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Vertebrate vision starts with light absorption by visual pigments in rod and cone photoreceptor cells of the retina. Rhodopsin, in rod cells, responds to dim light, whereas three types of cone opsins (red, green, and blue) function under bright light and mediate color vision. Cone opsins regenerate with retinal much faster than rhodopsin, but the molecular mechanism of regeneration is still unclear. Recent advances in the area pinpoint transient intermediate opsin conformations, and a possible secondary retinal-binding site, as determinant factors for regeneration. In this Review, we compile previous and recent findings to discuss possible mechanisms of ligand entry in cone opsins, involving a secondary binding site, which may have relevant functional and evolutionary implications

Bradykinin is produced in response to inflammation, trauma, burns, shock, allergy and some cardiovascular diseases. Actions of this peptide are mediated through two different G-protein coupled receptors, named B1 and B2 that have different pharmacological characteristics. The former is up-regulated during inflammation episodes or tissue trauma whereas, the latter is constitutively expressed in a variety of cell types. In a previous work we have characterized the molecular features that explain the observed structure-activity results for both receptors by means of molecular modeling studies, using diverse ligands for both receptors. These results were summarized in the form of two different pharmacophores that provided new insights to be used for the design of novel molecules with antagonistic profile. In the present work, we compare these pharmacophores to understand the features that characterize ligand selectivity to the two bradykinin receptors. The study shows that most of the residues involved in the binding pocket are similar in both receptors and consequently are the pharmacophores obtained. The main difference between the two pharmacophores remains on point #5 that involves a polar moiety for the B1 receptor and an aromatic ring for the B2 receptor. Accordingly, analysis of the prospective bound conformation of several non-selective small molecule ligands of the bradykinin receptors permits to conclude that fulfilment of point#5 is a requirement to produce selective ligands. However, the study also shows that this is a necessary condition only, since ligands need also to be bulky enough to avoid binding to these receptors in diverse poses. These results provide new insights for a better understanding of the molecular features that ligands are required to exhibit to be selective bradykinin ligands. © 2018

Human color vision is mediated by the red, green, and blue cone visual pigments. Cone opsins are G-protein-coupled receptors consisting of an opsin apoprotein covalently linked to the 11-cis-retinal chromophore. All visual pigments share a common evolutionary origin, and red and green cone opsins exhibit a higher homology, whereas blue cone opsin shows more resemblance to the dim light receptor rhodopsin. Here we show that chromophore regeneration in photoactivated blue cone opsin exhibits intermediate transient conformations and a secondary retinoid binding event with slower binding kinetics. We also detected a fine-tuning of the conformational change in the photoactivated blue cone opsin binding site that alters the retinal isomer binding specificity. Furthermore, the molecular models of active and inactive blue cone opsins show specific molecular interactions in the retinal binding site that are not present in other opsins. These findings highlight the differential conformational versatility of human cone opsin pigments in the chromophore regeneration process, particularly compared to rhodopsin, and point to relevant functional, unexpected roles other than spectral tuning for the cone visual pigments

G-protein coupled receptors (GPCRs) are the largest membrane protein superfamily encoded by the human genome and represent the largest class of drug targets for a wide range of pathological conditions. Two GPCRs members, the visual pigment rhodopsin associated with the retinal degenerative disease retinitis pigmentosa (RP), and the muscarinic acetylcholine (ACh) 3 receptor (M3R) associated with Alzheimer’s disease (AD), are studied in this thesis. Rhodopsin is the prototypical visual photoreceptor mediating scotopic vision and distributed throughout the retina. Upon illumination, the bound chromophore 11-cis-retinal isomerizes to all-trans-retinal and triggers the visual signaling cascade. Mutations found in rhodopsin, such as G90V and N55K, are responsible for the retinal degenerative disease RP. To counteract the low structural stability of these mutants, and to provide a deeper understanding of the molecular mechanisms leading to visual dysfunction, artificial membranes in the form of DMPC/DHPC bicelles and DDHA-PC liposomes were prepared. DMPC/DHPC bicelles and DDHA-PC liposomes provided a native-like bilayer environment, which preserved rhodopsin wild type and mutants structure and increased their thermal stability to varying degrees compared to the usual dodecyl maltoside (DM) detergent. Furthermore, chromophore regeneration of G90V and N55K mutants in DMPC/DHPC bicelles condition was enhanced compared to DM condition. Moreover, the kinetics for the active state metarhodopsin II (Meta II) decay indicated that retinal release rates of G90V and N55K mutants became faster in the presence of DMPC/DHPC bicelles and DDHA-PC liposomes compared to DM condition. The addition of hydroxylamine upon Meta II complete decay of WT and G90V in bicelles increased fluorescence intensity, suggesting that retinal can be retained inside the binding pocket. DMPC/DHPC bicelles and DDHA-PC liposomes provided stable conditions so that G90V opsin, obtained after Meta II completely decay, was able to regenerate upon the addition of exogenous retinal. On the other hand, N55K was not able to regenerate, indicating that the molecular mechanisms associated to this mutant has important differences which may be associated with their specific clinical phenotypes. The interactions between rhodopsin and arrestin, and between M3R and tau protein are studied in their association with the degenerative diseases, RP and AD respectively. Active rhodopsin bound R175E mutant arrestin and slowed down the retinal release from the binding pocket. Arrestin binding assays on mutants associated to RP would help uncover mechanisms related to visual cascade termination, not studied so far. M3R plays a role in muscarinic ACh signal transmission and on ion channels function especially in the central nervous system (CNS). In the M3R-tau interaction studies, M3R WT and mutants N132G and D518N did change the location of tau from the cytoplasm to the membrane when they were coexpressed in HEK293T cells. M3R mutants D518K and K523Q were affected when coexpressed with tau and trafficked from the membrane to the cytoplasm. These shifts in location likely result from the interaction between tau and M3R WT and mutants. This finding provides new clues about the specific tau binding/recognition sites on M3R and the possible involvement of such interaction in the pathophysiology of AD. Overall, the artificial membranes DMPC/DHPC bicelles and DDHA-PC liposomes systems provide a better bilayer environment to stabilize rhodopsin WT and mutants than DM detergent environment thus reverting their intrinsic thermal sensitivity. The different behavior of G90V and N55K in artificial membranes could be associated with their specific clinical phenotypes. On the other side, the results obtained on the interaction between GPCRs and other proteins provide a foundation for further studies associated with GPCRs mutants and degenerative diseases, Los receptores acoplados a proteína G (GPCRs) representan la mayor superfamilia de proteínas de membrana codificada por el genoma humano y también la mayor clase de dianas terapéuticas para diversas enfermedades. En esta tesis se estudian dos miembros de los GPCRs; el pigmento visual rodopsina, asociado con la enfermedad degenerativa de la retina retinitis pigmentosa (RP) y el receptor de acetilcolina (ACh) muscarínico 3 (M3R) asociado con la enfermedad de Alzheimer (AD). La rodopsina es el fotorreceptor visual prototípico responsable de la visión escotópica y se encuentra distribuido por toda la retina. Después de la iluminación, el cromóforo 11-cis-retinal se isomeriza a todo-trans-retinal y desencadena serie de reacciones intracelulares llegando al nervio óptico y permitiendo la unión. Mutaciones en rodopsina, tales como G90V y N55K son causantes de RP. Para entender más profundamente los mecanismos moleculares causantes de esta disfunción visual debido a estas mutaciones, se han preparado membranas artificiales tales como bicelas de DMPC/DHPC y liposomas de DDHA-PC. Estos sistemas lipídicos ofrecen un entorno bicapa más nativo, comparado con el que proporciona el detergente dodecil maltosido (DM), usado más tradicionalmente, lo que preserva la estructura de la rodopsina nativa y de los mutantes y aumenta su estabilidad térmica. La regeneración cromóforo para de G90V y N55K en bicelas de DMPC/DHPC es más elevada en comparación con el valor obtenido en DM. La cinética de decaimiento de la conformación activa metarodopsina II (Meta II) indica que la velocidad de liberación del retinal de los mutantes G90V y N55K es más alta en presencia de las bicelas de DMPC/DHPC, y de los liposomas de DDHA-PC, en comparación con las velocidades obtenidas en DM. La adición del reactivo hidroxilamina después del decaimiento completo de Meta II de WT y del mutante G90V en las bicelas provoca un incremento de la intensidad de fluorescencia, indicando que parte de Meta II aún mantiene el retinal en su sitio de unión. Las bicelas de DMPC/DHPC y los liposomas de DDHA-PC también estabilizan la opsina del mutante G90V, obtenida después del decaimiento de Meta II, pudiendo ser regenerada a rodopsina después de añadir retinal exógeno. Por lo contrario, el mutante N55K no regenera, lo que indica que los mutantes actúan con diferentes mecanismos, pudiéndose correlacionar este diferente comportamiento con los fenotipos clínicos específicos de cada mutante. Las interacciones entre la rodopsina y arrestina, y entre M3R y tau, han estudiado para entender el mecanismo funcional de las enfermedades degenerativas de RP y AD respectivamente. La unión de rodopsina activa con el mutante de arrestina R175E, ralentiza la liberación de retinal desde el sitio de unión. Estudios futuros de esta interacción con mutantes asociados a RP ayudarán a explorar los mecanismos de terminación de señal visual, y su conexión con las degeneraciones retinanas aspecto no muy estudiado hasta el momento. M3R desempeña funciones sobre la transmisión de la señal del muscarínico ACh y los canales ionicos, especialmente en el sistema nervioso central (SNC). Por otro lado, la interacción de M3R WT y los mutantes N132G y D518N, produce un cambio en la localización de tau desde el citoplasma a la membrana cuando se coexpresan en células HEK293T. Además, los mutantes M3R D518K y K523Q también son afectados cuando se coexpresan pasando de la membrana al citoplasma. Estos cambios en las localizaciones celulares tanto de tau como del receptor sugieren interacciones específicas entre ellos. Estos resultados proporcionan claves importantes sobre los sitios de unión de tau y M3R, así como la posible implicación de este complejo en la AD. En general, las membranas artificiales de DMPC/DHPC y DDHA-PC, proporcionan un mejor entorno para estabilizar la rodopsina WT y los mutantes asociados a RP. Las diferentes propiedades obtenidas de los mutantes G90V y N55K en estas membranas artificiales pueden estar asociadas a sus distintos fenotipos clínicos específicos. Por otra parte, los resultados obtenidos en el estudio de las interacciones entre los GPCR y otras proteínas proporcionan una base adicional a los estudios asociados a enfermedades degenerativas.

Visual rhodopsins are membrane proteins that function as light photoreceptors in the vertebrate retina. Specific amino acids have been positively selected in visual pigments during mammal evolution, which, as products of adaptive selection, would be at the base of important functional innovations. We have analyzed the top candidates for positive selection at the specific amino acids and the corresponding reverse changes (F13M, Q225R and A346S) in order to unravel the structural and functional consequences of these important sites in rhodopsin evolution. We have constructed, expressed and immunopurified the corresponding mutated pigments and analyzed their molecular phenotypes. We find that position 13 is very important for the folding of the receptor and also for proper protein glycosylation. Position 225 appears to be important for the function of the protein affecting the G-protein activation process, and position 346 would also regulate functionality of the receptor by enhancing G-protein activation and presumably affecting protein phosphorylation by rhodopsin kinase. Our results represent a link between the evolutionary analysis, which pinpoints the specific amino acid positions in the adaptive process, and the structural and functional analysis, closer to the phenotype, making biochemical sense of specific selected genetic sequences in rhodopsin evolution., This work was supported by Ministerio de Ciencia e Innovación, Spain (grants SAF2011-30216-C02-01) and a grant from Fundación Ramón Areces (to PG), and Grups de Recerca Consolidats de la Generalitat de Catalunya (2009 SGR 1402 to PG), Ministerio de Economía y Competitividad, Spain (BFU2013-43726-P) and Departament d’Economia i Coneixement de la Generalitat de Catalunya (GRC 2014 SGR 866 to JB). MAFS is the recipient of an FPI-UPC predoctoral fellowship and BMI has been the recipient of a predoctoral fellowship from AGAUR, Generalitat de Catalunya (PhD grant 2011 FI BI 00275).

Bradykinin (BK) is a member of the kinin family, released in response to inflammation, trauma, burns, shock, allergy and some cardiovascular diseases, provoking vasodilatation and increased vascular permeability among other effects. Their actions are mediated through at least two G-protein coupled receptors, B1 a receptor up-regulated during inflammation episodes or tissue trauma and B2 that is constitutively expressed in a variety of cell types. The goal of the present work is to carry out a structure–activity study of BK B2 antagonism, taking into account the stereochemical features of diverse non-peptide antagonists and the way these features translate into ligand anchoring points to complementary regions of the receptor, through the analysis of the respective ligand-receptor complex. For this purpose an atomistic model of the BK B2 receptor was built by homology modeling and subsequently refined embedded in a lipid bilayer by means of a 600 ns molecular dynamics trajectory. The average structure from the last hundred nanoseconds of the molecular dynamics trajectory was energy minimized and used as model of the receptor for docking studies. For this purpose, a set of compounds with antagonistic profile, covering maximal diversity were selected from the literature. Specifically, the set of compounds include Fasitibant, FR173657, Anatibant, WIN64338, Bradyzide, CHEMBL442294, and JSM10292. Molecules were docked into the BK B2 receptor model and the corresponding complexes analyzed to understand ligand-receptor interactions. The outcome of this study is summarized in a 3D pharmacophore that explains the observed structure–activity results and provides insight into the design of novel molecules with antagonistic profile. To prove the validity of the pharmacophore hypothesized a virtual screening process was also carried out. The pharmacophore was used as query to identify new hits using diverse databases of molecules. The results of this study revealed a set of new hits with structures not connected to the molecules used for pharmacophore development. A few of these structures were purchased and tested. The results of the binding studies show about a 33 % success rate with a correlation between the number of pharmacophore points fulfilled and their antagonistic potency. Some of these structures are disclosed in the present work.

Bradykinin (BK) is a nonapeptide involved in several pathophysiological conditions including among others, septic and haemorrhagic shock, anaphylaxis, arthritis, rhinitis, asthma, inflammatory bowel disease. Accordingly, BK antagonists have long been sought after for therapeutic intervention. Action of BK is mediated through two different G-protein coupled receptors known as B1 and B2. Although there are several B1 antagonists reported in literature, their pharmacological profile is not yet optimal so that new molecules need to be discovered. In the present work we have constructed an atomistic model of the B1 receptor and docked diverse available non-peptide antagonists in order to get a deeper insight into the structure-activity relationships involving binding to this receptor. The model was constructed by homology modelling using the chemokine CXC4 and bovine rhodopsin receptors as template. The model was further refined using molecular dynamics for 600 ns with the protein embedded in a POPC bilayer. From the refinement process we obtained an average structure that was used for docking studies using the Glide software. Antagonists selected for the docking studies include Compound 11, Compound 12, Chroman28, SSR240612, NPV-SAA164 and PS020990. The results of the docking study underline the role of specific receptor residues in ligand binding. The results of this study permitted to define a pharmacophore that describes the stereochemical requirements of antagonist binding, and can be used for the discovery of new compounds.

Muscarinic acetylcholine receptors expression and signaling in the human Jurkat T cell line were investigated. Semiquantitative real-time PCR and radioligand binding studies, using a wide set of antagonist compounds, showed the co-existence of M(3), M(4), and M(5) subtypes. Stimulation of these subpopulations caused a concentration and time- dependent activation of second messengers and ERK signaling pathways, with a major contribution of the M(3) subtype in a G(q/11)-mediated response. In addition, we found that T-cell stimulation leads to increased expression of M(3) and M(5) both at transcriptional and protein levels in a PLC/PKCθ dependent manner. Our data clarifies the functional role of AChR subtypes in Jurkat cells and pave the way to future studies on the potential cross-talk among these subpopulations and their regulation of T lymphocytes immune function.

Human M3 muscarinic acetylcholine receptor (M3R), present in both the central and the peripheral nervous system, is involved in several neurodegenerative and autoimmune diseases. Recently, M3R overexpression has been suggested to play a role in certain forms of cancer, showing promise as a new potential pharmacological target. However, the lack of structural information hampered to develop a new potent selective and potent antagonist. We describe here different strategies for overexpressing functional M3R on the perspective of future biophysical studies. To achieve this goal, four tagged M3R genes were engineered and codon optimized. Different heterologous expression systems, including mammalian cells and viral transfection, were employed to overexpress M3R. Although codon optimization resulted in only twofold to threefold increase of M3R expression, we found that epitope tagging of the synthetic M3R, especially with hemagglutinin and Flag epitope tags, could improve M3R expression levels. On the other hand, viral transfection led to a yield of 27 pmol/mg protein that is the highest level reported so far for this receptor subtype in mammalian cells. Taking together several of the strategies used can help increasing M3R expression, not only to start purification efforts but also for secondary structural analysis trial and functional analyses. V

The final publication is available at Springer via http://dx.doi.org/10.1007/s00018-015-2043-7 Human red and green visual pigments are seven transmembrane receptors of cone photoreceptor cells of the retina that mediate color vision. These pigments share a very high degree of homology and have been assumed to feature analogous structural and functional properties. We report on a different regeneration mechanism among red and green cone opsins with retinal analogs using UV–Vis/fluorescence spectroscopic analyses, molecular modeling and site-directed mutagenesis. We find that photoactivated green cone opsin adopts a transient conformation which regenerates via an unprotonated Schiff base linkage with its natural chromophore, whereas red cone opsin forms a typical protonated Schiff base. The chromophore regeneration kinetics is consistent with a secondary retinal uptake by the cone pigments. Overall, our findings reveal, for the first time, structural differences in the photoactivated conformation between red and green cone pigments that may be linked to their molecular evolution, and support the proposal of secondary retinal binding to visual pigments, in addition to binding to the canonical primary site, which may serve as a regulatory mechanism of dark adaptation in the phototransduction process.

La publicación de la primera estructura cristalográfica de resolución atómica de un miembro de la familia de los receptores acoplados a proteínas G (GPCR) como es la Rodopsina Bovina (Rho) (1)marcó el comienzo de los estudios sobre la relación entre la actividad biológica y la estructura de estas proteínas (1,2). Si bien se han llevado a cabo numerosos estudios de difracción de rayos X de las estructuras de GPCRs desde ese momento, dada la dificultad de cristalización de este tipo de proteínas, aun no se dispone hoy en día de la estructura tridimensional a nivel atómico de un gran número de GPCRs, entre ellos del receptor de Neuroquinina (NK1-R). Sin embargo, dado que los GPCRs comparten una estructura de 7 hélices de transmembrana enlazadas por loops se han podido producir modelos de diferentes receptores de membrana por homología de secuencia (2; 3; 4). El receptor NKI-R está involucrado en muchos eventos celulares de inflamación, dolor, trastornos neuronales, del sistema digestivo y del sistema inmune, incluyendo cáncer (4), un modelo 3D de este receptor puede ser utilizado para conocer con más detalle los principios básicos moleculares de su actividad biológica y ser utilizado en el diseño racional de fármacos. En el presente trabajo se ha obtenido un modelo del receptor NK1R mediante modelización por homología de secuencia (HM) y dinámica molecular (DM), utilizando la estructura 3D de alta resolución (1.8 Å) del receptor delta-opioide presentado el 2014. El modelo final fue utilizado para el anclaje de cinco antagonistas no peptídicos, confirmando las interacciones con ciertos residuos presentes en el sitio activo, en concordancia con estudios de mutagénesis dirigida. Estos resultados han permitido definir un farmacóforo 3D que cumplió con al menos dos puntos, al evaluar el docking con 5 diferentes antagonistas no peptídicos, conocidos por su eficacia terapéutica y su alta afinidad por el receptor. El modelo refinado del receptor NK1-R sugiere algunos residuos claves en el sitio de unión, de los cuales no se tiene referencia en estudios previos de mutagénesis dirigida, así mismo el farmacóforo obtenido presenta características útiles para el diseño racional de fármacos esto puede significar nuevos retos para futuras investigaciones. Los resultados y conclusiones obtenidos se detallan en las secciones respectivas del presente informe.

The type of serotonin receptor 1A (5-HT1A), the galanin type 1 (GalR1) and the orphan receptor 39 (GPR39) belong to the superfamily The serotonin receptor type 1A (5-HT1A), the galanin receptor type 1 (GalR1) and the orphan receptor 39 (GPR39) belong to the superfamily of G-protein-coupled receptors. All three GPCRs share, among other features, a relationship with the pathophysiology of unipolar depression associated with presence of zinc. The current paradigm with respect to these functional unities is that these receptors are not acting as monomeric forms but through complexs involving specific interactions with themselves or other receptors from the same family. This allows them to increase their functional possibilities exponentially, allowing versatility from a fixed number of receptors, and consequently also increases the number of pharmacological approachmes. The heterodimerization between 5-HT1A receptor and GalR1 has been previously described as an antagonistic interaction that could lead to unipolar depression. In this thesis we have shown that this interaction is avoided in the presence of zinc, giving a rational explanation for the antidepressant effect widely described for this cation. Although there was no published evidence regarding the interaction of these two receptors with GPR39, a receptor that is activated by zinc and whose expression depends on the concentration of this cation, in this thesis we have demonstrated the ability to interact of these three receptors in both forms GPR39-5-HT1A, 5-HT1A-GalR1 and the trimer GPR39-5-HT1A-GalR1. It has also been found that the functional capacity of the receptors is modified according the type of interaction in which these receptors are involved. So that monomeric and oligomeric forms have different signalling capacities. This would suggest that in the human brain all putative receptor configurations could be present, and that the presence of one or other would be regulated by zinc concentration. Deepening in the detailed molecular mechanism of the effect of zinc on these interactions should allow the development of new drugs for a disease with high prevalence in the nowadays society., El receptor de serotonina del tipus 1A (5-HT1A), el de galanina de tipus 1 (GalR1) i el receptor orfe 39 (GPR39) pertanyen a la superfamília dels receptors acoblats a proteïna G. Tots tres receptors comparteixen, entre d'altres característiques, una relació amb la fisiopatologia de la depressió unipolar associada amb la presència de zinc. El paradigma actual, respecte a la unitat funcional d'aquests receptors, és que no actuen en forma monomèrica sinó mitjançant complexos que involucren interaccions específiques amb ells mateixos o amb altres receptors de la mateixa família. Això els hi permet augmentar les seves possibilitats funcionals exponencialment, permetent una gran versatilitat a partir d'un nombre fix de receptors, i en conseqüència també augmenta el nombre de possibilitats d'abordatge farmacològic. L'heterodimerització entre el receptor 5-HT1A i el GalR1 s'ha descrit prèviament com una interacció antagònica que podria donar lloc a la depressió unipolar. En aquesta tesi s'ha demostrat que aquesta interacció s'evita en presència de zinc, donant una explicació racional a l'efecte antidepressiu àmpliament descrit per a aquest catió. Tot i que no hi havia cap evidència publicada respecte a la interacció d'aquests dos receptors amb el GPR39, un receptor que és activat pel zinc i l'expressió del qual depèn de la concentració del mateix, en aquesta tesi s'ha demostrat la capacitat d'interacció d'aquests tres receptors, tant en les formes GPR39-5-HT1A i 5-HT1A-GalR1 com en el trímer GPR39-5-HT1A-GalR1. A més, s'ha trobat que la capacitat funcional dels receptors es veu modificada segons el tipus d'interacció en la que aquests receptors participen, de manera que les formes monomèriques i oligomèriques presenten una capacitat de senyalització diferent. Això faria pensar que en el cervell humà podrien trobar-se totes les configuracions potencials de receptors, i que la presència d'unes o altres estaria regulada per la concentració de zinc. L'aprofundiment en el mecanisme molecular detallat de l'efecte del zinc en aquestes interaccions hauria de permetre el desenvolupament de nous fàrmacs per a una malaltia d'alta prevalença en la població mundial.

Allosteric receptor–receptor interactions in GPCR heteromers appeared to introduce an intermolecular allosteric mechanism contributing to the diversity and bias in the protomers. Examples of dopamine D2R heteromerization are given to show how such allosteric mechanisms significantly change the receptor protomer repertoire leading to diversity and biased recognition and signaling. In 1980s and 1990s, it was shown that neurotensin (NT) through selective antagonistic NTR–D2 like receptor interactions increased the diversity of DA signaling by reducing D2R-mediated dopamine signaling over D1R-mediated dopamine signaling. Furthermore, D2R protomer appeared to bias the specificity of the NTR orthosteric binding site toward neuromedin N vs. NT in the heteroreceptor complex. Complex CCK2R–D1R–D2R interactions in possible heteroreceptor complexes were also demonstrated further increasing receptor diversity. In D2R–5-HT2AR heteroreceptor complexes, the hallucinogenic 5-HT2AR agonists LSD and DOI were recently found to exert a biased agonist action on the orthosteric site of the 5-HT2AR protomer leading to the development of an active conformational state different from the one produced by 5-HT. Furthermore, as recently demonstrated allosteric A2A–D2R receptor–receptor interaction brought about not only a reduced affinity of the D2R agonist binding site but also a biased modulation of the D2R protomer signaling in A2A–D2R heteroreceptor complexes. A conformational state of the D2R was induced, which moved away from Gi/o signaling and instead favored ß-arrestin2-mediated signaling. These examples on allosteric receptor–receptor interactions obtained over several decades serve to illustrate the significant increase in diversity and biased recognition and signaling that develop through such mechanisms

Recientemente se han logrado grandes avances en la determinación de la estructura y función de los receptores acoplados a proteínas G (GPCR’s). Los GPCR´s constituyen gran familia de proteína integrales de membrana y se encuentran implicados en una gran variedad de procesos fisiológicos como la respuesta a hormonas y neurotransmisores, así como también son centrales en los sistemas sensoriales mediando las respuestas en los sentidos de la vista, olfato y el gusto. Entre ellos se encuentra la familia de los receptores muscarínicos de acetilcolina, compuesta por 5 subtipos de receptores (M1-M5), los cuales modulan la mayor parte de las acciones del neurotransmisor acetilcolina en el sistema nervioso central y en el sistema nervioso periférico. Los receptores muscarínicos han jugado un papel muy relevante en la compresión del funcionamiento de la neurotransmisión. En este trabajo nos centraremos únicamente en los receptores M1 y M3 debido a su potencial participación en el mecanismo molecular de la enfermedad de Alzheimer Hasta ahora se ha visto que estos receptores se encuentran implicados en varias patologías del sistema nervioso central, ya que modulan la señalización de diferentes sistemas neurotransmisores. Estos sistemas se ven alterados como en el caso de la enfermedad de Alzheimer, donde se ha visto que modulan la degradación de la APP (proteína precursora de amiloide) por la α-secretasa y de esta forma evitan la formación de las placas neuríticas conocidas como β-amiloides (Aβ) características de esta enfermedad. Además se ha sugerido que como resultado de su interacción con la proteína tau, pueden jugar un papel muy relevante en la pérdida de neuronas colinérgicas a través de un mecanismo que involucra la interacción intracelular y extracelular de la proteína tau con los receptores muscarínicos M1/M3 presentes en este tipo de neuronas. El objetivo de este trabajo es comprender y analizar detalladamente las propiedades biológicas y bioquímicas de las diferentes isoformas de la proteína tau (ya sean fragmentos o la proteína completa soluble) así como las posibles interacciones que puedan tener con los receptores muscarínicos en relación a su implicación con el Alzheimer. Para ello, el principal logro ha sido optimizar las metodologías de expresión y purificación de la proteína tau. Ello ha permitido, por primera vez en nuestro grupo, la obtención de la proteína tau purificada, así como también la obtención de los receptores muscarínicos mediante su expresión en líneas celulares apropiadas.

G protein-coupled receptors (GPCRs) play critical roles in cellular processes and signaling and have been shown to form heteromers with diverge biochemical and/or pharmacological activities that are different from those of the corresponding monomers or homomers. However, despite extensive experimental results supporting the formation of GPCR heteromers in heterologous systems, the existence of such receptor heterocomplexes in the brain remains largely unknown, mostly because of the lack of appropriate methodology. Herein, we describe the in situ proximity ligation assay procedure underlining its high selectivity and sensitivity to image GPCR heteromers with confocal microscopy in brain sections. We describe here how the assay is performed and discuss advantages and disadvantages of this method compared with other available techniques.

Una de las principales cuestiones en farmacología molecular de los GPCR es entender los mecanismos estructurales de las siete hélices transmembrana (TM) que se producen para estabilizar ya sea Rg o los diferentes estados R*. Para entender el mecanismo que cambia el equilibrio del conjunto a un estado activo R* se construyeron tres de los receptores de la serotonina (5-HT4, 5-HT6, y 5 HT7) sobre la base de su información más reciente de cristalografía de rayos X. Dando lugar a dos modelos de cada receptor: una inactiva y otra activa. Los modelos, mejorados y evaluados con la ayuda de datos farmacológicos y químicos se utilizaron principalmente para comprender la interacción entre un ligando y su receptor y su mecanismo de acción. Estos hallazgos estructurales pueden a su vez resultar útiles para el diseño de nuevos fármacos más eficaces y selectivos., One of the main questions in G protein coupled receptors (GPCRs) molecular pharmacology is to understand the structural arrangements of the seven transmembrane (TM) helices that occur to stabilize either the ground state (Rg) or different active states (R*) of the receptors. In order to understand the mechanism that shift the equilibrium of the ensemble to an active R* state models of the inactive and the active state of three serotonin receptors (5-HT4, 5-HT6, and 5-HT7) were built based on the latest information from X-ray crystallography. The resulting models were mainly used to understand the interaction between a ligand and its receptor and the mechanism of action. With the help of pharmacological and chemical data these models and complexes were improved and evaluated. These findings may prove valuable for structural based drug discovery efforts and facilitate the design of more effective and selective pharmaceuticals.

Rhodopsin is the visual photoreceptor responsible for dim light vision. This receptor is located in the rod cell of the retina and is a prototypical member of the G-protein-coupled receptor superfamily. The structural details underlying the molecular recognition event in transducin activation by photoactivated rhodopsin are of key interest to unravel the molecular mechanism of signal transduction in the retina. We constructed and expressed rhodopsin mutants in the second and third cytoplasmic domains of rhodopsin –where the natural amino acids were substituted by the human M3 acetylcholine muscarinic receptor homologous residues– in order to determine their potential involvement in G-protein recognition. These mutants showed normal chromophore formation and a similar photobleaching behavior than WT rhodopsin, but decreased thermal stability in the dark state. The single mutant V1383.53 and the multiple mutant containing V2275.62 and a combination of mutations at the cytoplasmic end of transmembrane helix 6 caused a reduction in transducin activation upon rhodopsin photoactivation. Furthermore, combination of mutants at the second and third cytoplasmic domains revealed a cooperative role, and partially restored transducin activation. The results indicate that hydrophobic interactions by V1383.53, V2275.62, V2506.33, V2546.37 and I2556.38 are critical for receptor activation and/or efficient rhodopsin–transducin interaction.

G Protein-coupled receptors (GPCRs) are integral cell membrane proteins of great relevance for pharmacology due to their role in transducing extracellular signals. The 3-D s tructure is unknown for most of them, and the investigation of their structure-function relationships usually relies on the construction of 3-D receptor models from amino acid sequence alignment onto those receptors of known structure. Sequence alignment risks the loss of relevant information. Different approaches have attempted the analysis of alignment-free sequences on the basis of amino acid physicochemical properties. In this paper, we use the Auto-Cross Covariance method and compare it to an amino acid composition representation. Novel semi-supervised manifold learning methods are then used to classify the several members of class C GPCRs on the basis of the transformed data. This approach is relevant because protein sequences are not always labeled and methods that provide robust classification for a limited amount of labels are required.

The final publication is available at Springer via http://dx.doi.org/10.1007/s12035-015-9153-y 5-Hydroxytryptamine 1A receptor and galanin receptor 1 belong to the G protein-coupled receptors superfamily, and they have been described to heterodimerize triggering an anomalous physiological state that would underlie depression. Zinc supplementation has been widely reported to improve treatment against major depressive disorder. Our work has focused on the study and characterization of these receptors and its relationships with zinc both under purified conditions and in cell culture. To this aim, we have designed a strategy to purify the receptors in a conformationally active state. We have used receptors tagged with the monoclonal Rho-1D4 antibody and employed ligand-assisted purification in order to successfully purify both receptors in a properly folded and active state. The interaction between both purified receptors has been analyzed by surface plasmon resonance in order to determine the kinetics of dimerization. Zinc effect on heteromer has also been tested using the same methodology but exposing the 5-hydroxytryptamine 1A receptor to zinc before the binding experiment. These results, combined with Förster resonance energy transfer (FRET) measurements, in the absence and presence of zinc, suggest that this ion is capable of disrupting this interaction. Moreover, molecular modeling suggests that there is a coincidence between zinc-binding sites and heterodimerization interfaces for the serotonin receptor. Our results establish a rational explanation for the role of zinc in the molecular processes associated with receptor-receptor interactions and its relationship with depression, in agreement with previously reported evidence for the positive effects of zinc in depression treatment, and the involvement of our target dimer in the same disease.

Besides some pharmacological, biochemical and biophysical evidences support the contention that muscarinic acetylcholine receptors can form homo- and heterodimers, the existence of specific M 3 and M 5 muscarinic receptors oligomers in living cells is a new concept. Interestingly, this phenomenon might have relevance in lymphocytic cholinergic function since both T- and B-cells naturally express high levels of these two receptor subtypes. Here, by means of co-immunoprecipitation and bioluminescence resonance energy transfer methods we demonstrated that M 3 and M 5 muscarinic receptors could form constitutive homo- and heterodimers in transiently transfected HEK-293T cells. Interestingly, this receptor–receptor interaction was unaltered by carbachol treatment but it was affected by the expression of a peptide corresponding to a portion of the third intracellular loop of the M 5 muscarinic receptor. In addition, the same peptide was able to abrogate the carbachol-induced mitogen-activated protein kinase phosphorylation and the carbachol-enhanced PHA-induced IL-2 production in derived lymphocytic T cells. Overall, these results suggest that the third intracellular loop of the M 5 muscarinic receptor might play a regulatory role in receptor function and heteromerization, thus providing the molecular framework for a potential cholinergic-based therapeutic intervention of the immune system.