Adenosine A2A receptors : characterization, localization, G protein coupling and interactions with dopamine D2 receptors

University dissertation from Stockholm : Karolinska Institutet, Department of Physiology and Pharmacology

Abstract: Adenosine not only has a role in cellular energy metabolism but is recognized as an intercellular messenger with an important function in physiology and pathophysiology. It exerts its effects via a family of G protein coupled receptors of which four members have been cloned from several species: A, A2A, A2B and A3. For the first three receptors, sequence homology between species is high. However, it is known that there are significant differences in pharmacology between species homologues of A, adenosine receptors despite only minor differences in amino acid composition. It was therefore important to determine to what extent functional data obtained in rats can be extrapolated to man. All of the human adenosine receptor subtypes were stably expressed in Chinese hamster ovary (CHO) cells and their pharmacological profiles were characterized using radioligand binding studies and functional assays. The human A2A receptor was also characterized using a novel A2A receptor antagonist and compared with the rat A2A receptor expressed in PC12 cells using radioligand binding studies. In addition, cyclic AMP accumulation in intact cells was used as a functional assay. In general, the potencies and affinities detemined for the selected agonists and antagonists exhibit the typical pharmacology of the respective subtypes, with some species-specific characteristics. These differences were more marked when functional assays (cAMP accumulation and adenylyl cyclase activity) were used than in radioligand binding assays. Using double labeling with riboprobes it was established that the A2A receptors are predominantly expressed in striatum, where they are co-localized with dopamine D2 receptors in striatal mediumsized neurons. This is the morphological basis for behavioral, functional and biochemical data showing an antagonistic interaction between the two receptors in the brain. To further characterize the interactions between A2A receptors and D2 receptors we have co-transfected these receptors in two different cell lines. The interaction can occur at several different levels. First, we confirm the finding that activation of A2A receptors decreases the affinity of dopamine to D2 receptors in binding experiments. Secondly, we showed that the ability of D2 agonists to counteract A2A receptor mediated responses may be quantitatively more important. The present study also showed that the addition of GTP significantly increased antagonist radioligand binding at A, receptors, both in whole-hemisphere sections from human brain and in membranes from CHO cells expressing human A, receptors, probably by decreasing cryptic binding of endogenous adenosine. No such effect was detected for an antagonist radioligand at human A2A receptors. There were differences in the density of the two receptors in rat and human brain that appeared related to differences in the affinity of the endogenous agonist. Finally, it is shown directly that adenosine A2A receptor mRNA and Golf mRNA are co-localized in striatal medium-sized neurons to a much higher extent than with Gs mRNA. Using a photoaffinity labeling method we show that A2A receptors are not only co-localized with Golf in striatum, but also functionally coupled to it. Thus, A,, receptors may activate Golf rather than G. in striatum. In conclusion, these studies emphasize the importance of examining receptors from the appropriate species, and they also underline the importance of using functional assays as adjuncts to binding assays when examining agonists. The ability of dopamine to inhibit tonic activation of A2A receptors may be at least as important as the ability of adenosine to alter the activity of dopamine on D2 receptors. A2A receptors may couple to different G proteins in different locations.

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