A Transmembrane Single-Polypeptide-Chain (sc) Linker to Connect the Two G-Protein–Coupled Receptors in Tandem and the Design for an In Vivo Analysis of Their Allosteric Receptor- Receptor Interactions

A transmembrane (TM) single-polypeptide-chain (sc) linker can connect two G-protein–coupled receptors (GPCRs) in tandem. The priority of a gene-fusion strategy for any two class A GPCRs has been demonstrated. In the striatal function, dopamine (DA) plays a critical role. In the striatum, how the GPCR for adenosine, subtype A2A (A2AR), contributes to the DA neurotransmission in the “volume transmission”/dual-transmission model has been studied extensively. In addition to the fusion receptor, i.e., the prototype scA2AR/D2R complex (the GPCR for DA, subtype D2), several types were created and tested experimentally. To further elucidate this in vivo, we designed a new molecular tool, namely, the supermolecule scA2AR/D2R. Here, no experiments on its expression were done. However, the TM linker to connect the nonobligate dimer as the transient class A GPCR nanocluster that has not been identified at the cell surface membrane deserves discussion through scA2AR/D2R. Supramolecular designs, are experimentally testable and will be used to confirm in vivo the functions of the two GPCRs interactive in such a low specific signal to the nonspecific noise (S/N) ratio in the neurotransmission in the brain. The sc also has, at last, become straightforward in the field of GPCRs, similar to in the field of antibody

Potential of caveolae in the therapy of cardiovascular and neurological diseases.

Caveolae are membrane micro-domains enriched in cholesterol, sphingolipids and caveolins, which are transmembrane proteins with a hairpin-like structure. Caveolae participate in receptor-mediated trafficking of cell surface receptors and receptor-mediated signaling. Furthermore, caveolae participate in clathrin-independent endocytosis of membrane receptors. On the one hand, caveolins are involved in vascular and cardiac dysfunction. Also, neurological abnormalities in caveolin-1 knockout mice and a link between caveolin-1 gene haplotypes and neurodegenerative diseases have been reported. The aim of this article is to present the rationale for considering caveolae as potential targets in cardiovascular and neurological diseases

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

Adenosine A2A receptor ligand recognition and signaling is blocked by A2B receptors

The adenosine receptor (AR) subtypes A2A and A2B are rhodopsin-like Gs protein-coupled receptors whose expression is highly regulated under pathological, e.g. hypoxic, ischemic and inflammatory conditions. Both receptors play important roles in inflammatory and neurodegenerative diseases, are blocked by caffeine, and have now become major drug targets in immuno-oncology. By Förster resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), bimolecular fluorescence complementation (BiFC) and proximity ligation assays (PLA) we demonstrated A2A-A2BAR heteromeric complex formation. Moreover we observed a dramatically altered pharmacology of the A2AAR when co-expressed with the A2BAR (A2B ≥ A2A) in recombinant as well as in native cells. In the presence of A2BARs, A2A-selective ligands lost high affinity binding to A2AARs and displayed strongly reduced potency in cAMP accumulation and dynamic mass redistribution (DMR) assays. These results have major implications for the use of A2AAR ligands as drugs as they will fail to modulate the receptor in an A2A-A2B heteromer context. Accordingly, A2A-A2BAR heteromers represent novel pharmacological targets

Brain dopamine transmission in health and Parkinson's disease: modulation of synaptic transmission and plasticity through volume transmission and dopamine heteroreceptors.

This perspective article provides observations supporting the view that nigro-striatal dopamine neurons and meso-limbic dopamine neurons mainly communicate through short distance volume transmission in the um range with dopamine diffusing into extrasynaptic and synaptic regions of glutamate and GABA synapses. Based on this communication it is discussed how volume transmission modulates synaptic glutamate transmission onto the D1R modulated direct and D2R modulated indirect GABA pathways of the dorsal striatum. Each nigro-striatal dopamine neuron was first calculated to form large numbers of neostriatal DA nerve terminals and then found to give rise to dense axonal arborizations spread over the neostriatum, from which dopamine is released. These neurons can through DA volume transmission directly influence not only the striatal GABA projection neurons but all the striatal cell types in parallel. It includes the GABA nerve cells forming the island-/striosome GABA pathway to the nigral dopamine cells, the striatal cholinergic interneurons and the striatal GABA interneurons. The dopamine modulation of the different striatal nerve cell types involves the five dopamine receptor subtypes, D1R to D5R receptors, and their formation of multiple extrasynaptic and synaptic dopamine homo and heteroreceptor complexes. These features of the nigro-striatal dopamine neuron to modulate in parallel the activity of practically all the striatal nerve cell types in the dorsal striatum, through the dopamine receptor complexes allows us to understand its unique and crucial fine-tuning of movements, which is lost in Parkinson's disease. Integration of striatal dopamine signals with other transmitter systems in the striatum mainly takes place via the receptor-receptor interactions in dopamine heteroreceptor complexes. Such molecular events also participate in the integration of volume transmission and synaptic transmission. Dopamine modulation of the glutamate synapses on the dorsal striato-pallidal GABA pathway involves D2R heteroreceptor complexes such as D2R-NMDAR, A2AR-D2R, and NTSR1-D2R heteroreceptor complexes. The dopamine modulation of glutamate synapses on the striato-entopeduncular/nigral pathway takes place mainly via D1R heteroreceptor complexes such as D1R-NMDAR, A2R-D1R, and D1R-D3R heteroreceptor complexes. Dopamine modulation of the island/striosome compartment of the dorsal striatum projecting to the nigral dopamine cells involve D4R-MOR heteroreceptor complexes. All these receptor-receptor interactions have relevance for Parkinson's disease and its treatment

Human M3 muscarinic acetylcholine receptor protein-protein interactions: roles in receptor signaling and regualation

Muscarinic acetylcholine receptors (mAChRs) have been shown to mediate various functions in the central and peripheral nervous systems. These include modulation of exocrine glandular secretion, vasodilatation and smooth muscle contraction, cell proliferation or survival, neural development and synaptic plasticity. mAChRs are activated by both endogenously produced acetylcholine and exogenously administered muscarinic compounds. Pharmacological, anatomical and molecular studies have demonstrated the existence of five muscarinic receptor subtypes, denoted as muscarinic M1, M2, M3, M4 and M5, which belong to class I family of heptahelical, transmembrane G-protein coupled receptors (GPCRs). Each receptor subtypes are characterized by a distinct selectivity for heterotrimeric G protein coupling. Thus, M1, M3 and M5 are coupled to Gq/11 proteins and stimulate phospholipase C activity, resulting in the generation of the second messengers inositol (1,4,5)-trisphosphate (IP3) and diacylglycerol (DAG), the mobilization of intracellular Ca2+ and the activation of protein kinase C (PKC). On the other hand, M2 and M4 are coupled to Gi/0 proteins, which results in the inhibition of adenilate cyclase, as well as prolonging potassium channel, non-selective cation channel, and transient receptor potential channel opening. By means of this differential set of G protein partners, mAChRs can initiate distinct signalling pathways within a same cell in order to trigger diverse, even opposed, functional outcomes in response to the same stimuli. It has been proven as well that mAChRs regulate a baste network of signalling intermediates, including small GTPase Rho, phospholipase D, phosphoinositide-3 kinase, non-receptor kinases and mitogenactivated protein kinases. Although the first proteins found to have functional interactions with mAChRs were, of course, G proteins, an increasing amount of evidence in the field suggests that this simplistic model defined as “one receptor -one G protein -one effector no longer exists. A great number of proteins have been identified as interacting with mAChRs, including GPCRs, kinases, and scaffolding proteins such as arrestin. Determining in part the signalling efficiency/specificity for mAChRs. Thus, receptors are now considered as complex signalling units, or signalosomes, that dynamically couple to multiple G proteins or other molecular entities or scaffold proteins in a temporally and spatially regulated manner, and even can form homodimers or heterodimers with distinct GPCRs or other non-GPCR membrane receptors, resulting in pharmacologically and functionally distinct receptor populations. In this work “Human M3 muscarinic acetylcholine receptor protein-protein interactions: roles in receptor signalling and regulation”, it is discuss novel mAChRs interacting partners that link the receptors to alternative signalling pathways beyond G proteins. Emphases on explaining how mAChRs regulate signal transduction pathways mediated by these proteins, including receptor dimerization have been putting out. It has been demonstrated by different approach (from resonance energy transfer to tandem affinity purification and mass spectrometry) the active role of interacting protein in mAChRs regulation and signalling. We shown that a particular complex is not necessarily of invariable composition, nor are all its building blocks uniquely associated with that specific complex. One complex may be the result not only of physical interaction between the receptor and the partners’ protein, but also of the participation of many non-“direct” associations resulting in the formation of a network that interconnects the receptor with a number of other pathways, determining receptor specificities. This allows us to address some fundamental questions concerning the importance of molecular mechanisms hidden behind the pharmacology properties for each receptor subtype

5HT1AR-FGFR1 Heteroreceptor Complexes Differently Modulate GIRK Currents in the Dorsal Hippocampus and the Dorsal Raphe Serotonin Nucleus of Control Rats and of a Genetic Rat Model of Depression

The midbrain raphe serotonin (5HT) neurons provide the main ascending serotonergic projection to the forebrain, including hippocampus, which has a role in the pathophysiology of depressive disorder. Serotonin 5HT1A receptor (R) activation at the soma-dendritic level of serotonergic raphe neurons and glutamatergic hippocampal pyramidal neurons leads to a decrease in neuronal firing by activation of G protein-coupled inwardly-rectifying potassium (GIRK) channels. In this raphe-hippocampal serotonin neuron system, the existence of 5HT1AR-FGFR1 heteroreceptor complexes has been proven, but the functional receptor–receptor interactions in the heterocomplexes have only been investigated in CA1 pyramidal neurons of control Sprague Dawley (SD) rats. In the current study, considering the impact of the receptor interplay in developing new antidepressant drugs, the effects of 5HT1AR-FGFR1 complex activation were investigated in hippocampal pyramidal neurons and in midbrain dorsal raphe serotonergic neurons of SD rats and of a genetic rat model of depression (the Flinders Sensitive Line (FSL) rats of SD origin) using an electrophysiological approach. The results showed that in the raphe-hippocampal 5HT system of SD rats, 5HT1AR-FGFR1 heteroreceptor activation by specific agonists reduced the ability of the 5HT1AR protomer to open the GIRK channels through the allosteric inhibitory interplay produced by the activation of the FGFR1 protomer, leading to increased neuronal firing. On the contrary, in FSL rats, FGFR1 agonist-induced inhibitory allosteric action at the 5HT1AR protomer was not able to induce this effect on GIRK channels, except in CA2 neurons where we demonstrated that the functional receptor–receptor interaction is needed for producing the effect on GIRK. In keeping with this evidence, hippocampal plasticity, evaluated as long-term potentiation induction ability in the CA1 field, was impaired by 5HT1AR activation both in SD and in FSL rats, which did not develop after combined 5HT1AR-FGFR1 heterocomplex activation in SD rats. It is therefore proposed that in the genetic FSL model of depression, there is a significant reduction in the allosteric inhibition exerted by the FGFR1 protomer on the 5HT1A protomer-mediated opening of the GIRK channels in the 5HT1AR-FGFR1 heterocomplex located in the raphe-hippocampal serotonin system. This may result in an enhanced inhibition of the dorsal raphe 5HT nerve cell and glutamatergic hippocampal CA1 pyramidal nerve cell firing, which we propose may have a role in depression