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Background: Adherence to adjuvant endocrine therapy (AET) is low in women with breast cancer. Negative beliefs about the necessity of AET and high concerns are barriers to adherence.Purpose: To use the multiphase optimization strategy to optimize the content of an information leaflet intervention, to support AET beliefs. Methods: We conducted an online screening experiment using a 2^5 factorial design to optimize the leaflet. The leaflet had five components, each with two levels; 1) diagrams about AET mechanisms (on/off); 2) infographics displaying AET benefits (enhanced/basic); 3) AET side-effects (enhanced/basic); 4) answers to AET concerns (on/off); 5) breast cancer survivor (patient) input: quotes and photographs (on/off). Healthy adult women (n=1604), recruited via a market research company, were randomized to one of 32 experimental conditions, which determined the levels of components received. Participants completed the beliefs about medicines questionnaire before and after viewing the leaflet. Results: There was a significant main effect of patient input on beliefs about medication (β=0.063, p

The current situation of water resources, within the framework of the global pandemic COVID-19, centers the starting point of this doctoral thesis. The treatments applied in the fight against the SARS-CoV-2 virus, have led to intensive consumption of the drugs dexamethasone (DEX), amoxicillin (AMX), paracetamol (PAR) and sertraline (STR), which finally end in the aquatic medium as part of the group of emerging contaminants (ECs). In this context, advanced oxidation processes (AOPs) are an attractive option for their removal, standing out electrochemical oxidation (EOX) because its high effectiveness. Nevertheless, previous studies have demonstrated that under the application of some AOPs, it may be possible that intermediate and derivative compounds can be generated, possibly more toxic than the initial ones, like polychlorinated dibenzo-p-dioxin and furans (PCDD/Fs). To corroborate this hypothesis, this doctoral thesis has assessed, in an experimental and theoretical way (computational chemistry) the formation of PCDD/Fs and the final toxicity of the samples, after applying EOX to matrices containing the COVID-19 drugs, dexamethasone (DEX), amoxicillin (AMX), paracetamol (PAR) and sertraline (STR). RESUMEN: La situación actual de los recursos hídricos, en el marco de la pandemia mundial COVID-19, es el punto de partida de esta tesis doctoral. El tratamiento aplicado en la lucha contra el virus SARS-CoV-2, ha llevado al consumo intensivo de los fármacos dexametasona (DEX), amoxicilina (AMX), paracetamol (PAR) y sertralina (STR), que finalmente acaban en el medio acuático formando parte del grupo de contaminantes emergentes (CEs). En este contexto, los procesos de oxidación avanzada (POAs) se presentan como una opción atractiva para su eliminación, destacando la oxidación electroquímica (EOX) por su alta eficacia. Sin embargo, estudios previos han demostrado, que, bajo la aplicación de algunos POAs, es posible que se generen compuestos intermedios derivados, que pueden ser más tóxicos que los iniciales, como las dibenzo-p-dioxinas policloradas y los furanos (PCDD/Fs). Para corroborar esta hipótesis, esta tesis doctoral ha evaluado, experimental y teóricamente (química computacional) la formación de PCDD/Fs y la toxicidad final de las muestras, tras aplicar EOX a matrices que contienen los fármacos COVID-19, dexametasona (DEX), amoxicilina (AMX), paracetamol (PAR) y sertralina (STR). This research was financially supported by the Spanish Ministry of Science, Innovation and Universities through the Projects CTM2014-58029-R (MINECO/FEDER,UE), “Identificación y cuantificación de las variables responsables de la potencial formación de PCDD/Fs en procesos de oxidación avanzada”; CTM2017-87740-R (AEI/FEDER, UE) “Aplicación de Tecnologías Ambientales a Matrices Líquidas Conteniendo Contaminantes Orgánicos Emergentes Precursores de la Formación de Derivados Clorados (PCDD/Fs).

Background: Women with a family history of breast cancer have an increased lifetime risk of the disease. Delay in symptom presentation can lead to poorer outcomes. Low awareness of breast cancer symptoms and help-seeking barriers have been associated with delay in presentation in the general population. Symptom awareness and help-seeking barriers among women at increased risk of breast cancer are unknown. Methods: We conducted a secondary analysis of survey data which included women with moderate and high risk of breast cancer from 20 secondary and tertiary care clinics in England (n=408). Women completed a validated survey assessing breast cancer symptom awareness, barriers to help-seeking and anticipated delay in help-seeking. Results: Women recognised an average of 9.1/11 breast cancer symptoms (SD=2.1). Nipple rash was the least recognised symptom (51.0%). Women educated to at least degree level had higher awareness than those with lower education (β= 0.14, 95% CI 0.13, 0.99, p = 0.011). Women at lower socioeconomic status (SES) had lower awareness than those at higher SES (β= -0.13, 95% CI -1.09, -0.07, p=0.027). Women reported several anticipated help-seeking barriers (mean=4.0/11, SD=2.8). Most women (376/408; 92.2%) reported that they would seek medical help within 2 weeks of discovering a breast cancer symptom. Conclusion: Interventions to increase awareness of non-lump breast cancer symptoms and reduce help-seeking barriers are needed, particularly in less educated and more deprived women.

Background: The National Institute for Health and Care Excellence (NG151) recommends considering daily aspirin for people with Lynch syndrome to reduce colorectal cancer risk. However, patients and their healthcare providers considering preventive therapy need to negotiate complex decisions that weigh up potential benefits and harms.Method: We conducted semi-structured interviews to explore the barriers and facilitators to using aspirin for preventive therapy. We recruited 15 people with Lynch syndrome, and 23 healthcare providers across multiple professions in primary and specialist care in the United Kingdom. Interview schedules were informed by the Theoretical Domains Framework.Results: There were three themes: 1) Considering potential harms and benefits; 2) Healthcare pathway; 3) Patients’ level of interest in aspirin. All healthcare providers, across primary and specialist care, viewed general practitioners (GPs) as being responsible for prescribing and overseeing the use of aspirin. However, GPs were unfamiliar with aspirin for preventive therapy, and concerned about prescribing at higher doses (300-600mg). To support decision-making, GPs wanted clarification from specialist clinicians on the evidence and dose to prescribe. Not all participants with Lynch syndrome received information on aspirin from their healthcare provider, and several were unsure who to discuss aspirin with. GPs were more inclined to prescribe aspirin for patients with expressed preferences for the medication, however several patients were uncertain and wanted further guidance.Conclusions: Coordinated and multilevel strategies are needed, addressing the needs of both GPs and people with Lynch syndrome, to ensure consistent implementation of national guidance on aspirin for preventive therapy.

International audience; There is increasing support for water molecules playing a role in signal propagation through G protein-coupled receptors (GPCRs). However, exploration of the hydration features of GPCRs is still in its infancy. Here, we combined site-specific labeling with unnatural amino acids to molecular dynamics to delineate how local hydration of the ghrelin receptor growth hormone secretagogue receptor (GHSR) is rearranged upon activation. We found that GHSR is characterized by a specific hydration pattern that is selectively remodeled by pharmacologically distinct ligands and by the lipid environment. This process is directly related to the concerted movements of the transmembrane domains of the receptor. These results demonstrate that the conformational dynamics of GHSR are tightly coupled to the movements of internal water molecules, further enhancing our understanding of the molecular bases of GPCR-mediated signaling.

International audience; GHSR controls, among others, growth hormone and insulin secretion, adiposity, feeding and glucose metabolism. Therefore, an inverse agonist ligand capable of selectively targeting GHSR and reducing its high constitutive activity appears to be a good candidate for the treatment of obesity-related metabolic diseases. In this context, we present a study that led to the development of several highly potent and selective inverse agonists of GHSR based on the 1,2,4-triazole scaffold. We demonstrate that, depending on the nature of the substituents on positions 3, 4 and 5, 2 this scaffold leads to ligands that exert an intrinsic inverse agonist activity on GHSR-catalyzed G protein activation through the stabilization of a specific inactive receptor conformation. Thanks to an in vivo evaluation, we also show that one of the most promising ligands not only exerts an effect on insulin secretion in rat pancreatic islets but also affects the orexigenic effects of ghrelin in mice.

Liver-expressed antimicrobial peptide 2 (LEAP2) was recently recognized as an endogenous ligand for the growth hormone secretagogue receptor (GHSR), which also is a receptor for the hormone ghrelin. LEAP2 blocks ghrelin-induced activation of GHSR and inhibits GHSR constitutive activity. Since fluorescence-based imaging and pharmacological analyses to investigate the biology of GHSR require reliable probes, we developed a novel fluorescent GHSR ligand based on the N-terminal LEAP2 sequence, hereafter named F-LEAP2. In vitro, F-LEAP2 displayed binding affinity and inverse agonism to GHSR similar to LEAP2. In a heterologous expression system, F-LEAP2 labeling was specifically observed in the surface of GHSR-expressing cells, in contrast to fluorescent ghrelin labeling that was mainly observed inside the GHSR-expressing cells. In mice, centrally-injected F-LEAP2 reduced ghrelin-induced food intake, in a similar fashion to LEAP2, and specifically labeled cells in GHSR-expressing brain areas. Thus, F-LEAP2 represents a valuable tool to study the biology of GHSR in vitro and in vivo.

International audience

International audience; The ghrelin receptor or growth hormone secretagogue receptor (GHSR) is a G-protein-coupled receptor that controls growth hormone and insulin secretion, food intake, and reward-seeking behaviors. Liver-expressed antimicrobial peptide 2 (LEAP2) was recently described as an endogenous antagonist of GHSR. Here, we present a study aimed at delineating the structural determinants required for LEAP2 activity toward GHSR. We demonstrate that the entire sequence of LEAP2 is not necessary for its actions. Indeed, the N-terminal part alone confers receptor binding and activity to LEAP2. We found that both LEAP2 and its N-terminal part behave as inverse agonists of GHSR and as competitive antagonists of ghrelin-induced inositol phosphate production and calcium mobilization. Accordingly, the N-terminal region of LEAP2 is able to inhibit ghrelin-induced food intake in mice. These data demonstrate an unexpected pharmacological activity for LEAP2 that is likely to have an important role in the control of ghrelin response under normal and pathological conditions.

The growth hormone secretagogue receptor (GHSR) and dopamine receptor (D2R) have been shown to oligomerize in hypothalamic neurons with a significant effect on dopamine signaling, but the molecular processes underlying this effect are still obscure. We used here the purified GHSR and D2R to establish that these two receptors assemble in a lipid environment as a tetrameric complex composed of two each of the receptors. This complex further recruits G proteins to give rise to an assembly with only two G protein trimers bound to a receptor tetramer. We further demonstrate that receptor heteromerization directly impacts on dopamine-mediated Gi protein activation by modulating the conformation of its α-subunit. Indeed, association to the purified GHSR:D2R heteromer triggers a different active conformation of Gαi that is linked to a higher rate of GTP binding and a faster dissociation from the heteromeric receptor. This is an additional mechanism to expand the repertoire of GPCR signaling modulation that could have implications for the control of dopamine signaling in normal and physiopathological conditions.

RESUMEN: Los métodos de tratamiento convencionales empleados en la gestión de efluentes ácidos con contenido metálico, presentan ciertas limitaciones como la falta de selectividad, limitada eficacia para llegar a los límites de concentración requeridos, generación de residuos, etc. que hacen necesario la búsqueda de soluciones alternativas para su gestión. Además de buscar alternativas que permitan reducir el contenido metálico, su alto valor añadido justifica el desarrollo de nuevas tecnologías de separación que permitan su recuperación para su reutilización. Los ácidos agotados se caracterizan por ser corrientes residuales con elevada concentración de metales con valor añadido como zinc, cobre, níquel, cromo, etc. en presencia de altas concentraciones de hierro y ácidos minerales, habitualmente sulfúrico o clorhídrico. El presente Trabajo Fin de Máster (TFM) incide en la búsqueda de alternativas de tratamiento para la gestión de ácidos agotados en medio sulfato con la siguiente composición: 6,5 g Ni2+ L-1, 3,3 g Cu2+ L-1, 22,4 g Fe2+ L-1 y una acidez libre en torno al 6% (pH≈0,55). Actualmente dicha corriente se trata mediante precipitación química con el objetivo de reducir la acidez y el contenido iónico. Sin embargo, la presencia de elevadas concentraciones de níquel en la torta de filtración, le confiere características de peligrosidad que hacen necesario su depósito en vertederos de residuos peligrosos incrementando los costes globales de gestión. Este TFM tiene como objetivo general reducir la concentración de níquel de ácidos agotados en medio sulfato suministrados por un gestor autorizado de residuos. Como objetivo secundario se plantea la recuperación selectiva de níquel y en su caso de aquellos metales con valor añadido. Para ello se ha seleccionado la tecnología de intercambio iónico empleando una resina quelante de la casa comercial Purolite®, que posee el grupo funcional bis-picolilamina. Los experimentos se han realizado en discontinuo empleando un tanque agitado con control de pH el cual se fijó en un valor de 2,0 ya que es el mínimo valor para el que se demostró que el agente de separación sigue siendo activo. Se realizaron varios grupos de experimentos tanto de carga como de regeneración empleando disoluciones sintéticas de níquel y níquel-cobre y ácidos agotados reales con altas concentraciones de hierro. Las resinas se regeneraron en dos etapas; primero con ácido sulfúrico para recuperar el níquel captado, y a continuación con hidróxido amónico para recuperar el cobre captado. Finalmente, se ha estudiado la estabilidad de la resina cuando se somete a ciclos consecutivos de carga-descarga. Como principal resultado cabe mencionar que las resinas empleadas captan de forma simultánea níquel y cobre siendo este último, el que se adsorbe de forma preferencial. Las cinéticas de eliminación de ambos metales son muy rápidas cuando se emplean disoluciones sintéticas, consiguiéndose reducir sus concentraciones iniciales alrededor de un 90% en los primeros 10 minutos de operación. Los porcentajes de eliminación aumentan hasta un 95-99% cuando se alcanzan las condiciones de estado estacionario. La cinética de eliminación de níquel se ve ralentizada cuando se trabaja con disoluciones reales en presencia de hierro debido a posibles problemas de precipitación local o competición, obteniéndose porcentajes de eliminación del 90% tras 50 minutos de operación. En cuanto a la regeneración de la resina, en disoluciones monocomponente y bicomponente se ha conseguido recuperar el 100% del níquel a los 4 ciclos de experimentación, de dos horas cada uno, empleándose ácido sulfúrico 1 mol L-1, mientras que en las aguas reales no es capaz de liberar todo el contenido de níquel para el mismo tiempo de experimentación, ya que se recupera el ≈80% del níquel en disolución. En cuanto al cobre, se consigue recuperar el 80% para aguas reales, y en disoluciones sintéticas, el 40% no es liberado de la resina tras tres ciclos de experimentación con hidróxido amónico 2 mol L-1 de una hora cada uno, para ambos casos. Por último, en cuanto a la efectividad de la resina a lo largo del tiempo, se puede confirmar su eficacia para la captación de níquel para el intervalo de trabajo analizado. Se han realizado 8 ciclos de experimentación, en los que se ha capturado prácticamente el 100% de níquel para todos los experimentos independientemente de la disolución utilizada, sin disminuir la eficacia del proceso. Se confirma que la tecnología de intercambio iónico con resinas quelantes es una alternativa eficaz para mejorar la eficacia de la gestión de ácidos agotados en medio sulfato con contenido en níquel y cobre. Una vez analizada la viabilidad de las etapas de carga y regeneración, se ha comprobado la estabilidad del agente de separación mediante la realización de ciclos experimentales en los que no se observaron pérdidas de eficacia significativa. A pesar de los prometedores resultados obtenidos hasta el momento, la viabilidad del cambio de escala queda condicionada a un estudio más detallado en materia de competición entre especies metálicas, optimización de las condiciones de regeneración, integración de la tecnología en el proceso de gestión, etc. ABSTRACT: The conventional treatment methods used in the management of metal-containing acidic effluents present some limitations such as lack of selectivity, limited efficiency to reach the required concentration limits, waste generation, etc. which make it necessary to find alternative solutions for its management. In addition to seeking alternatives to reduce metal content, its high added value promotes the development of new separation technologies that allow its recovery for reuse. Spent acids are characteristic because they are residual solutions with high concentration of high value metals such as zinc, copper, nickel, chromium, etc. in the presence of high concentrations of iron and mineral acids, usually sulfuric or hydrochloric acids. The aim of the present work is to find alternative treatments for the management of spent acids in sulphate medium with the following composition: 6,5 g Ni2+ L-1, 3,3 g Cu2+ L-1, 22,4 g Fe2+ L-1 and a free acidity around 6% (pH≈0.55). This stream is currently treated by chemical precipitation in order to reduce acidity and ionic content. However, the high concentration of nickel in the filtration product makes necessary to be deposited in hazardous waste landfills, increasing the overall management costs. This work has the main objective of reducing the nickel concentration in spent acid, supplied by an authorized waste enterprise. A secondary objective is to obtain the selective recovery of nickel, and other high value metals when it proceeds. For this work, the ion exchange technology has been selected using a chelating resin of Purolite®, which has the bis-picolylamine functional group. Batch experiments were carried out using a stirred tank with pH control, that was set at a value of 2, corresponding to the minimum pH value in which the separation agent stays active. Several groups of experiments were carried out on both loading and regeneration, using synthetic solutions of nickel and nickel-copper and real spent acids with high concentrations of iron. The resins were regenerated in two steps; first with sulfuric acid to recover the nickel collected, and then with ammonium hydroxide to recover the captured copper. Finally, the stability of the resin has been studied through consecutive load-discharge cycles. As a main result, it should be mentioned that the resins used simultaneously captured nickel and copper. Resins have a preference for copper. The elimination kinetics of both metals was very rapid when using synthetic solutions, and reduced their initial concentrations by about 90% in the first 10 minutes of operation. Elimination rates increased up to 95-99% when steady-state conditions were reached. Nickel elimination kinetics were slowed down when iron was presented in the real solutions due to potential precipitation or competition problems among the elements, resulting in removal percentages of 90% after 50 minutes of operation. As for the regeneration of the resin, it has been possible to recover 100% of the nickel at the 4 cycles of experimentation, of two hours each, using 1 mol/L sulfuric acid, whereas in the real waters it was not able to release all the nickel content for the same experimentation time as it recovered ≈80% of the nickel in solution. As for copper, it was possible to recover 80% for real water, and in synthetic solutions, 40% was not released from the resin after three cycles of experimentation with ammonium hydroxide 2 mol/L of one hour each, for both cases. Finally, regarding the effectiveness of the resin over time, its effectiveness for the uptake of nickel for the series of experiments analyzed was confirmed. Eight cycles of experimentation have been performed, in which almost 100% of nickel has been captured for all experiments regardless of the solution used, without reducing the efficiency of the process. It was confirmed that ion exchange technology with chelating resins is an effective alternative to improve the efficiency of the management of spent acids in sulphate medium containing nickel and copper. Once the viability of the loading and regeneration steps has been analyzed, the stability of the separation agent has been checked by performing experimental cycles in which no significant loss of efficiency was observed. In spite of the promising results obtained so far, the feasibility of the scale change is conditioned to a more detailed study on competition between metal species, optimization of regeneration conditions, integration of technology in the management process, etc. Máster en Ingeniería Química

Introducing a second chiral center on our previously described 1,2,4-triazole, allowed us to increase diversity and elongate the 'C-terminal part' of the molecule. Therefore, we were able to explore mimics of the substance P analogs described as inverse agonists. Some compounds presented affinities in the nanomolar range and potent biological activities, while one exhibited a partial inverse agonist behavior similar to a Substance P analog.

International audience; The dynamic character of G protein-coupled receptors is essential to their function. However, the details of how ligands stabilize a particular conformation to selectively activate a signaling pathway and how signaling proteins affect this conformational repertoire remain unclear. Using a prototypical peptide-activated class A G protein-coupled receptor (GPCR), the ghrelin receptor, reconstituted as a monomer into lipid discs and labeled with a fluorescent conformational reporter, we demonstrate that ligand efficacy and functional selectivity are directly related to different receptor conformations. Of importance, our data bring direct evidence that distinct effector proteins affect the conformational landscape of the ghrelin receptor in different ways. Whereas G proteins affect the balance between active and inactive receptor substates in favor of the active state, agonist-induced arrestin recruitment is accompanied by a marked change in the structural features of the receptor that adopt a conformation different from that observed in the absence of arrestin. In contrast to G proteins and arrestins, μ-AP2 has no significant effect on the organization of the transmembrane core of the receptor. Such a modulation of a GPCR conformational landscape by pharmacologically distinct ligands and effectors provides insights into the structural bases that decisively affect ligand efficacy and subsequent biological responses. This is also likely to have major implications for the design of drugs activating specific GPCR-associated signaling pathways.

Despite its central role in signaling and the potential therapeutic applications of inverse agonists, the molecular mechanisms underlying G protein-coupled receptor (GPCR) constitutive activity remain largely to be explored. In this context, ghrelin receptor GHS-R1a is a peculiar receptor in the sense that it displays a strikingly high, physiologically relevant, constitutive activity. To identify the molecular mechanisms responsible for this high constitutive activity, we have reconstituted a purified GHS-R1a monomer in a lipid disc. Using this reconstituted system, we show that the isolated ghrelin receptor per se activates Gq in the absence of agonist, as assessed through guanosine 5′-O-(thiotriphosphate) binding experiments. The measured constitutive activity is similar in its extent to that observed in heterologous systems and in vivo. This is the first direct evidence for the high constitutive activity of the ghrelin receptor being an intrinsic property of the protein rather than the result of influence of its cellular environment. Moreover, we show that the isolated receptor in lipid discs recruits arrestin-2 in an agonist-dependent manner, whereas it interacts with μ-AP2 in the absence of ligand or in the presence of ghrelin. Of importance, these differences are linked to ligand-specific GHS-R1a conformations, as assessed by intrinsic fluorescence measurements. The distinct ligand requirements for the interaction of purified GHS-R1a with arrestin and AP2 provide a new rationale to the differences in basal and agonist-induced internalization observed in cells.

There is compelling evidence that G protein-coupled receptors exist as homo- and heterodimers, but the way these assemblies function at the molecular level remains unclear. We used here the purified leukotriene B4 receptor BLT1 stabilized in its dimeric state to analyze how a receptor dimer activates G proteins. For this, we produced heterodimers between the wild-type BLT1 and a BLT1/ALXR chimera. The latter is no longer activated by leukotriene B4 but is still activated by ALXR agonists. In this heterodimer, agonist binding to either one of the two protomers induced asymmetric conformational changes within the receptor dimer. Of importance, no G protein activation was observed when using a dimer where the ligand-loaded protomer was not able to trigger GDP/GTP exchange due to specific mutations in its third intracellular loop, establishing that the conformation of the agonist-free protomer is not competent for G protein activation. Taken together, these data indicate that although ligand binding to one protomer in the heterodimer is associated with cross-conformational changes, a trans-activation mechanism where the ligand-free subunit would trigger GDP/GTP exchange cannot be considered in this case for G protein activation. This observation sheds light into the way GPCR dimers, in particular heterodimers, could activate their cognate G proteins.

G protein-coupled receptors (GPCRs) are integral membrane proteins that play a pivotal role in signal transduction. Understanding their dynamics is absolutely required to get a clear picture of how signaling proceeds. Molecular characterization of GPCRs isolated in detergents nevertheless stumbles over the deleterious effect of these compounds on receptor function and stability. We explored here the potential of a styrene-maleic acid polymer to solubilize receptors directly from their lipid environment. To this end, we used two GPCRs, the melatonin and ghrelin receptors, embedded in two membrane systems of increasing complexity, liposomes and membranes from Pichia pastoris. The styrene-maleic acid polymer was able, in both cases, to extract membrane patches of a well-defined size. GPCRs in SMA-stabilized lipid discs not only recognized their ligand but also transmitted a signal, as evidenced by their ability to activate their cognate G proteins and recruit arrestins in an agonist-dependent manner. Besides, the purified receptor in lipid discs undergoes all specific changes in conformation associated with ligand-mediated activation, as demonstrated in the case of the ghrelin receptor with fluorescent conformational reporters and compounds from distinct pharmacological classes. Altogether, these data highlight the potential of styrene-maleic stabilized lipid discs for analyzing the molecular bases of GPCR-mediated signaling in a well-controlled membrane-like environment.

International audience; The G protein-coupled receptor GHS-R1a mediates ghrelin-induced growth hormone secretion, food intake, and reward-seeking behaviors. GHS-R1a signals through Gq, Gi/o, G13, and arrestin. Biasing GHS-R1a signaling with specific ligands may lead to the development of more selective drugs to treat obesity or addiction with minimal side effects. To delineate ligand selectivity at GHS-R1a signaling, we analyzed in detail the efficacy of a panel of synthetic ligands activating the different pathways associated with GHS-R1a in HEK293T cells. Besides β-arrestin2 recruitment and ERK1/2 phosphorylation, we monitored activation of a large panel of G protein subtypes using a bioluminescence resonance energy transfer-based assay with G protein-activation biosensors. We first found that unlike full agonists, Gq partial agonists were unable to trigger β-arrestin2 recruitment and ERK1/2 phosphorylation. Using G protein-activation biosensors, we then demonstrated that ghrelin promoted activation of Gq, Gi1, Gi2, Gi3, Goa, Gob, and G13 but not Gs and G12. Besides, we identified some GHS-R1a ligands that preferentially activated Gq and antagonized ghrelin-mediated Gi/Go activation. Finally, we unambiguously demonstrated that in addition to Gq, GHS-R1a also promoted constitutive activation of G13. Importantly, we identified some ligands that were selective inverse agonists toward Gq but not of G13. This demonstrates that bias at GHS-R1a signaling can occur not only with regard to agonism but also to inverse agonism. Our data, combined with other in vivo studies, may facilitate the design of drugs selectively targeting individual signaling pathways to treat only the therapeutically relevant function.

International audience; How G protein-coupled receptor conformational dynamics control G protein coupling to trigger signaling is a key but still open question. We addressed this question with a model system composed of the purified ghrelin receptor assembled into lipid discs. Combining receptor labeling through genetic incorporation of unnatural amino acids, lanthanide resonance energy transfer, and normal mode analyses, we directly demonstrate the occurrence of two distinct receptor:Gq assemblies with different geometries whose relative populations parallel the activation state of the receptor. The first of these assemblies is a preassembled complex with the receptor in its basal conformation. This complex is specific of Gq and is not observed with Gi. The second one is an active assembly in which the receptor in its active conformation triggers G proteinactivation. The active complex is present even in the absence of agonist, in a direct relationship with the high constitutive activity of the ghrelin receptor. These data provide direct evidence of a mechanism for ghrelin receptor-mediated Gq signaling in which transition of the receptor from an inactive to an active conformation is accompanied by a rearrangement of a preassembled receptor: G protein complex, ultimately leading to G protein activation and signaling.

RESUMEN: El presente trabajo tuvo como objetivo el estudio del acoplamiento que existe entre los lazos de control de una columna de flotación piloto a escala laboratorio. Este trabajo se centró específicamente en el trabajo con los lazos de control del flujo de colas con el nivel, y del lazo del flujo de aire con el holdup. Primero se realizaron en el laboratorio diversos saltos en escalón a lazo abierto con el objetivo de obtener unas gráficas que reflejasen dichos saltos, y a partir de ellas se hallaron las funciones de transferencia que permitieron identificar el sistema. La siguiente etapa se realizó en simulación en Simulink® y constó de las siguientes partes: una vez obtenidas las funciones de transferencia en laboratorio, se procedió a la sintonía del controlador PID. Después, se realizaron diferentes pruebas de sintonía entre los lazos de control de la columna, es decir, se sintonizaron ambos lazos con diferentes parámetros del PID (más lentos o más rápidos) y se probaron diferentes configuraciones, con la finalidad de intentar reducir el acoplamiento entre los lazos de flujo de colas y flujo de aire. Se estudiaron los parámetros del tiempo de estabilización y la integral del error para las diferentes configuraciones y se llegó a la conclusión de que la mejor configuración es con ambos lazos sintonizados de manera lenta (los parámetros del PID tienen valores bajos). Los resultados obtenidos en simulación en estas etapas descritas se validaron en laboratorio. Se procedió a la identificación de la existencia de interacciones entre los lazos mediante el método de la matriz de ganancias relativas (RGA). Una vez realizado esto, se calcularon los desacopladores estáticos manualmente, y se procedieron a aplicar en la simulación, obteniéndose que los resultados no son los esperados, ya que los valores de la integral del error y del tiempo de estabilización son peores al ser aplicados. ABSTRACT: This work´s aim was the study of the accomplishment that exists between control loops of a pilot flotation column, laboratory scale. This work was specifically focused on the work with control loops of tails flux with level, and the air flux with holdup. First, jump steps in open loops were made at the laboratory, with the aim to obtain some graphs which reflected that jumps, and from them, transfer functions that allow identifying the system were found. The next step was made by simulation in Simulink®, and it had the following parts: once the transfer functions were obtained in laboratory, the tuning of the PID controller was made. After that, different kind of tuning between column control loops were made (slower or faster) and different configurations were tried, with the purpose of trying to reduce the accomplishment between tails flux loop and air flux loop. The parameters of stabilization time and error integral were studied for the different configurations, and it was concluded that the best configuration was with both slower tuned loops (the PID parameters were lower). The results obtained in simulations in these described stages were validated in the laboratory. One of the last stages was the identification of the existence of interactions between the control loops by the method of the relative gain array (RGA). Once this step was made, the decoupling was calculated by hand, and it was applied in simulation. The results were not the expected ones. This is why the values of the integral error and the stabilization time were the worst ones to be applied. Grado en Ingeniería Química

Cadherin-mediated cell-cell adhesion is a dynamic process that is regulated during embryonic development, cell migration, and differentiation. Different cadherins are expressed in specific tissues consistent with their roles in cell type recognition. In this study, we examine the formation of N-cadherin–dependent cell-cell contacts in fibroblasts and myoblasts. In contrast to E-cadherin, both endogenous and ectopically expressed N-cadherin shuttles between an intracellular and a plasma membrane pool. Initial formation of N-cadherin–dependent cell-cell contacts results from the recruitment of the intracellular pool of N-cadherin to the plasma membrane. N-cadherin also localizes to the Golgi apparatus and both secretory and endocytotic vesicles. We demonstrate that the intracellular pool of N-cadherin is tightly associated with the microtubule (MT) network and that junction formation requires MTs. In addition, localization of N-cadherin to the cortex is dependent on an intact F-actin cytoskeleton. We show that N-cadherin transport requires the MT network as well as the activity of the MT-associated motor kinesin. In conclusion, we propose that N-cadherin distribution is a regulated process promoted by cell-cell contact formation, which controls the biogenesis and turnover of the junctions through the MT network.

International audience; In this study we show that expression of active Cdc42Hs and Rac1 GTPases, two Rho family members, leads to the reorganization of the vimentin intermediate filament (IF) network, showing a perinuclear collapse. Cdc42Hs displays a stronger effect than Rac1 as 90% versus 75% of GTPase-expressing cells show vimentin collapse. Similar vimentin IF modifications were observed when endogenous Cdc42Hs was activated by bradykinin treatment, endogenous Rac1 by platelet-derived growth factor/epidermal growth factor, or both endogenous proteins upon expression of active RhoG. This reorganization of the vimentin IF network is not associated with any significant increase in soluble vimentin. Using effector loop mutants of Cdc42Hs and Rac1, we show that the vimentin collapse is mostly independent of CRIB (Cdc42Hs or Rac-interacting binding)-mediated pathways such as JNK or PAK activation but is associated with actin reorganization. This does not result from F-actin depolymerization, because cytochalasin D treatment or Scar-WA expression have merely no effect on vimentin organization. Finally, we show that genistein treatment of Cdc42 and Rac1-expressing cells strongly reduces vimentin collapse, whereas staurosporin, wortmannin, LY-294002, R(p)-cAMP, or RII, the regulatory subunit of protein kinase A, remain ineffective. Moreover, we detected an increase in cellular tyrosine phosphorylation content after Cdc42Hs and Rac1 expression without modification of the vimentin phosphorylation status. These data indicate that Cdc42Hs and Rac1 GTPases control vimentin IF organization involving tyrosine phosphorylation events.

Ascorbic acid (AA) plays a significant role in preventing photobiological damage in human skin which could lead to cutaneous disorders such as premature aging or skin cancer. The aim of this work was to assess AA concentrations in human dermis by a microdialysis technique associated with gas chromatography-mass spectrometry (GC-MS). Due to the non-volatility of AA, it was necessary to derivatize this acid. Three methods, one methylation and two silylations, were validated and compared to determine the most sensitive. To validate the methods, calibration curves were plotted from AA concentrations ranging from 34 to 500 nmol mL−1. The calibration curves were linear with a good correlation coefficient ( r ≥ 0.99). Repeatability and reproducibility were significant with a coefficient of variation of less than 10%. The accuracy of the techniques was meaningful as the bias was low (ranging from −5.6 to 5.0%). According to our results, silylation was the most sensitive method to assess AA. Thus, this method was performed to determine AA concentrations in microdialysates. In order to study AA in human dermis, a microdialysis method was used to sample AA and the GC-MS technique used to assess this acid in the microdialysates. Microdialysis can only partially collect AA from human dermis. An ex vivo recovery of AA was 30 ± 2% ( n = 7). The average concentration of AA in microdialysates was 250 ± 66 nmol mL−1 (Mean ± SD, n = 5). In view of the AA recovery, AA concentrations in human skin ranged from 759 to 891 nmol mL−1.

Glutamate-induced glutamate release may be involved in the delayed neuronal death induced by N-methyl-D-aspartate (NMDA). In order to examine a possible modulatory effect of the presynaptic group III mGluRs on glutamate excitotoxicity, the effect of L-2-amino-4-phosphonobutyrate (L-AP4) was examined on NMDA-induced delayed death of mouse cerebellar granule neurons in culture. We found that L-AP4, at high concentration (in the millimolar range), inhibited in a non-competitive manner the NMDA-induced toxicity. This effect was mimicked by high concentration of L-serine-o-phosphate (L-SOP), and was inhibited by pertussis toxin (PTX) indicating the involvement of a Gi/o protein. This suggests the involvement of mGluR7 in the L-AP4 effect, and this was consistent with the detection of both mGluR7 protein and mRNA in these cultured neurons. To examine the mechanism of the L-AP4-induced protection from excitotoxic damage, the effect of L-AP4 on glutamate release was examined. L-AP4 (> or = 1 mM) noncompetitively inhibited by more than 60% the glutamate release induced by NMDA during the insult. We also observed that the 10-min NMDA receptor stimulation resulted in a dramatic increase in the extracellular glutamate concentration reaching 6000% of the control value 24 h after the insult. This large increase was also inhibited when NMDA was applied in the presence of > or = 1 mM L-AP4. Part of the L-AP4-induced protection from excitotoxic damage of granule neurons may therefore result from the inhibition of the vicious cycle: dying cells release glutamate, glutamate induced cell death. The present results add to the hypothesis that presynaptic mGluRs, probably mGluR7, may be the targets of drugs decreasing glutamate release and then neuronal death observed in some pathological situations.

Metabotropic glutamate receptors (mGlu receptors), the Ca2+-sensing receptor, gamma-aminobutyric acid type B receptors, and one group of pheromone receptors constitute a unique family (also called family 3) of heptahelical receptors. This original family shares no sequence similarity with any other G protein-coupled receptors. The identification and comparison of the molecular determinants of receptor/G protein coupling within the different receptor families may help identify general rules involved in this protein/protein interaction. In order to detect possible contact sites important for coupling selectivity between family 3 receptors and the G protein alpha-subunits, we examined the coupling of the cyclase-inhibiting mGlu2 and mGlu4 receptors to chimeric alphaq-subunits bearing the 5 extreme C-terminal amino acid residues of either Galphai, Galphao, or Galphaz. Whereas mGlu4 receptor activated all three chimeric G proteins, mGlu2 receptor activated Galphaqi and Galphaqo but not Galphaqz. The mutation of isoleucine -4 of Galphaqz into cysteine was sufficient to recover coupling of the mutant G protein to mGlu2 receptor. Moreover, the mutation of cysteine -4 of Galphaqo into isoleucine was sufficient to suppress the coupling to mGlu2 receptor. Mutations at positions -5 and -1 had an effect on coupling efficiency, but not selectivity. Our results emphasize the importance of the residue -4 of the alpha-subunits in their specific interaction to heptahelical receptors by extending this finding on the third family of G protein-coupled receptors.

G protein-coupled receptors are at a central node of all cell communications. Investigating their molecular functioning is therefore crucial for both academic purposes and drug design. However, getting the receptors as isolated, stable and purified proteins for such studies still stumbles over their instability out of the membrane environment. Different membrane-mimicking environments have been developed so far to increase the stability of purified receptors. Among them are amphipols. These polymers not only preserve the native fold of receptors purified from membrane fractions but they also allow specific applications such as folding receptors purified from inclusion bodies back to their native state. Of importance, amphipol-trapped G protein-coupled receptors essentially maintain their pharmacological properties so that they are perfectly adapted to further investigate the molecular mechanisms underlying signaling processes. We review here how amphipols have been used to refold and stabilize detergent-solubilized purified receptors and what are the main subsequent molecular pharmacology analyses that were performed using this strategy.

The dynamic character of GPCRs (G-protein-coupled receptors) is essential to their function. However, the details of how ligands and signalling proteins stabilize a receptor conformation to trigger the activation of a given signalling pathway remain largely unexplored. Multiple data, including recent results obtained with the purified ghrelin receptor, suggest a model where ligand efficacy and functional selectivity are directly related to different receptor conformations. Importantly, distinct effector proteins (G-proteins and arrestins) as well as ligands are likely to affect the conformational landscape of GPCRs in different manners, as we show with the isolated ghrelin receptor. Such modulation of the GPCR conformational landscape by pharmacologically distinct ligands and effector proteins has major implications for the design of new drugs that activate specific signalling pathways.

Heterodimerization of G protein-coupled receptors has an impact on their signaling properties, but the molecular mechanisms underlying heteromer-directed selectivity remain elusive. Using purified monomers and dimers reconstituted into lipid discs, we explored how dimerization impacts the functional and structural behavior of the ghrelin receptor. In particular, we investigated how a naturally occurring truncated splice variant of the ghrelin receptor exerts a dominant negative effect on ghrelin signaling upon dimerization with the full-length receptor. We provide direct evidence that this dominant negative effect is due to the ability of the non-signaling truncated receptor to restrict the conformational landscape of the full-length protein. Indeed, associating both proteins within the same disc blocks all agonist- and signaling protein-induced changes in ghrelin receptor conformation, thus preventing it from activating its cognate G protein and triggering arrestin 2 recruitment. This is an unambiguous demonstration that allosteric conformational events within dimeric assemblies can be directly responsible for modulation of signaling mediated by G protein-coupled receptors.

Two proteins with seven transmembrane-spanning domains typical of guanosine-nucleotide-binding-protein-coupled receptors have been identified as cannabinoid receptors; the central cannabinoid receptor, CB1, and the peripheral cannabinoid receptor, CB2, initially described in rat brain and spleen, respectively. Here, we report the distribution patterns for both CB1 and CB2 transcripts in human immune cells and in several human tissues, as analysed using a highly sensitive and quantitative PCR-based method. CB1 was mainly expressed in the central nervous system and, to a lower extent, in several peripheral tissues such as adrenal gland, heart, lung, prostate, uterus, ovary, testis, bone marrow, thymus and tonsils. In contrast, the CB2 gene, which is not expressed in the brain, was particularly abundant in immune tissues, with an expression level 10-100-fold higher than that of CB1. Although CB2 mRNA was also detected in some other peripheral tissues, its level remained very low. In spleen and tonsils, the CB2 mRNA content was equivalent to that of CB1 mRNA in the central nervous system. Among the main human blood cell subpopulations, the distribution pattern of the CB2 mRNA displayed important variations. The rank order of CB2 mRNA levels in these cells was B-cells > natural killer cells >> monocytes > polymorphonuclear neutrophil cells > T8 cells > T4 cells. The same rank order was also established in human cell lines belonging to the myeloid, monocytic and lymphoid lineages. The prevailing expression of the CB2 gene in immune tissues was confirmed by Northern-blot analysis. In addition, the expression of the CB2 protein was demonstrated by an immunohistological analysis performed on tonsil sections using specific anti-(human CB2) IgG; this experiment showed that CB2 expression was restricted to B-lymphocyte-enriched areas of the mantle of secondary lymphoid follicles. These results suggest that (a) CB1 and CB2 can be considered as tissue-selective antigens of the central nervous system and immune system, respectively, and (b) cannabinoids may exert specific receptor-mediated actions on the immune system through the CB2 receptor.

International audience; Nonionic amphipols (NAPols) synthesized by homotelomerization of an amphiphatic monomer are able to keep membrane proteins (MPs) stable and functional in the absence of detergent. Some of their biochemical and biophysical properties and applications have been examined, with particular attention being paid to their complementarity with the classical polyacrylate-based amphipol A8-35. Bacter-iorhodopsin (BR) from Halobacterium salinarum and the cytochrome b 6 f complex from Chlamydomonas reinhardtii were found to be in their native state and highly stable following complexation with NAPols. NAPol-trapped BR was shown to undergo its complete photocycle. Because of the pH insensitivity of NAPols, solution nuclear magnetic resonance (NMR) two-dimensional 1 H− 15 N heteronuclear single-quantum coherence spectra of NAPol-trapped outer MP X from Escherichia coli (OmpX) could be recorded at pH 6.8. They present a resolution similar to that of the spectra of OmpX/A8-35 complexes recorded at pH 8.0 and give access to signals from solvent-exposed rapidy exchanging amide protons. Like A8-35, NAPols can be used to fold MPs to their native state as demonstrated here with BR and with the ghrelin G protein-coupled receptor GHS-R1a, thus extending the range of accessible folding conditions. Following NAPol-assisted folding, GHS-R1a bound four of its specific ligands, recruited arrestin-2, and activated binding of GTPγS by the G αq protein. Finally, cell-free synthesis of MPs, which is inhibited by A8-35 and sulfonated amphipols, was found to be very efficient in the presence of NAPols. These results open broad new perspectives on the use of amphipols for MP studies. S

Despite its central role in signaling and the potential therapeutic applications of inverse agonists, the molecular mechanisms underlying G protein-coupled receptor (GPCR) constitutive activity remain largely to be explored. In this context, ghrelin receptor GHS-R1a is a peculiar receptor in the sense that it displays a strikingly high, physiologically relevant, constitutive activity. To identify the molecular mechanisms responsible for this high constitutive activity, we have reconstituted a purified GHS-R1a monomer in a lipid disc. Using this reconstituted system, we show that the isolated ghrelin receptor per se activates G(q) in the absence of agonist, as assessed through guanosine 5'-O-(thiotriphosphate) binding experiments. The measured constitutive activity is similar in its extent to that observed in heterologous systems and in vivo. This is the first direct evidence for the high constitutive activity of the ghrelin receptor being an intrinsic property of the protein rather than the result of influence of its cellular environment. Moreover, we show that the isolated receptor in lipid discs recruits arrestin-2 in an agonist-dependent manner, whereas it interacts with μ-AP2 in the absence of ligand or in the presence of ghrelin. Of importance, these differences are linked to ligand-specific GHS-R1a conformations, as assessed by intrinsic fluorescence measurements. The distinct ligand requirements for the interaction of purified GHS-R1a with arrestin and AP2 provide a new rationale to the differences in basal and agonist-induced internalization observed in cells.

International audience; The growth hormone secretagogue receptor type 1a (GHS-R1a) belongs to class A G-protein-coupled receptors (GPCR). This receptor mediates pleiotropic effects of ghrelin and represents a promising target for dysfunctions of growth hormone secretion and energy homeostasis including obesity. Identification of new compounds which bind GHS-R1a is traditionally achieved using radioactive binding assays. Here we propose a new fluorescence-based assay, called Tag-lite binding assay, based on a fluorescence resonance energy transfer (FRET) process between a terbium cryptate covalently attached to a SNAP-tag fused GHS-R1a (SNAP-GHS-R1a) and a high-affinity red fluorescent ghrelin ligand. The long fluorescence lifetime of the terbium cryptate allows a time-resolved detection of the FRET signal. The assay was made compatible with high-throughput screening by using prelabeled cells in suspension under a 384-well plate format. Ki values for a panel of 14 compounds displaying agonist, antagonist, or inverse agonist properties were determined using both the radioactive and the Tag-lite binding assays performed on the same batches of GHS-R1a-expressing cells. Compound potencies obtained in the two assays were nicely correlated. This study is the first description of a sensitive and reliable nonradioactive binding assay for GHS-R1a in a format amenable to high-throughput screening.

G protein-coupled receptors (GPCRs) regulate numerous physiological functions. The primary difficulty presented by their study in vitro is to obtain them in sufficient amounts under a functional and stable form. Escherichia coli is a host of choice for producing recombinant proteins for structural studies. However, the insertion of GPCRs into its plasma membrane usually results in bacterial death. An alternative approach consists of targeting recombinant receptors to inclusion bodies, where they accumulate without affecting bacterial growth, and then folding them in vitro. This approach, however, stumbles over the very low folding yields typically achieved, whether in detergent solutions or in detergent-lipid mixtures. Here, we show that synthetic polymers known as amphipols provide a highly efficient medium for folding GPCRs. Using a generic protocol, we have folded four class A GPCRs to their functional state, as evidenced by the binding of their respective ligands. This strategy thus appears to have the potential to be generalized to a large number of GPCRs. These data are also of interest from a more fundamental point of view: they indicate that the structural information stored in the sequence of these four receptors allows them to reach their correct three-dimensional structure in an environment that bears no similarity, beyond the amphiphilic character, to lipid bilayers.

Previous studies have shown that glutathione S-transferases (GSTs) can operate in synergy with efflux transporters, multi-drug resistance proteins (MRPs), to confer resistance to several carcinogens, mutagens and anticancer drugs. To address the poorly documented role of the GSTM1 in cancer chemoresistance, we used CAL1 human melanoma cells expressing no endogenous GSTM1 and a high level of MRP1. Cells were transfected with an expression vector containing the GSTM1 cDNA, and different clones were selected expressing different levels of GSTM1 (RT-PCR, Western blot, and enzyme activity). Cells overexpressing GSTM1 displayed a 3- to 4-fold increase in resistance to anticancer drugs vincristine (VCR) and chlorambucil (CHB) in proliferation, cytotoxic, and clonogenic survival assays. Inhibitors of MRP1 (sulfinpyrazone, verapamil) and GST (dicumarol, curcumin) completely reversed the GSTM1-associated resistance to VCR, indicating that a MRP efflux function is necessary to potentiate GSTM1-mediated resistance to VCR. Conversely, MRP1 inhibitors had no effect on the sensitivity to CHB. Using immunofluorescence assay, GSTM1 was also shown to protect microtubule network integrity from VCR-induced inhibition of microtubule polymerization. In conclusion, these results show that GSTM1 alone is involved in melanoma resistance to CHB, whereas it can act in synergy with MRP1 to protect cells from toxic effects of VCR.

8-Methoxypsoralen (8-MOP) is currently used in PUVA therapy (psoralen+UVA) to treat dermatological diseases such as psoriasis, vitiligo and atopic dermatitis. The aim of this work was to validate a method for collecting 8-MOP from patient dermis by a non invasive technique, microdialysis, and then to assess this molecule by gas chromatography–mass spectrometry (GC–MS). 5-Methoxypsoralen (5-MOP) was used as an internal standard. The calibration curve demonstrated a linear relationship between the peak areas of 8-MOP and 5-MOP over a wide range of 8-MOP concentrations (0.9–100 ng/ml). Within- and between-run precisions were measured, using four different 8-MOP concentrations, which varied from 98.0 to 102.0% and from 98.5 to 101.8%, respectively. The limits of detection and quantification were 0.29 and 0.52 ng/ml, respectively. The method was validated and then applied to determine the pharmacokinetic of 8-MOP in ten psoriatic patient dermis, after oral intake of this drug. The results demonstrated that the association of microdialysis with the GC–MS method was an efficient procedure to collect and assess 8-MOP in human dermis, in vivo.

Until recently, the determination of metallic elements concentrations in normal skin, in vivo, was rare due to the lack of non-invasive techniques. Microdialysis has the advantage of being slightly invasive when applied to the collection in vivo of endogenous or exogenous substances from the skin. Iron is an active element in different cutaneous disorders. The aim of this work was to assess iron by atomic absorption spectrometry (AAS) after the collection of samples by microdialysis from human dermis. A first essential step, before determining the in vivo iron concentration in human dermis, was to establish an experimental protocol applicable to ex vivo as well as in vivo conditions. For this reason, this work deals only with the assessment of iron in ex vivo human dermis. A skin microdialysis technique and a calibration method, the No Net Flux, were used to quantify basal iron concentrations in human dermis and the same method was also used to determine in vitro and ex vivo iron recoveries. No differences were detected between in vitro and ex vivo recoveries. Ex vivo basal iron dermis concentrations ranged from 3.6 to 7.7 microg/l. This study shows that non-invasive microdialysis is an efficient method for sampling iron from human skin. A sensitive and accurate AAS technique was able to assess low iron concentrations in human dermis. The strategy adopted for this work was efficient and appropriate for the determination of iron in human skin and experiments will be carried out in vivo.

Among phospholipase C-coupled metabotropic glutamate receptors (mGluRs), some have a surprisingly long carboxyl-terminal intracellular domain (mGluR1a, -5a, and -5b), and others have a short one (mGluR1b, -1c, and -1d). All mGluR1 sequences are identical up to 46 residues following the 7th transmembrane domain, followed by 313, 20, 11, and 26 specific residues in mGluR1a, mGluR1b, mGluR1c, and mGluR1d, respectively. Several functional differences have been described between the long isoforms (mGluR1a, -5a, and -5b) and the short ones (mGluR1b, -1c, and -1d). Compared with the long receptors, the short ones induce slower increases in intracellular Ca2+, are activated by higher concentration of agonists, and do not exhibit constitutive, agonist-independent activity. To identify the residues responsible for these functional properties, a series of truncated, chimeric, and mutated receptors were constructed. We found that the deletion of the last 19 carboxyl-terminal residues in mGluR1c changed its properties into those of mGluR1a. Moreover, the exchange of the long carboxyl-terminal domain of mGluR5a with that of mGluR1c generated a chimeric receptor that possessed functional properties similar to those of mGluR1c. Mutagenesis of specific residues within the 19 carboxyl-terminal residues of mGluR1c revealed the importance of a cluster of 4 basic residues in defining the specific properties of this receptor. Since this cluster is part of the sequence common to all mGluR1 variants, we conclude that the long carboxyl-terminal domain of mGluR1a suppresses the inhibitory action of this sequence element.

Three splice variants of the rat metabotropic glutamate receptor 1 (mGlu1a, 1b and 1c receptors) have been characterized so far. All have the same sequence up to the 46th residue following the 7th transmembrane domain, followed by different carboxyl-terminal tails. Whereas mGlu1b and mGlu1c receptors possess a short intracellular carboxyl-terminal tail, the mGlu1a receptor has a very long one. Compared to cells expressing mGlu1b or mGlu1c receptors, a higher agonist potency and basal phospholipase C activity were detected in cells expressing mGlu1a receptors. Another variant with a short carboxyl-terminal tail, the HmGlu1d receptor, has been recently isolated from human brain. Here we show that the mGlu1d receptor variant also exists in the rat. Like all rat mGlu1 receptor variants, the mGlu1d receptor activates phospholipase C upon stimulation with mGlu1 receptor agonists. Although the rank order of agonist potency is the same on mGlu1a and mGlu1d receptors, agonists are less potent in stimulating phospholipase C in mGlu1d receptor-expressing cells than in cells expressing mGlu1a receptors. Moreover, like the other short variants it has no significant constitutive activity. These results indicate that the mGlu1d receptor shares similar functional properties with the other short mGlu1 receptor splice variants, and further suggests that the long carboxyl-terminal tail of the mGlu1a receptor increases phospholipase C coupling efficacy.

The effects of the phenylglycine derivatives, α-methyl-4-carboxyphenylglycine (MCPG), 4-carboxyphenylglycine (4CPG), 4-carboxy-3-hydroxyphenylglycine (4C3HPG), 3-hydroxyphenylglycine (3HPG) and 3,5-dihydrohyphenylglycine (DHPG) were tested on LLC-PK1 cells transiently expressing the rat mGluR1a or mGluR5a receptors. As previously reported by others, ( S )-3HPG and ( RS )-DHPG were found to be partial agonists at mGluR1a, whereas (+)-MCPG, ( S )-4CPG and ( S )-4C3HPG competitively antagonized the effect of Glu. Surprisingly, the 4-carboxy derivatives of phenylglycine antagonized the effect of 1 S ,3 R -ACPD on mGluR1a with lower K B values. On mGluR5a, ( S )-3HPG and ( RS )-DHPG are also partial agonists. However, in contrast to their effects on mGluR1a, ( S )-4CPG did not inhibit the effect of Glu or 1 S ,3 R -ACPD, and ( S )-4C3HPG acted as an agonist at high concentration. Whereas no significant antagonism of the Glu effect on mGluR5a was observed with 1 mM (+)-MCPG, this compound was found to potently and competitively antagonize the effect of 1 S ,3 R -ACPD. Finally, the effect of 4CPG was also examined on cultured cortical and cerebellar neurons that express mGluR5 and mGluR1 mRNA, respectively. 4CPG inhibited 1 S ,3 R -ACPD-stimulated IP production in cerebellar neurons only. These results (1) demonstrate that phenylglycine derivatives can be used to discriminate between effects mediated by mGluR1 and mGluR5 and (2) suggest that the apparent potency of phenylglycine antagonists depends on the agonist used to activate these receptors.