microtubule binding protein, astrin, respectively. In the proposal I present evidence that (i) the receptor becomes ubiquitinated and that USP4 prevents receptor ubiquitination. USP4 activity rescues the receptor from degradation and increases the proportion of functional receptors bound to the plasma membrane. (ii) If the interaction with astrin is disrupted receptor distribution to neurites becomes inefficient and the receptor is restricted to the base of nerve cell extensions. Thus, I propose to examine the mode of action by which USP4 and astrin impinge on the regulation of the A 2A- adenosine receptor. In addition, the recognition sites for USP4 and astrin, respectively, in the receptor carboxyl terminus will be defined in order to generate receptor mutants with minimal structural alterations which are not recognized by these accessory proteins. For a definitive evaluation I propose to generate mice in which the wild-type receptor is replaced by the mutant forms. The mice will be subjected to behavioral and cardiovascular tests and this will be complemented by characterizing receptor localization and regulation in isolated tissue. The projected work is intended to clarify the basis of A 2A-adenosine receptor regulation in vivo. It is surmised that in addition, it will be possible to make more general inferences on the substrate recognition properties of USP4 and on the targeting function of astrin in nerve cells.

The A2A-adenosine receptor is a pharmacological target in the central nervous system and the vasculature. Targeted deletion of the receptor gene causes behavioral defects in mice including anxiety and psychomotor hyperactivity, increased blood pressure and platelet aggregation. We found that truncation of the A 2A-receptor carboxyl terminus leads to altered receptor regulation in cell culture. (i) Degradation of the receptor by the proteasome is abolished and (ii) subcellular localization in nerve cells is different from that of the full-length receptor. Our working hypothesis is that the truncated receptor fails to interact with USP4, a ubiquitin-specific protease, and with the microtubule binding protein, astrin, respectively. In the proposal I present evidence that (i) the receptor becomes ubiquitinated and that USP4 prevents receptor ubiquitination. USP4 activity rescues the receptor from degradation and increases the proportion of functional receptors bound to the plasma membrane. (ii) If the interaction with astrin is disrupted receptor distribution to neurites becomes inefficient and the receptor is restricted to the base of nerve cell extensions. Thus, I propose to examine the mode of action by which USP4 and astrin impinge on the regulation of the A2A-adenosine receptor. In addition, the recognition sites for USP4 and astrin, respectively, in the receptor carboxyl terminus will be defined in order to generate receptor mutants with minimal structural alterations which are not recognized by these accessory proteins. For a definitive evaluation I propose to generate mice in which the wild-type receptor is replaced by the mutant forms. The mice will be subjected to behavioral and cardiovascular tests and this will be complemented by characterizing receptor localization and regulation in isolated tissue. The projected work is intended to clarify the basis of A2A-adenosine receptor regulation in vivo. It is surmised that in addition, it will be possible to make more general inferences on the substrate recognition properties of USP4 and on the targeting function of astrin in nerve cells.