Dissecting the conserved NPxxY motif of the M₃ muscarinic acetylcholine receptor: critical role of Asp-7.49 for receptor signaling and multiprotein complex formation

Acetylcholine challenge produces M-3 muscarinic acetylcholine receptor activation and accessory/scaffold proteins recruitment into a signalsome complex. The dynamics of such a complex is not well understood but a conserved NPxxY motif located within transmembrane 7 and juxtamembrane helix 8 of the receptor was found to modulate G protein activation. Here by means of receptor mutagenesis we unravel the role of the conserved M-3 muscarinic acetylcholine receptor NPxxY motif on ligand binding, signaling and multiprotein complex formation. Interestingly, while a N7.49D receptor mutant showed normal ligand binding properties a N7.49A mutant had reduced antagonist binding and increased affinity for carbachol. Also, besides this last mutant was able to physically couple to G alpha(q/11) after carbachol challenge it was neither capable to activate phospholipase C nor phospholipase D. On the other hand, we demonstrated that the Asn-7.49 is important for the interaction between M3R and ARF1 and also for the formation of the ARF/Rho/beta gamma signaling complex, a complex that might determine the rapid activation and desensitization of PLD. Overall, these results indicate that the NPxxY motif of the M-3 muscarinic acetylcholine receptor acts as key conformational switch for receptor signaling and multiprotein complex formation

Posttranslational modifications of human M3 muscarinic acetylcholine receptor: zooming in its functional implications

The human M3 muscarinic acetylcholine receptor (M3R) regulates many important physiological roles in the central and peripheral nervous systems, and it is involved in the pathophysiology of several neurodegenerative and autoimmune diseases, representing attractive potential pharmacological target for intervention. However, the lack of structural information on this receptor hampered the development of new potent antagonist with increased selectivity and lower side effects. Such structural information can be only achieved by means of experimental biophysical techniques, which require large quantities of pure receptor. Considering that under physiological conditions the expression of G-protein coupled receptors (GPCRs) is relatively low, optimization of the receptor overexpression is a pre-requisite for structural studies efforts to be performed. In addition, although is has well established that GPCR undergo post-translational and increase evidences support that these are tight links to receptor roles, little progress has been made in the post-translational modifications field in some GPCRs, such as the case of M3R. In this study, we provide some strategies to improve muscarinic receptor heterologous expression in mammalian cells guaranteeing proper post-translational modifications. In addition, we have been able to extract high levels of functional receptor from COS-7 cells using a detergent combination tested, and to purify the receptor to near homogeneity-keeping the full wild type receptor properties- by means of different affinity purification methods. Regarding the post-translational modifications studied, our findings provide the first evidence of the critical role that N-glycan chains play in determining muscarinic receptor distribution and localization, as well as in cell integrity. Furthermore, our data reveal a role for palmitoylation in determining M3R residence within lipid raft, as well as in receptor internalization and down-regulationEl receptor muscarínico de acetilcolina subtipo M3 humano (M3R) regula importantes funciones en el sistema nervioso central y periférico, y está implicado en la fisiopatología de varias enfermedades neurodegenerativas y autoinmunes, lo que representa una atractiva diana terapéutica para la intervención farmacológica. Sin embargo, la falta de la información estructural sobre este receptor obstaculizado el desarrollo de nuevos y potente fármacos de gran selectividad y bajo efecto secundario. Tal información estructural, puede lograrse por medio de la experimentación con técnicas biofísicas que requieren grandes cantidades de receptor puro. Teniendo en cuenta que en condiciones fisiológicas la expresión de receptores acoplados a proteínas G (GPCR) es baja, la sobreproducción del receptor es un pre-requisito para que los estudios estructurales puedan ser realizados. Además, aunque se ha establecido que los GPCR sufren modificaciones post-translationales y que en los últimos años un significante número de reportes sugieren que estas modificaciones están estrechamente vinculadas a las funciones del receptor, poco se ha avanzado en el estudio de estas modificaciones en el campo de algunos GPCRs, como es el caso de M3R. En este estudio, nosotros describimos algunas estrategias para mejorar la expresión de los receptores muscarínicos en células de mamíferos garantizando unas correctas modificaciones post-translacionales. Además, hemos sido capaces de extraer altos niveles de receptor funcional a partir de células COS-7 con una combinación de detergentes, purificamos el receptor M3R cerca de la homogeneidad, mantenimiento de la totalidad de las propiedades biológicas encontradas en el receptor silvestre. En relación a las modificaciones post-translationales estudiadas, nuestros resultados proporcionan la primera evidencia del papel crítico de las cadenas de N-glicanos en la determinación de la localización de estos receptores, así como en la integridad celular. Además, nuestros datos revelan un importante papel de las modificaciones lipídicas de M3R en relación a la distribución del receptor en microdominios resistente a detergentes, así como en la regulación del receptor. En resumen, las estrategias utilizadas pueden contribuir al incremento de la expresión M3R. De esta forma los esfuerzos para la purificación del receptor a gran escala pueden ser iniciados. Para ellos, nosotros revelamos una posible estrategia. Además, proponemos los posibles sitios de N-glicosilación y S-acilación en el M3R expresado en células COS-7, y proporcionamos evidencias experimentales que avalan la implicación funcional de estas modificaciones en el papel del receptor

The existence of FGFR1-5-HT1A receptor heterocomplexes in midbrain 5-HT neurons of the rat: relevance for neuroplasticity

The ascending midbrain 5-HT neurons to the forebrain may be dysregulated in depression and have a reduced trophic support. With in situ proximity ligation assay (PLA) and supported by coimmunoprecipitation and colocation of the FGFR1 and 5-HT1A immunoreactivities in the midbrain raphe cells, evidence for the existence of FGFR1-5-HT1A receptor heterocomplexes in the dorsal and median raphe nuclei of the Sprague Dawley rat as well as in the rat medullary raphe RN33B cells has been obtained. Especially after combined FGF-2 and 8-OH-DPAT treatment, a marked and significant increase in PLA clusters was found in the RN33B cells. Similar results were reached with the FRET technique in HEK293T cells, where TM-V of the 5HT1A receptor was found to be part of the receptor interface. The combined treatment with FGF-2 and the 5-HT1A agonist also synergistically increased FGFR1 and ERK1/2 phosphorylation in the raphe midline area of the midbrain and the RN33B cells as well as their differentiation, as seen from development of the increased number and length of extensions per cell and their increased 5-HT immunoreactivity. These signaling and differentiation events were dependent on the receptor interface since they were blocked by incubation with TM-V but not by TM-II. Together, the results indicate that the 5-HT1A autoreceptors by being part of a FGFR1-5-HT1A receptor heterocomplex in the midbrain raphe 5-HT nerve cells appear to have a trophic role in the central 5-HT neuron systems in addition to playing a key role in reducing the firing of these neuron

On the existence and function of galanin receptor heteromers in the central nervous system

Galanin receptor (GalR) subtypes 1-3 linked to central galanin neurons may form heteromers with each other and other types of G protein-coupled receptors in the central nervous system (CNS). These heteromers may be one molecular mechanism for galanin peptides and their N-terminal fragments (gal 1-15) to modulate the function of different types of glia-neuronal networks in the CNS, especially the emotional and the cardiovascular networks. GalR-5-HT1A heteromers likely exist with antagonistic GalR-5-HT1A receptor-receptor interactions in the ascending midbrain raphe 5-HT neuron systems and their target regions. They represent a novel target for antidepressant drugs. Evidence is given for the existence of GalR1-5-HT1A heteromers in cellular models with trans-inhibition of the protomer signaling. A GalR1-GalR2 heteromer is proposed to be a galanin N-terminal fragment preferring receptor (1-15) in the CNS. Furthermore, a GalR1-GalR2-5-HT1A heterotrimer is postulated to explain why only galanin (1-15) but not galanin (1-29) can antagonistically modulate the 5-HT1A receptors in the dorsal hippocampus rich in gal fragment binding sites. The results underline a putative role of different types of GalR-5-HT1A heteroreceptor complexes in depression. GalR antagonists may also have therapeutic actions in depression by blocking the antagonistic GalR-NPYY1 receptor interactions in putative GalR-NPYY1 receptor heteromers in the CNS resulting in increases in NPYY1 transmission and antidepressant effects. In contrast the galanin fragment receptor (a postulated GalR1-GalR2 heteromer) appears to be linked to the NPYY2 receptor enhancing the affinity of the NPYY2 binding sites in a putative GalR1-GalR2-NPYY2 heterotrimer. Finally, putative GalR-α2-adrenoreceptor heteromers with antagonistic receptor-receptor interactions may be a widespread mechanism in the CNS for integration of galanin and noradrenaline signals also of likely relevance for depressio

Dissecting the conserved NPxxY motif of the M₃ muscarinic acetylcholine receptor: critical role of Asp-7.49 for receptor signaling and multiprotein complex formation

Acetylcholine challenge produces M-3 muscarinic acetylcholine receptor activation and accessory/scaffold proteins recruitment into a signalsome complex. The dynamics of such a complex is not well understood but a conserved NPxxY motif located within transmembrane 7 and juxtamembrane helix 8 of the receptor was found to modulate G protein activation. Here by means of receptor mutagenesis we unravel the role of the conserved M-3 muscarinic acetylcholine receptor NPxxY motif on ligand binding, signaling and multiprotein complex formation. Interestingly, while a N7.49D receptor mutant showed normal ligand binding properties a N7.49A mutant had reduced antagonist binding and increased affinity for carbachol. Also, besides this last mutant was able to physically couple to G alpha(q/11) after carbachol challenge it was neither capable to activate phospholipase C nor phospholipase D. On the other hand, we demonstrated that the Asn-7.49 is important for the interaction between M3R and ARF1 and also for the formation of the ARF/Rho/beta gamma signaling complex, a complex that might determine the rapid activation and desensitization of PLD. Overall, these results indicate that the NPxxY motif of the M-3 muscarinic acetylcholine receptor acts as key conformational switch for receptor signaling and multiprotein complex formation

Dissecting the conserved NPxxY motif of the M₃ muscarinic acetylcholine receptor: critical role of Asp-7.49 for receptor signaling and multiprotein complex formation

Acetylcholine challenge produces M-3 muscarinic acetylcholine receptor activation and accessory/scaffold proteins recruitment into a signalsome complex. The dynamics of such a complex is not well understood but a conserved NPxxY motif located within transmembrane 7 and juxtamembrane helix 8 of the receptor was found to modulate G protein activation. Here by means of receptor mutagenesis we unravel the role of the conserved M-3 muscarinic acetylcholine receptor NPxxY motif on ligand binding, signaling and multiprotein complex formation. Interestingly, while a N7.49D receptor mutant showed normal ligand binding properties a N7.49A mutant had reduced antagonist binding and increased affinity for carbachol. Also, besides this last mutant was able to physically couple to G alpha(q/11) after carbachol challenge it was neither capable to activate phospholipase C nor phospholipase D. On the other hand, we demonstrated that the Asn-7.49 is important for the interaction between M3R and ARF1 and also for the formation of the ARF/Rho/beta gamma signaling complex, a complex that might determine the rapid activation and desensitization of PLD. Overall, these results indicate that the NPxxY motif of the M-3 muscarinic acetylcholine receptor acts as key conformational switch for receptor signaling and multiprotein complex formation

Acute Cocaine Enhances Dopamine D2R Recognition and Signaling and Counteracts D2R Internalization in Sigma1R-D2R Heteroreceptor Complexes

The current study was performed to establish the actions of nanomolar concentrations of cocaine, not blocking the dopamine transporter, on dopamine D2 receptor (D2R)-sigma 1 receptor (delta 1R) heteroreceptor complexes and the D2R protomer recognition, signaling and internalization in cellular models. We report the existence of D2R-delta 1R heteroreceptor complexes in subcortical limbic areas as well as the dorsal striatum, with different distribution patterns using the in situ proximity ligation assay. Also, through BRET, these heteromers were demonstrated in HEK293 cells. Furthermore, saturation binding assay demonstrated that in membrane preparations of HEK293 cells coexpressing D2R and delta 1R, cocaine (1 nM) significantly increased the D2R B-max values over cells singly expressing D2R. CREB reporter luc-gene assay indicated that coexpressed delta 1R significantly reduced the potency of the D2R-like agonist quinpirole to inhibit via D2R activation the forskolin induced increase of the CREB signal. In contrast, the addition of 100 nM cocaine was found to markedly increase the quinpirole potency to inhibit the forskolin-induced increase of the CREB signal in the D2R-delta 1R cells. These events were associated with a marked reduction of cocaine-induced internalization of D2R protomers in D2R-delta 1R heteromer-containing cells vs D2R singly expressing cells as studied by means of confocal analysis of D2R-delta 1R trafficking and internalization. Overall, the formation of D2R-delta 1R heteromers enhanced the ability of cocaine to increase the D2R protomer function associated with a marked reduction of its internalization. The existence of D2R-delta 1R heteromers opens up a new understanding of the acute actions of cocaine

G Protein-Coupled Receptor heterodimerization in the brain

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

Nuevas técnicas didácticas en educación sexual

Se plantean diferentes metodologías para trabajar la educación afectiva y sexual de una manera creativa. En concreto, se muestra como el cine, la música, la fotografía, la literatura, el estudio de casos e Internet aportan elementos que pueden ser usados para desarrollar actividades de educación sexual. Además, se incluyen fuentes de información para la búsqueda de información científica sobre sexualidad humana .MadridBiblioteca de Educación del Ministerio de Educación, Cultura y Deporte; Calle San Agustín 5 -3 Planta; 28014 Madrid; Tel. +34917748000; [email protected]

Diversity and bias through receptor–receptor interactions in GPCR heteroreceptor complexes. Focus on examples from dopamine D2 receptor heteromerization

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 mechanismsPeer Reviewe