Modulation of adenosine A(2A) receptor function by interacting proteins. New targets for Huntington’s disease / Modulación de las funciones del receptor A2A de adenosina por interacción con otras proteínas. Nuevas dianas para la enfermedad de Huntington.

Resumen

In this dissertation we studied the pharmacological and functional consequences of adenosine A2A receptor interaction with other proteins, as other neurotransmitter receptores localized in the human brain and an important enzyme regulating the extracellular concentration of adenosine, the ecto-ADA (adenosine desaminase). The first aim of this thesis was to study the molecular and functional interaction of A(2A)Rwith ADA. We found out that A(2A)Racted as a membrane anchoring protein of ADA, more exactly, that ADA bound to A2A receptor homomers and induced a strong modification of their quaternary structure in the way it behaved as a positive allosteric ligand. In addition at the functional level, ADA markedly enhanced A2A receptor signalling, increasing the A2A receptor agonist-induced ERK 1/2 phosphorylation. This powerful regulation of A2A function exerted by ADA might have important implications in the physiology and pharmacology of neuronal A2A receptors that are implicated in the striatal motor regulation. The second aim of this thesis was to search for a compound possibly useful in the treatment Huntington´s disease (among other neurological diseases), concretely a more selective antagonist of A2A receptor for presynaptic A1-A2A receptor heteromers versus postsynaptic A2A-D2 receptor heteromers. Applying in vitro and in vivo approaches, we discovered that the A2A receptor antagonists SCH-442416 showed a presynaptically preferential profile and, on the other hand, the KW-6002 behaved as a postsynaptically preferential A(2A)Rantagonist. Other analysed compounds ZM-241385, MSX-2, SCH-420814, and SCH-58261 showed no clear presynaptic or postsynaptic preference, i.e. presented a mixed profiles. The presynaptic preference of SCH-442416 was due to a strong negative cooperativity induced by the physical presence of dopamine D2 receptor in the A2A-D2 receptor heteromer that was detected by the compound SCH-442416. This cooperativity also indicates that A2A-A2A receptor homodimers are present in the A2A-D2 receptor heteromers. In summary, on the basis of their preferential pre- versus postsynaptic actions, SCH-442416 can be used as a lead compound in the development of antidyskinetic drugs in Huntington’s disease, meanwhile KW-6002 confirms to be possibly beneficial in Parkinson’s disease. The third aim of this thesis consisted in investigation of pharmacological and functional properties of A2A receptors in the A2A-CB1 receptor heteromers and determination whether selective A2A receptor antagonists show different selectivity for A2A receptors or A2A-CB1 receptor heteromers. We observed that adenosine A2A receptor changed its G-protein coupling from stimulatory Gs to inhibitory Gi protein when it formed heteromer with CB1 receptor and a synergistic cross-talk in G-protein activation was observed when both receptors were co-activated. At the same time, we saw that CB1 receptor mainly controled the ERK 1/2 signalling under the A2A-CB1 receptor heteromer. The A2A-CB1 receptor heteromers did not show allosteric effects at the ligand binding level. The two specific A2A receptor antagonist, KW-6002 and VER-7835 lost affinity for A2A receptors when expressed in A2A-CB1 receptor heteromers. This all means that A2A-CB1 receptor heteromers constitute a singular unit for adenosine and cannabinoids signalling, introducing diversity in A2A receptor signalling that can be therapeutically relevant in neurological diseases involving striatal neurons. In summary, the results presented in this Thesis show the importance of GPCR heteromers in the brain striatum and their physiological importance for the treatment of neurological diseases.