Biased receptor functionality versus biased agonism in G-protein-coupled receptors

Functional selectivity is a property of G-protein-coupled receptors (GPCRs) by which activation by different agonists leads to different signal transduction mechanisms. This phenomenon is also known as biased agonism and has attracted the interest of drug discovery programs in both academy and industry. This relatively recent concept has raised concerns as to the validity and real translational value of the results showing bias; firstly biased agonism may vary significantly depending on the cell type and the experimental constraints, secondly the conformational landscape that leads to biased agonism has not been defined. Remarkably, GPCRs may lead to differential signaling even when a single agonist is used. Here we present a concept that constitutes a biochemical property of GPCRs that may be underscored just using one agonist, preferably the endogenous agonist. 'Biased receptor functionality' is proposed to describe this effect with examples based on receptor heteromerization and alternative splicing. Examples of regulation of final agonist-induced outputs based on interaction with β-arrestins or calcium sensors are also provided. Each of the functional GPCR units (which are finite in number) has a specific conformation. Binding of agonist to a specific conformation, i.e. GPCR activation, is sensitive to the kinetics of the agonist-receptor interactions. All these players are involved in the contrasting outputs obtained when different agonists are assayed

Neuronal calcium and cAMP cross-talk mediated by cannabinoid CB1 receptor and EF-hand calcium sensor interactions.

Endocannabinoids are important players in neural development and function. They act via receptors, whose activation inhibits cAMP production. The aim of the paper was to look for calcium- and cAMP-signaling cross-talk mediated by cannabinoid CB1 receptors (CB1R) and to assess the relevance of EF-hand CaM-like calcium sensors in this regard. Using a heterologous expression system, we demonstrated that CB1R interacts with calneuron-1 and NCS1 but not with caldendrin. Furthermore, interaction motives were identified in both calcium binding proteins and the receptor, and we showed that the first two sensors competed for binding to the receptor in a Ca2+-dependent manner. Assays in neuronal primary cultures showed that, CB1R-NCS1 complexes predominate at basal Ca2+ levels, whereas in the presence of ionomycin, a calcium ionophore, CB1R-calneuron-1 complexes were more abundant. Signaling assays following forskolin-induced intracellular cAMP levels showed in mouse striatal neurons that binding of CB1R to NCS1 is required for CB1R-mediated signaling, while the binding of CB1R to calneuron-1 completely blocked Gi-mediated signaling in response to a selective receptor agonist, arachidonyl-2-chloroethylamide. Calcium levels and interaction with calcium sensors may even lead to apparent Gs coupling after CB1R agonist challenge

The sigma-1 receptor as key common factor in cocaine and food seeking behaviors

Addiction and eating disorders involve brain reward circuits. A previous history of binge eating predisposes to addictive behavior, while the cessation of exposure to drugs of abuse leads to reward activities, including intake of tasty foods. Cocaine use is associated with a decrease in food intake, with reversal after the drug use is stopped. Exciting new findings show that receptors for the 'hunger' hormone, ghrelin, directly interact with the sigma-1 receptors (1R), which is a target of cocaine. 1R are key players in regulating dopaminergic neurotransmission and ghrelin-mediated actions. This review focuses on the 1 receptor as general neuroendocrine regulator by directly interacting with neuronal G-protein-coupled receptors. This review also covers the early mechanisms by which cocaine binding to 1 blocks the food-seeking behavior triggered by ghrelin. Such new findings appear as fundamental to understand common mechanisms in drug addiction and eating disorders

Cocaine Effects on Dopaminergic Transmission Depend on a Balance between Sigma-1 and Sigma-2 Receptor Expression.

gma σ1 and σ2 receptors are targets of cocaine. Despite sharing a similar name, the two receptors are structurally unrelated and their physiological role is unknown. Cocaine increases the level of dopamine, a key neurotransmitter in CNS motor control and reward areas. While the drug also affects dopaminergic signaling by allosteric modulations exerted by σ1R interacting with dopamine D1 and D2 receptors, the potential regulation of dopaminergic transmission by σ2R is also unknown. We here demonstrate that σ2R may form heteroreceptor complexes with D1 but not with D2 receptors. Remarkably σ1, σ2, and D1 receptors may form heterotrimers with particular signaling properties. Determination of cAMP levels, MAP kinase activation and label-free assays demonstrate allosteric interactions within the trimer. Importantly, the presence of σ2R induces bias in signal transduction as σ2R ligands increase cAMP signaling whereas reduce MAP kinase activation. These effects, which are opposite to those exerted via σ1R, suggest that the D1 receptor-mediated signaling depends on the degree of trimer formation and the differential balance of sigma receptor and heteroreceptor expression in acute versus chronic cocaine consumption. Although the physiological role is unknown, the heteroreceptor complex formed by σ1, σ2, and D1 receptors arise as relevant to convey the cocaine actions on motor control and reward circuits and as a key factor in acquisition of the addictive habit. KEYWORDS: ERK1/2 phosphorylation; acute; addiction; cAMP; chronic; dopamine D1 and D2 receptors; label-free; signalin

Trypanosoma brucei aquaglyceroporin 2 is a high-affinity transporter for pentamidine and melaminophenyl arsenic drugs and the main genetic determinant of resistance to these drugs.

OBJECTIVES: Trypanosoma brucei drug transporters include the TbAT1/P2 aminopurine transporter and the high-affinity pentamidine transporter (HAPT1), but the genetic identity of HAPT1 is unknown. We recently reported that loss of T. brucei aquaglyceroporin 2 (TbAQP2) caused melarsoprol/pentamidine cross-resistance (MPXR) in these parasites and the current study aims to delineate the mechanism by which this occurs. METHODS: The TbAQP2 loci of isogenic pairs of drug-susceptible and MPXR strains of T. brucei subspecies were sequenced. Drug susceptibility profiles of trypanosome strains were correlated with expression of mutated TbAQP2 alleles. Pentamidine transport was studied in T. brucei subspecies expressing TbAQP2 variants. RESULTS: All MPXR strains examined contained TbAQP2 deletions or rearrangements, regardless of whether the strains were originally adapted in vitro or in vivo to arsenicals or to pentamidine. The MPXR strains and AQP2 knockout strains had lost HAPT1 activity. Reintroduction of TbAQP2 in MPXR trypanosomes restored susceptibility to the drugs and reinstated HAPT1 activity, but did not change the activity of TbAT1/P2. Expression of TbAQP2 sensitized Leishmania mexicana promastigotes 40-fold to pentamidine and >1000-fold to melaminophenyl arsenicals and induced a high-affinity pentamidine transport activity indistinguishable from HAPT1 by Km and inhibitor profile. Grafting the TbAQP2 selectivity filter amino acid residues onto a chimeric allele of AQP2 and AQP3 partly restored susceptibility to pentamidine and an arsenical. CONCLUSIONS: TbAQP2 mediates high-affinity uptake of pentamidine and melaminophenyl arsenicals in trypanosomes and TbAQP2 encodes the previously reported HAPT1 activity. This finding establishes TbAQP2 as an important drug transporter

Differential effect of amphetamine over the corticotropin-releasing factor CRF2 receptor, the orexin OX1 receptor and the CRF2-OX1 heteroreceptor complex

Stress is one of the factors underlying drug seeking behavior that often goes in parallel with loss of appetite. We here demonstrate that orexin 1 receptors (OX1R) may form complexes with the corticotropin releasing factor CRF2 receptor. Two specific features of the heteromer were a cross-antagonism and a blockade by CRF2 of OX1R signaling. In cells expressing one of the receptors, agonist-mediated signal transduction mechanisms were potentiated by amphetamine. Sigma 1 (σ1) and 2 (σ2) receptors are targets of drugs of abuse and, despite sharing a similar name, the two receptors are structurally unrelated and their physiological role is not known. We here show that σ1 receptors interact with CRF2 receptors and that σ2 receptors interact with OX1R. Moreover, we show that amphetamine effect on CRF2 receptors was mediated by σ1R whereas the effect on OX1 receptors was mediated by σ2R. Amphetamine did potentiate the negative cross-talk occurring within the CRF2-OX1 receptor heteromer context, likely by a macromolecular complex involving the two sigma receptors and the two GPCRs. Finally, in vivo microdialysis experiments showed that amphetamine potentiated orexin A-induced dopamine and glutamate release in the ventral tegmental area (VTA). Remarkably, the in vivo orexin A effects were blocked by a selective CRF2R antagonist. These results show that amphetamine impacts on the OX1R-, CRF2R- and OX1R/CRF2R-mediated signaling and that cross-antagonism is instrumental for in vivo detection of GPCR heteromers

Molecular and functional interaction between GPR18 and cannabinoid CB2 G-protein-coupled receptors. Relevance in neurodegenerative diseases

GPR18, still considered an orphan receptor, may respond to endocannabinoids, whose canonical receptors are CB1 and CB2. GPR18 and CB2 receptors share a role in peripheral immune response regulation and are co-expressed in microglia, which are immunocompetent cells in the central nervous system (CNS). We aimed at identifying heteroreceptor complexes formed by GPR18 and CB1R or CB2R in resting and activated microglia. Receptor-receptor interaction was assessed using energy-transfer approaches, and receptor function by determining cAMP levels and ERK1/2 phosphorylation in heterologous cells and primary cultures of microglia. Heteroreceptor identification in primary cultures of microglia was achieved by in situ proximity ligation assays. Energy transfer results showed interaction of GPR18 with CB2R but not with CB1R. CB2-GPR18 heteroreceptor complexes displayed particular functional properties (heteromer prints) often consisting of negative cross-talk (activation of one receptor reduces signaling arising from the partner receptor) and cross-antagonism (the response of one of the receptors is blocked by a selective antagonist of the partner receptor). Activated microglia showed the heteromer print (negative cross-talk and bidirectional cross-antagonism) and increased expression of CB2R and GPR18. Due to the important role of CB2R in neuroprotection, we further investigated heteroreceptor occurrence in primary cultures of microglia from transgenic mice overexpressing human APPSw,Ind, an Alzheimer's disease model. Microglial cells from transgenic mice showed the heteromer print and functional interactions that were similar to those found in cells from wild-type animals that were activated by treatment with lipopolysaccharide and interferon-ɤ. Our results show that GPR18 and its heteromers may play important roles in neurodegenerative processes

Cross-communication between Gi and Gs in a G-protein-coupled receptor heterotetramer guided by a receptor C-terminal domain

BACKGROUND: G-protein-coupled receptor (GPCR) heteromeric complexes have distinct properties from homomeric GPCRs, giving rise to new receptor functionalities. Adenosine receptors (A1R or A2AR) can form A1R-A2AR heteromers (A1-A2AHet), and their activation leads to canonical G-protein-dependent (adenylate cyclase mediated) and -independent (β-arrestin mediated) signaling. Adenosine has different affinities for A1R and A2AR, allowing the heteromeric receptor to detect its concentration by integrating the downstream Gi- and Gs-dependent signals. cAMP accumulation and β-arrestin recruitment assays have shown that, within the complex, activation of A2AR impedes signaling via A1R. RESULTS: We examined the mechanism by which A1-A2AHet integrates Gi- and Gs-dependent signals. A1R blockade by A2AR in the A1-A2AHet is not observed in the absence of A2AR activation by agonists, in the absence of the C-terminal domain of A2AR, or in the presence of synthetic peptides that disrupt the heteromer interface of A1-A2AHet, indicating that signaling mediated by A1R and A2AR is controlled by both Gi and Gs proteins. CONCLUSIONS: We identified a new mechanism of signal transduction that implies a cross-communication between Gi and Gs proteins guided by the C-terminal tail of the A2AR. This mechanism provides the molecular basis for the operation of the A1-A2AHet as an adenosine concentration-sensing device that modulates the signals originating at both A1R and A2AR

Trypanosoma brucei aquaglyceroporin 2 is a high-affinity transporter for pentamidine and melaminophenyl arsenic drugs and the main genetic determinant of resistance to these drugs

Objectives Trypanosoma brucei drug transporters include the TbAT1/P2 aminopurine transporter and the high-affinity pentamidine transporter (HAPT1), but the genetic identity of HAPT1 is unknown. We recently reported that loss of T. brucei aquaglyceroporin 2 (TbAQP2) caused melarsoprol/pentamidine cross-resistance (MPXR) in these parasites and the current study aims to delineate the mechanism by which this occurs. Methods The TbAQP2 loci of isogenic pairs of drug-susceptible and MPXR strains of T. brucei subspecies were sequenced. Drug susceptibility profiles of trypanosome strains were correlated with expression of mutated TbAQP2 alleles. Pentamidine transport was studied in T. brucei subspecies expressing TbAQP2 variants. Results All MPXR strains examined contained TbAQP2 deletions or rearrangements, regardless of whether the strains were originally adapted in vitro or in vivo to arsenicals or to pentamidine. The MPXR strains and AQP2 knockout strains had lost HAPT1 activity. Reintroduction of TbAQP2 in MPXR trypanosomes restored susceptibility to the drugs and reinstated HAPT1 activity, but did not change the activity of TbAT1/P2. Expression of TbAQP2 sensitized Leishmania mexicana promastigotes 40-fold to pentamidine and >1000-fold to melaminophenyl arsenicals and induced a high-affinity pentamidine transport activity indistinguishable from HAPT1 by Km and inhibitor profile. Grafting the TbAQP2 selectivity filter amino acid residues onto a chimeric allele of AQP2 and AQP3 partly restored susceptibility to pentamidine and an arsenical. Conclusions TbAQP2 mediates high-affinity uptake of pentamidine and melaminophenyl arsenicals in trypanosomes and TbAQP2 encodes the previously reported HAPT1 activity. This finding establishes TbAQP2 as an important drug transporte

Interacción molecular y funcional entre receptores involucrados en la ingesta de alimentos y el consumo de drogas de abuso

[spa] Actualmente en la comunidad científica aún existe debate acerca de la heteromerización entre receptores GPCR y el papel que esta tiene en la señalización y funcionalidad celular. Tradicionalmente estos receptores han sido considerados entidades monoméricas, No obstante, en las últimas décadas han sido obtenidos resultados que apoyan la teoría de que estos receptores pueden formar homómeros y heterómeros. Entender cómo funcionan los receptores al formar complejos heteroméricos con otros receptores, u otras proteínas, es de vital importancia para dar explicación a muchos mecanismos fisiológicos, así como para el diseño de nuevos fármacos. Entender cómo se establecen las modulaciones entre unos receptores y otros posibilitará también en un futuro el desarrollo de tratamientos preventivos para distintos trastornos psicológicos, como la adicción a drogas. Uno de los principales objetivos de esta tesis ha sido estudiar y aportar nuevos datos para entender el funcionamiento de la adicción a sustancias psicoestimulantes, concretamente, la cocaína. En esta Tesis ha sido puesto de manifiesto la importancia de la modulación que pueden ejercer las interacciones que afectan a los GPCR en relación a la adicción a cocaína. En primer lugar, ha sido demostrada la capacidad de los receptores sigma-1 y sigma-2 para interaccionar con distintos GPCR, los receptores D1 y D2 de dopamina, el heterómero de receptores CRF1R-OX1R o el receptor de grelina, GHS-R1a. La cocaína, al unirse a los receptores sigma, modifica, en todos los casos, la señalización normal inducida por estos GPCRs, contribuyendo a la adicción a la droga de abuso, provocando una recaída en el comportamiento de búsqueda de cocaína en respuesta a una situación de estrés o bloqueando de la sensación de apetito tras el consumo de la droga. Al producirse las interacciones, los complejos formados pueden ajustar su actividad a distintos ambientes o necesidades celulares que los receptores, por sí solo, no serían capaces de detectar. En esta misma línea está el papel que juegan las proteínas sensoras de calcio en la modulación de la actividad de algunos GPCR. El calcio es un mensajero imprescindible, las fluctuaciones en los niveles de este ion pueden ir desde niveles submicromolares hasta milimolares, siendo uno de los mecanismos de regulación más importantes en la célula. Distintas concentraciones de calcio pueden inducir diferentes señalizaciones a través de un mismo heterómero de GPCR. Ha sido descrita la capacidad del heterómero A2A-D2 para unir diferentes proteínas sensoras de calcio, NCS-1 y calneurona-1, las cuales tienen diferentes afinidades por el ion calcio y regularan el heterómero de manera distinta en función de las bajas (unión de NCS-1) o altas (unión de calneurona-1) concentraciones de calcio celular, dándole versatilidad a un mismo heterómero. Por último, han sido investigadas las interacciones entre GPCR a nivel estructural con la finalidad de elucidar las regiones de los receptores implicadas en la formación de los heterómeros. Ha sido propuesto un nuevo modelo estructural tetramérico formado por un homodímero de receptores A1 de adenosina y un homodímero de receptores A2A de adenosina. En este complejo son las regiones transmembrana TM4/5 las responsables de la homodimerización y las regiones TM5/6 las responsables de la heteromerización, formando una estructura romboidal a en la que se acoplan dos proteínas G, Gi y Gs. Ha sido demostrada la importancia de la comunicación entre las proteínas G en un heterómero de GPCR, explicando, por primera vez, el mecanismo por el que el heterómero A1R-A2AR actúa como sensor de la concentración de adenosina pudiendo dar respuestas opuestas, vía proteína Gi o Gs, en función de la concentración extracelular de adenosina.[eng] Currently in the scientific community there is still debate about the heteromerization between GPCR and the role it has in cell signaling and functionality. Traditionally, these receptors have been considered monomeric entities. However, in recent decades new results have been obtained supporting the theory that these receptors can form oligomers. Understand how receptors interact when forming heteromeric complexes is crucial to explain many physiological mechanisms as well as in the design of new drugs. To elucidate how the modulation between receptors works will also enable the development of treatments for different disorders, such as drug addiction. One of the main objectives of this Thesis has been to contribute with new data to understand the underlying mechanisms of psychostimulant substances addiction. The importance of the modulation on GPCR exerted by heteromers in relation to cocaine addiction has been shown. The ability of the cocaine-binding receptors sigma-1R and sigma-2R to interact with different GPCRs, dopamine D1R and D2R, CRF1R-OX1R or GHS-R1a-GHS-R1b heteromers, has been demonstrated. Cocaine, when bound to the sigma receptors modifies the normal signaling induced by these GPCRs, contributing to drug addiction, relapse in cocaine seeking behavior or blocking the appetite sensation after the drug consumption. When interactions occur, the formed complexes can adjust their functionality to different cellular environments that these receptors, by themselves, would not be able to detect. One example is the modulating role exerted by the calcium sensing proteins on certain GPCR. Calcium is one of the most important regulatory mechanisms in the cell. Different calcium concentration may induce different signaling through the same GPCR heteromer. The ability of heteromers to bind different calcium sensors has been described. These calcium sensors have different affinities for the calcium ion and regulate differently the heteromer, depending on the low or high calcium concentrations, thus providing versatility to the complex. Finally, interactions between GPCRs at the structural level have been investigated in order to elucidate the receptor domains involved in the heteromers formation. A new tetrameric structural model formed by A1R-A1R-A2AR-A2AR adenosine receptors has been proposed, forming a rhomboidal structure in which two G proteins, Gi and Gs, are coupled. It has also been explained the mechanisms by which this heteromer acts as a adenosine concentration sensor giving opposite responses, via Gi or Gs, depending on the extracellular adenosine concentration