Identification and characterization of Adenosine A(2A) heteromers in the CNS = Identificació i caracterització d’heteròmers d’Adenosina A2A al SNC

The aims of this thesis are: Aim 1. The involvement of A(1)R-A(2A)R heteromer in glial cells and its study at a molecular level. Aim 2. To find evidence for allosteric interactions between partner receptors in the A(2A)RD(2)R receptor heteromer which confer specific pharmacological characteristics to the heteromer. Aim 3. Search for selective antagonists of A(2A)R for presynaptic A(1)R-A(2A)R heteromers versus postsynaptic A(2A)R-D(2)R heteromers that can be useful for treatment of neurological disorder’s treatment, particularly Huntington’s disease. Aim 4. Investigate the pharmacological and functional properties of A(2A)R in the A(2A)CB1 heteromer. Aim 5. Compound screening of different A(2A)R antagonists in stable CHO cell lines expressing A(2A)R, A(1)R-A(2A)R, A(2A)R-D(2)R or A(2A)R-CB(1)R heteromers. The conclusions of the objectives are: Conclusions derived from the first aim: The involvement of A(1)R-A(2A)R heteromer in glial cells and its study at a molecular level. - Upon GABA uptake, adenosine has a biphasic effect, which is mediated by A(1)RA(2A)R heteromers coupled to both Gi/0 and Gs proteins. Extracellular adenosine acting on these A(1)R-A(2A)R functional units operates in a concerted way to balance a PKA-dependent action on GABA uptake. The neural output would thus be inhibitory at low firing rates and facilitatory at high firing rates. - Adenosine by acting on adenosine receptors in astrocytes may significantly contribute to neurotransmission in a dual manner, which depends on the concentration of the nucleoside that is in turn dependent on neuronal firing activity. - BRET and single molecule tracking with TIRF microscope show that the minimal GPCR heteromer unit may consist of four protomers and two G proteins. The strong similarity between GPCRs suggests that the molecular model proposed could apply to other receptors. - These heteromers can be formed in the plasma membrane and are stable on the order of minutes. Such stability suggests that designing ways to target these heteromers may indeed be a viable strategy. - The orientation of the alpha-subunits of the G proteins is on the distal receptors, suggesting that G proteins cross-talk could occur via receptors across the heteromer complex. - The heteromeric unit described, with its dynamic and structural limitations, provides the molecular framework to understand why heteromers are functionally distinct units and not merely the aggregation of two entities with independent functions. Conclusions derived from the second aim: To find an evidence for allosteric interactions between partner receptors in the A(2A)R-D(2)R receptor heteromer which confer specific pharmacological characteristics to the heteromer. In cell culture, the agonist and antagonist binding to the adenosine A(2A)R diminish the affinity of dopamine D(2)R agonists and antagonists. - Those negative interactions between ligands are consequence of allosteric interactions between both receptors conforming the A(2A)R-D(2)R heteromer and constitute a unique biochemical property of this heteromer. - In ex vivo tissue, using these allosteric interactions as a heteromer fingerprint, it has been demonstrated the expression of A(2A)R-D(2)R heteromer in human striatum. - The fact that the A(2A)R antagonists are able to modulate dopamine D(2)R pharmacology has to be taken into account to understand pathologies such as Parkinson’s disease or for human PET neuroimaging. Conclusions derived from the third aim: Search for selective antagonists of A(2A)R for presynaptic A(1)R-A(2A)R heteromers versus postsynaptic A(2A)R-D(2)R heteromers that can be useful for neurological disorder’s treatment, particularly Huntington’s disease. - The physical presence of dopamine D(2)R in the A(2A)R-D(2)R heteromer induced a strong negative cooperativity in the A(2A)R that was detected by SCH-442416. This cooperativity indicates that A(2A)R-A(2A)R homodimers are present in the A(2A)R-D(2)R heteromer. - Based on in vitro and in vivo approaches, the compound SCH-442416 was classified as a preferential presynaptic A(2A)R antagonist, and the compound KW- 6002 was classified as a preferential postsynaptic A(2A)R antagonist. Considering this, SCH-442416 can be used as a lead compound in the development of antidyskinetic drugs in Huntington’s disease; meanwhile KW-6002 can be beneficial in Parkinson’s disease. Conclusions derived from the fourth aim: Investigate the pharmacological and functional properties of A(2A)R in the A(2A)R-CB1R heteromer. - Adenosine A(2A)R changes its G-protein coupling from stimulatory Gs to inhibitory Gi when it forms heteromer with CB1R and a synergistic cross-talk in G-protein activation is observed when both receptors are coactivated. - CB1R mainly controls the ERK1/2 signaling under the A(2A)R-CB1R heteromer. - The A(2A)R-CB1R heteromer does not show allosteric effects at the ligand binding level. Conclusions derived from the fifth aim: Compound screening of different A(2A)R antagonists in stable CHO cell lines expressing A(2A)R, A(1)R-A(2A)R, A(2A)R-D(2)R or A(2A)R-CB1R heteromers. - Compound number 9 could be a good candidate to treat Parkinson’s disease due to its preferential binding to A(2A)R forming A(2A)R-D(2)R heteromer.