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Title: Noncanonical Neurotransmitter Activation of Catecholamine Receptors
Author: Sánchez Soto, Marta
Director: Casadó, Vicent
Ferré, Sergi
Keywords: Dopamina
Proteïnes G
G Proteins
Issue Date: 16-Jan-2017
Publisher: Universitat de Barcelona
Abstract: [eng] From the D2‐like receptors (D2,D3, D4), only D4 receptor (D4R) has been described as “promiscuous” since it can be activated both by dopamine (DA) and norepinephrine (NE) and function as a noradrenergic receptor in the brain. However, there is no evidence for the other D2‐like receptors. In addition, D4R has a large number of polymorphisms, one of the variants, D4.7, has been associated with several neuropsychiatric disorders. The first aim of this thesis was to study the possible activation of D2‐like recptors (including the three main D4 variants, D2 short, D2 long and D3) by radiolabeled ligand binding and BRET‐based functional assays. Our results indicate that first, NE binds and activates D2‐like receptors and second, there are no differences in the signaling of D4R variants. In addition, the potency of DA and NE depends not only on the receptor but also on the G protein subtype. The catecholamine NE is implicated in important brain functions and it binds to three receptor families. The α2A receptor (α2AR) is expressed in many brain regions and is particularly enriched in the striatum together with α2C (α2CR). The low levels of NE in the striatum led us to question what is the role of α2R there and hypothesize whether DA could provide the endogenous neurotransmitter of α2R in the striatum. Therefore, the second aim of this thesis was to study the activation of α2AR and α2CR by DA and dopaminergic ligands through radiolabeled binding experiments, activation of different subtypes of G protein and adenylyl cyclase inhibition. Surprisingly, the potencies of α2AR and α2CR for DA are very similar or even higher than for some D2‐like receptors. Thus, we can hypothesize that the DA in the striatum should reach enough concentration to activate α2AR and α2CR. In addition, these receptors are also targets for compounds previously described as D2‐like agonists. Particularly striking is the ability of the D3R and D4R agonists, 7‐OH‐PIPAT and RO‐105824 to bind and activate with high affinity α2AR i α2CR. Similar to the previous part, the efficacy and potency of ligands depends on the receptor and the G protein subtype. Finally, is its well accepted that G protein‐coupled receptors (GPCRs) form homo‐, heteromers and high order oligomers. Since we had shown that DA and NE bind and activate D2‐like receptors and α2R, we wanted to study the activation of G protein mediated by the complexes formed by D4R variants, D4.4R and D4.7R, and D2R or α2R. In order to do that we used CODA‐RET (complemented donor‐acceptor resonance energy transfer) a technique that allows the study of the function of a signaling complex formed by two defined GPCRs and a subunit of the heterotrimeric G protein. Our results indicate that, although they physically interact, D4.7R is not implicated in the G protein activation when is forming complexes with D2R or α2AR. Also, there are no differences in the potency or efficacy of the endogenous neurotransmitters DA and NE with D4.4R and D4.7R monomers or homodimers, but the association with D2R or 2AR discloses differences between the two D4R variants. Finally, we found important differences in the potency and efficacy dopaminergic ligands for the different receptor complexes that could be important for some neuropsychiatric diseases.
[cat] Dels receptors D2‐like de dopamina (D2, D3, D4) només el D4 (D4R) s’ha descrit com a “promiscu” ja que pot ser activat tant per dopamina (DA) com per noradrenalina (NE); en canvi, no hi ha evidències de que això sigui cert per la resta de receptors D2‐like. A més, el D4R és un receptor molt polimòrfic i una de les seves variants, la D4.7, s’ha associat a desordres psiquiàtrics. El primer objectiu va ser estudiar la possible activació dels receptors D2‐like (D2S, D2L, D3 i les tres variants més prevalents de D4) per NE mitjançant estudis d’unió de radiolligands i assajos funcionals de BRET. Els resultats mostren que primer, la NE s’uneix i activa els receptors D2‐like i segon, no hi ha diferències en la senyalització de les variants de D4R. El receptor α2A (α2AR) adrenèrgic s’expressa en moltes zones cerebrals i està particularment enriquit a l’estriat junt amb el receptor α2C (α2CR). Els baixos nivells de NE a l’estriat donen peu a la possibilitat de que la DA sigui el neurotransmissor endogen de α2AR i α2CR. Per tant, el segon objectiu de la tesi va ser estudiar l’activació de α2AR i α2CR per DA i lligands dopaminèrgics mitjançant estudis d’unió de radiolligands, activació de diferent proteïnes G i inhibició d’adenilat ciclasa. Sorprenentment les potències de α2AR i α2CR per la DA són molt similars o inclús més altes que per alguns receptors D2‐ like. Per tant és probable que els nivells de DA a l’estriat siguin suficients per activar α2R. A més, aquests receptors també són activats per compostos prèviament descrits com agonistes dels receptors D2‐like com ara el 7‐OH‐PIPAT (D3R) i el RO‐105824 (D4R). A més, tant l’eficàcia com la potència dels lligands depèn del receptor i del subtipus de proteïna G. Per últim, els GPCRs poden formar dímers, heteròmers o entitats superiors. A la tercera part vam voler comparar l’activació de proteïna G per DA i NE mitjançant complexes entre dues de les variants de D4R, D4.4R i D4.7R amb D2R i α2AR. Els resultats indiquen que hi ha diferències en l’activació dels diferents complexes que podria tenir importància en malalties com RLS i Parkinson. A més, donen suport a la teoria de que les diferències entre les variants de D4R es troben en la seva interacció amb altres receptors.
Appears in Collections:Tesis Doctorals - Departament - Bioquímica i Biomedicina Molecular

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