Meritxell Canals

Presynaptic Control of Striatal Glutamatergic Neurotransmission by Adenosine A1–A2A Receptor Heteromers

The functional role of heteromers of G-protein-coupled receptors is a matter of debate. In the present study, we demonstrate that heteromerization of adenosine A1 receptors (A1Rs) and A2A receptors (A2ARs) allows adenosine to exert a fine-tuning modulation of glutamatergic neurotransmission. By means of coimmunoprecipitation, bioluminescence and time-resolved fluorescence resonance energy transfer techniques, we showed the existence of A1R–A2AR heteromers in the cell surface of cotransfected cells. Immunogold detection and coimmunoprecipitation experiments indicated that A1R and A2AR are colocalized in the same striatal glutamatergic nerve terminals. Radioligand-binding experiments in cotransfected cells and rat striatum showed that a main biochemical characteristic of the A1R–A2AR heteromer is the ability of A2AR activation to reduce the affinity of the A1R for agonists. This provides a switch mechanism by which low and high concentrations of adenosine inhibit and stimulate, respectively, glutamate release. Furthermore, it is also shown that A1R–A2AR heteromers constitute a unique target for caffeine and that chronic caffeine treatment leads to modifications in the function of the A1R–A2AR heteromer that could underlie the strong tolerance to the psychomotor effects of caffeine.

Structural basis for modulation of a G-protein-coupled receptor by allosteric drugs.

The design of G-protein-coupled receptor (GPCR) allosteric modulators, an active area of modern pharmaceutical research, has proved challenging because neither the binding modes nor the molecular mechanisms of such drugs are known. Here we determine binding sites, bound conformations and specific drug–receptor interactions for several allosteric modulators of the M2 muscarinic acetylcholine receptor (M2 receptor), a prototypical family A GPCR, using atomic-level simulations in which the modulators spontaneously associate with the receptor. Despite substantial structural diversity, all modulators form cation–π interactions with clusters of aromatic residues in the receptor extracellular vestibule, approximately 15 Å from the classical, ‘orthosteric’ ligand-binding site. We validate the observed modulator binding modes through radioligand binding experiments on receptor mutants designed, on the basis of our simulations, either to increase or to decrease modulator affinity. Simulations also revealed mechanisms that contribute to positive and negative allosteric modulation of classical ligand binding, including coupled conformational changes of the two binding sites and electrostatic interactions between ligands in these sites. These observations enabled the design of chemical modifications that substantially alter a modulator’s allosteric effects. Our findings thus provide a structural basis for the rational design of allosteric modulators targeting muscarinic and possibly other GPCRs.

Orexin-1 Receptor-Cannabinoid CB1 Receptor Heterodimerization Results in Both Ligand-dependent and -independent Coordinated Alterations of Receptor Localization and Function

Following inducible expression in HEK293 cells, the human orexin-1 receptor was targeted to the cell surface but became internalized following exposure to the peptide agonist orexin A. By contrast, constitutive expression of the human cannabinoid CB1 receptor resulted in a predominantly punctate, intracellular distribution pattern consistent with spontaneous, agonistindependent internalization. Expression of the orexin-1 receptor in the presence of the CB1 receptor resulted in both receptors displaying the spontaneous internalization phenotype. Single cell fluorescence resonance energy transfer imaging indicated the two receptors were present as heterodimers/oligomers in intracellular vesicles. Addition of the CB1 receptor antagonist SR-141716A to cells expressing only the CB1 receptor resulted in re-localization of the receptor to the cell surface. Although SR-141716A has no significant affinity for the orexin-1 receptor, in cells co-expressing the CB1 receptor, the orexin-1 receptor was also re-localized to the cell surface by treatment with SR-141716A. Treatment of cells co-expressing the orexin-1 and CB1 receptors with the orexin-1 receptor antagonist SB-674042 also resulted in re-localization of both receptors to the cell surface. Treatment with SR-141716A resulted in decreased potency of orexin A to activate the mitogen-activated protein kinases ERK1/2 only in cells co-expressing the two receptors. Treatment with SB-674042 also reduced the potency of a CB1 receptor agonist to phosphorylate ERK1/2 only when the two receptors were co-expressed. These studies introduce an entirely novel pharmacological paradigm, whereby ligands modulate the function of receptors for which they have no significant inherent affinity by acting as regulators of receptor heterodimers.

Adenosine A2A and dopamine D2 heteromeric receptor complexes and their function

The existence of A2A-D2 heteromeric complexes is based on coimmunoprecipitation studies and on fluorescence resonance energy transfer and bioluminescence resonance energy transfer analyses. It has now become possible to show that A2A and D2 receptors also coimmunoprecipitate in striatal tissue, giving evidence for the existence of A2A-D2 heteromeric receptor complexes also in rat striatal tissue. The analysis gives evidence that these heteromers are constitutive, as they are observed in the absence of A2A and D2 agonists. The A2A-D2 heteromers could either be A2A-D2 heterodimers and/or higher-order A2A-D2 hetero-oligomers. In striatal neurons there are probably A2A-D2 heteromeric complexes, together with A2A-D2 homomeric complexes in the neuronal surface membrane. Their stoichiometry in various microdomains will have a major role in determining A2A and D2 signaling in the striatopallidal GABA neurons. Through the use of D2/D1 chimeras, evidence has been obtained that the fifth transmembrane (TM) domain and/or the 13 of the D2 receptor are part of the A2A-D2 receptor interface, where electrostatic epitope-epitope interactions involving the N-terminal part of 13 of the D2 receptor (arginine-rich epitope) play a major role, interacting with the carboxyl terminus of the A2A receptor. Computerized modeling of A2A-D2 heteromers are in line with these findings. It seems likely that A2A receptor-induced reduction of D2 receptor recognition, G protein coupling, and signaling, as well as the existence of A2A-D2 co-trafficking, are the consequence of the existence of an A2A-D2 receptor heteromer. The relevance of A2A-D2 heteromeric receptor complexes for Parkinson’s disease and schizophrenia is emphasized as well as for the treatment of these diseases. Finally, recent evidence for the existence of antagonistic A2A-D3 heteromeric receptor complexes in cotransfected cell lines has been summarized.

The α1b-adrenoceptor exists as a higher-order oligomer: effective oligomerization is required for receptor maturation, surface delivery and function

Approaches to identify G protein-coupled receptor oligomers rather than dimers have been lacking. Using concatamers of fluorescent proteins, we established conditions to monitor sequential three-color fluorescence resonance energy transfer (3-FRET) and used these to detect oligomeric complexes of the α1b-adrenoceptor in single living cells. Mutation of putative key hydrophobic residues in transmembrane domains I and IV resulted in substantial reduction of sequential 3-FRET and was associated with lack of protein maturation, prevention of plasma membrane delivery, and elimination of signaling function. Although these mutations prevented cell surface delivery, bimolecular fluorescence complementation studies indicated that they did not ablate protein-protein interactions and confirmed endoplasmic reticulum/Golgi retention of the transmembrane domain I plus transmembrane domain IV mutated receptor. The transmembrane domain I plus transmembrane domain IV mutated receptor was a “dominant-negative” in blocking cell surface delivery of the wild-type receptor. Mutations only in transmembrane domain I did not result in a reduction in 3-FRET, whereas restricting mutation to transmembrane domain IV did result in reduced 3-FRET. Mutations in either transmembrane domain I or transmembrane domain IV, however, were sufficient to eliminate cell surface delivery. Terminal N-glycosylation is insufficient to determine cell surface delivery because both transmembrane domain I and transmembrane domain IV mutants matured as effectively as the wild-type receptor. These data indicate that the α1b-adrenoceptor is able to form oligomeric rather than only simple dimeric complexes and that disruption of effective oligomerization by introducing mutations into transmembrane domain IV has profound consequences for cell surface delivery and function.

Pharmacological modulation of chemokine receptor function

G protein‐coupled chemokine receptors and their peptidergic ligands are interesting therapeutic targets due to their involvement in various immune‐related diseases, including rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, chronic obstructive pulmonary disease, HIV‐1 infection and cancer. To tackle these diseases, a lot of effort has been focused on discovery and development of small‐molecule chemokine receptor antagonists. This has been rewarded by the market approval of two novel chemokine receptor inhibitors, AMD3100 (CXCR4) and Maraviroc (CCR5) for stem cell mobilization and treatment of HIV‐1 infection respectively. The recent GPCR crystal structures together with mutagenesis and pharmacological studies have aided in understanding how small‐molecule ligands interact with chemokine receptors. Many of these ligands display behaviour deviating from simple competition and do not interact with the chemokine binding site, providing evidence for an allosteric mode of action. This review aims to give an overview of the evidence supporting modulation of this intriguing receptor family by a range of ligands, including small molecules, peptides and antibodies. Moreover, the computer‐assisted modelling of chemokine receptor–ligand interactions is discussed in view of GPCR crystal structures. Finally, the implications of concepts such as functional selectivity and chemokine receptor dimerization are considered.

Intramembrane receptor-receptor interactions: a novel principle in molecular medicine

Summary.In 1980/81 Agnati and Fuxe introduced the concept of intramembrane receptor–receptor interactions and presented the first experimental observations for their existence in crude membrane preparations. The second step was their introduction of the receptor mosaic hypothesis of the engram in 1982. The third step was their proposal that the existence of intramembrane receptor–receptor interactions made possible the integration of synaptic (WT) and extrasynaptic (VT) signals. With the discovery of the intramembrane receptor–receptor interactions with the likely formation of receptor aggregates of multiple receptors, so called receptor mosaics, the entire decoding process becomes a branched process already at the receptor level in the surface membrane. Recent developments indicate the relevance of cooperativity in intramembrane receptor–receptor interactions namely the presence of regulated cooperativity via receptor–receptor interactions in receptor mosaics (RM) built up of the same type of receptor (homo-oligomers) or of subtypes of the same receptor (RM type1). The receptor–receptor interactions will to a large extent determine the various conformational states of the receptors and their operation will be dependent on the receptor composition (stoichiometry), the spatial organization (topography) and order of receptor activation in the RM. The biochemical and functional integrative implications of the receptor–receptor interactions are outlined and long-lived heteromeric receptor complexes with frozen RM in various nerve cell systems may play an essential role in learning, memory and retrieval processes. Intramembrane receptor–receptor interactions in the brain have given rise to novel strategies for treatment of Parkinson’s disease (A2A and mGluR5 receptor antagonists), schizophrenia (A2A and mGluR5 agonists) and depression (galanin receptor antagonists). The A2A/D2, A2A/D3 and A2A/mGluR5 heteromers and heteromeric complexes with their possible participation in different types of RM are described in detail, especially in the cortico-striatal glutamate synapse and its extrasynaptic components, together with a postulated existence of A2A/D4 heteromers. Finally, the impact of intramembrane receptor–receptor interactions in molecular medicine is discussed outside the brain with focus on the endocrine, the cardiovascular and the immune systems.

Cannabinoid Receptor Type 1 Protects against Age- Related Osteoporosis by Regulating Osteoblast and Adipocyte Differentiation in Marrow Stromal Cells

Age-related osteoporosis is characterized by reduced bone formation and accumulation of fat in the bone marrow compartment. Here, we report that the type 1 cannabinoid receptor (CB1) regulates this process. Mice with CB1 deficiency (CB1(-/-)) had increased peak bone mass due to reduced bone resorption, but developed age-related osteoporosis with reduced bone formation and accumulation of adipocytes in the bone marrow space. Marrow stromal cells from CB1(-/-) mice had an enhanced capacity for adipocyte differentiation, a reduced capacity for osteoblast differentiation, and increased expression of phosphorylated CREB (pCREB) and PPARgamma. Pharmacological blockade of CB1 receptors stimulated adipocyte differentiation, inhibited osteoblast differentiation, and increased cAMP and pCREB in osteoblast and adipocyte precursors. The CB1 receptor is therefore unique in that it regulates peak bone mass through an effect on osteoclast activity, but protects against age-related bone loss by regulating adipocyte and osteoblast differentiation of bone marrow stromal cells.

A Monod-Wyman-Changeux mechanism can explain G protein-coupled receptor (GPCR) allosteric modulation.

Background: The Monod-Wyman-Changeux (MWC) mechanism is the preeminent conformational selection model for allosteric proteins. Results: The novel allosteric ligand, BQCA, behaves according to a two-state MWC mechanism at the M1 muscarinic GPCR. Conclusion: Chemical biological properties of GPCR allosteric ligands can be rationalized by the MWC model. Significance: Application of our experimental framework to allosteric GPCR modulators can assist ligand classification and drug discovery. The Monod-Wyman-Changeux (MWC) model was initially proposed to describe the allosteric properties of regulatory enzymes and subsequently extended to receptors. Yet despite GPCRs representing the largest family of receptors and drug targets, no study has systematically evaluated the MWC mechanism as it applies to GPCR allosteric ligands. We reveal how the recently described allosteric modulator, benzyl quinolone carboxylic acid (BQCA), behaves according to a strict, two-state MWC mechanism at the M1 muscarinic acetylcholine receptor (mAChR). Despite having a low affinity for the M1 mAChR, BQCA demonstrated state dependence, exhibiting high positive cooperativity with orthosteric agonists in a manner that correlated with efficacy but negative cooperativity with inverse agonists. The activity of BQCA was significantly increased at a constitutively active M1 mAChR but abolished at an inactive mutant. Interestingly, BQCA possessed intrinsic signaling efficacy, ranging from near-quiescence to full agonism depending on the coupling efficiency of the chosen intracellular pathway. This latter cellular property also determined the difference in magnitude of positive cooperativity between BQCA and the orthosteric agonist, carbachol, across pathways. The lack of additional, pathway-biased, allosteric modulation by BQCA was confirmed in genetically engineered yeast strains expressing different chimeras between the endogenous yeast Gpa1 protein and human Gα subunits. These findings define a chemical biological framework that can be applied to the study and classification of allosteric modulators across different GPCR families.

Working memory deficits in transgenic rats overexpressing human adenosine A2A receptors in the brain

Adenosine receptors in the central nervous system have been implicated in the modulation of different behavioural patterns and cognitive functions although the specific role of A(2A) receptor (A(2A)R) subtype in learning and memory is still unclear. In the present work we establish a novel transgenic rat strain, TGR(NSEhA2A), overexpressing adenosine A(2A)Rs mainly in the cerebral cortex, the hippocampal formation, and the cerebellum. Thereafter, we explore the relevance of this A(2A)Rs overexpression for learning and memory function. Animals were behaviourally assessed in several learning and memory tasks (6-arms radial tunnel maze, T-maze, object recognition, and several Morris water maze paradigms) and other tests for spontaneous motor activity (open field, hexagonal tunnel maze) and anxiety (plus maze) as modification of these behaviours may interfere with the assessment of cognitive function. Neither motor performance and emotional/anxious-like behaviours were altered by overexpression of A(2A)Rs. TGR(NSEhA2A) showed normal hippocampal-dependent learning of spatial reference memory. However, they presented working memory deficits as detected by performance of constant errors in the blind arms of the 6 arm radial tunnel maze, reduced recognition of a novel object and a lack of learning improvement over four trials on the same day which was not observed over consecutive days in a repeated acquisition paradigm in the Morris water maze. Given the interdependence between adenosinic and dopaminergic function, the present results render the novel TGR(NSEhA2A) as a putative animal model for the working memory deficits and cognitive disruptions related to overstimulation of cortical A(2A)Rs or to dopaminergic prefrontal dysfunction as seen in schizophrenic or Parkinsons disease patients.