Wilber Romero-Fernandez - Academia.edu (original) (raw)

Papers by Wilber Romero-Fernandez

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

Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 2011

Acetylcholine challenge produces M(3) muscarinic acetylcholine receptor activation and accessory/... more 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 Gα(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 M(3)R and ARF1 an...

Research paper thumbnail of Dopamine D-4 receptor oligomerization: contribution to receptor biogenesis

archive.ugent.be

BRET, bioluminescence resonance energy transfer; CHO, Chinese hamster ovary; DAPI, 4¢,6-diamidino... more BRET, bioluminescence resonance energy transfer; CHO, Chinese hamster ovary; DAPI, 4¢,6-diamidino-2-phenylindole; D n R, dopamine D n receptor; ER, endoplasmic reticulum; GPCR, G protein-coupled receptor; HRP, horseradish peroxidase; MP, milk powder; YFP, yellow fluorescent protein.

Research paper thumbnail of Dopamine D2 and D4 receptor heteromerization and its allosteric receptor–receptor interactions

Biochemical and Biophysical Research Communications, 2011

Dopamine D2 and D4 receptors partially codistribute in the dorsal striatum and appear to play a f... more Dopamine D2 and D4 receptors partially codistribute in the dorsal striatum and appear to play a fundamental role in complex behaviors and motor function. The discovery of D2R–D4.xR (D4.2R, D4.4R or D4.7R) heteromers has been made in cellular models using co-immunoprecipitation, in situ Proximity Ligation Assays and BRET1 techniques with the D2R and D4.7R receptors being the least effective in

Research paper thumbnail of Moonlighting Proteins and Protein–Protein Interactions as Neurotherapeutic Targets in the G Protein-Coupled Receptor Field

Neuropsychopharmacology, 2014

There is serious interest in understanding the dynamics of the receptor-receptor and receptor-pro... more There is serious interest in understanding the dynamics of the receptor-receptor and receptor-protein interactions in space and time and their integration in GPCR heteroreceptor complexes of the CNS. Moonlighting proteins are special multifunctional proteins because they perform multiple autonomous, often unrelated, functions without partitioning into different protein domains. Moonlighting through receptor oligomerization can be operationally defined as an allosteric receptor-receptor interaction, which leads to novel functions of at least one receptor protomer. GPCR-mediated signaling is a more complicated process than previously described as every GPCR and GPCR heteroreceptor complex requires a set of G protein interacting proteins, which interacts with the receptor in an orchestrated spatio-temporal fashion. GPCR heteroreceptor complexes with allosteric receptor-receptor interactions operating through the receptor interface have become major integrative centers at the molecular level and their receptor protomers act as moonlighting proteins. The GPCR heteroreceptor complexes in the CNS have become exciting new targets for neurotherapeutics in Parkinson's disease, schizophrenia, drug addiction, and anxiety and depression opening a new field in neuropsychopharmacology.

Research paper thumbnail of Muscarinic receptor family interacting proteins: Role in receptor function

Journal of Neuroscience Methods, 2011

a b s t r a c t G protein-coupled receptors constitute one of the most important families of memb... more a b s t r a c t G protein-coupled receptors constitute one of the most important families of membrane receptors through which cells respond to extracellular stimuli. Receptors of this superfamily likely function as signal transduction complexes. The identification and analysis of their components provide new insights into a better understanding of these receptors' function and regulation. We used tandem-affinity purification and mass spectrometry as a systematic approach to characterize multiprotein complexes in the acetylcholine muscarinic receptor subfamily. To overcome the limitations associated with membrane protein receptor solubilization with detergents, we developed a strategy in which receptors are co-expressed with a cytoplasmic minigene construct, encoding the third intracellular loop and the C-terminal tail tagged to the tandem-affinity-cassette of each receptor subtype. Numerous protein complexes were identified, including many new interactions in various signalling pathways. Systematic identification data set together with protein interactions reported in the literature revealed a high degree of connectivity. These allow the proposal, for the first time, of an outline of the muscarinic interactome as a network of protein complexes and a context for a more reasoned and informed approach to drug discovery and muscarinic receptor subtype specificities.

Research paper thumbnail of Differential expression of muscarinic acetylcholine receptor subtypes in Jurkat cells and their signaling

Journal of Neuroimmunology, 2011

Muscarinic acetylcholine receptors expression and signaling in the human Jurkat T cell line were ... more Muscarinic acetylcholine receptors expression and signaling in the human Jurkat T cell line were investigated. Semiquantitative real-time PCR and radioligand binding studies, using a wide set of antagonist compounds, showed the co-existence of M 3 , M 4 , and M 5 subtypes. Stimulation of these subpopulations caused a concentration and time-dependent activation of second messengers and ERK signaling pathways, with a major contribution of the M 3 subtype in a G q/11 -mediated response. In addition, we found that T-cell stimulation leads to increased expression of M 3 and M 5 both at transcriptional and protein levels in a PLC/ PKCθ dependent manner. Our data clarifies the functional role of AChR subtypes in Jurkat cells and pave the way to future studies on the potential cross-talk among these subpopulations and their regulation of T lymphocytes immune function.

Research paper thumbnail of On the Existence of a Possible A2A–D2–β-Arrestin2 Complex: A2A Agonist Modulation of D2 Agonist-Induced β-Arrestin2 Recruitment

Journal of Molecular Biology, 2011

Given that coactivation of adenosine A 2A (A 2A R) and dopamine D 2 (D 2 R) receptors results in ... more Given that coactivation of adenosine A 2A (A 2A R) and dopamine D 2 (D 2 R) receptors results in the coaggregation, cointernalization, and codesensitization of the A 2A R and D 2 R and the role of scaffolding protein β-arrestin2 in the desensitization, internalization, and signaling of G-protein-coupled receptors, in this study we explored the ability of the A 2A R agonist CGS21680 in A 2A R-D 2 R-coexpressing cells to modulate the D 2 R agonistinduced recruitment of β-arrestin2 to the D 2 R by means of proximity-based bioluminescence resonance energy transfer (BRET 2 ) and co-trafficking analysis. We found evidence that CGS21680 can increase the maximal BRET 2 signal between β-arrestin2 RLuc and D 2L R GFP2 upon D 2 R activation, by increasing the potency of the D 2 R agonist to exert this action. In addition, this change was associated with an increased formation of cytoplasmic clusters containing β-arrestin2 GFP2 and D 2L R YFP as seen from the cotrafficking analysis. Furthermore, the A 2A R agonist advanced the time for the increase in Akt phosphorylation obtained with the D 2 R agonist. Finally, using a novel bioinformatics approach to predict the protein-protein interface, we have also found that amino acid pro-triplets TNY, LLS, RAF, and VSR may be crucial for the -induced β-arrestin2 recruitment by A 2A R-D 2 R heteromers. Taken together, the results indicate that the antagonistic A 2A R-D 2 R allosteric receptor-receptor interaction in A 2A R-D 2 R heteromers favors β-arrestin2 recruitment to the D 2L R protomer with subsequent cointernalization associated with a reduced time onset of Akt phosphorylation followed by a rapid dephosphorylation. Thus, β-arrestin2 action becomes more rapid and short-lasting and, in this way, mimics G-proteinmediated signaling.

Research paper thumbnail of Altered trafficking and unfolded protein response induction as a result of M3 muscarinic receptor impaired N-glycosylation

Glycobiology, 2011

The human M 3 muscarinic acetylcholine receptor is present in both the central and peripheral ner... more The human M 3 muscarinic acetylcholine receptor is present in both the central and peripheral nervous system, and it is involved in the pathophysiology of several neurodegenerative and autoimmune diseases. We suggested a possible N-glycosylation map for the M 3 muscarinic receptor expressed in COS-7 cells. Here, we examined the role that N-linked glycans play in the folding and in the cell surface trafficking of this receptor. The five potential asparagine-linked glycosylation sites in the muscarinic receptor were mutated and transiently expressed in COS-7 cells. The elimination of N-glycan attachment sites did not affect the cellular expression levels of the receptor. However, proper receptor localization to the plasma membrane was affected as suggested by reduced [ 3 H]-N-methylscopolamine binding. Confocal microscopy confirmed this observation and showed that the nonglycosylated receptor was primarily localized in the intracellular compartments. The mutant variant showed an increase in phosphorylation of the α-subunit of eukaryote initiation factor 2, and other well-known endoplasmic reticulum stress markers of the unfolded protein response pathway, which further supports the proposal of the improper intracellular accumulation of the nonglycosylated receptor. The receptor devoid of glycans showed more susceptibility to events that culminate in apoptosis reducing cell viability. Our findings suggest up-regulation of pro-apoptotic Bax protein, down-regulation of anti-apoptotic Bcl-2, and cleavage of caspase-3 effectors. Collectively, our data provide experimental evidence of the critical role that N-glycan chains play in determining muscarinic receptor distribution, localization, as well as cell integrity.

Research paper thumbnail of Volume transmission and its different forms in the central nervous system

Chinese Journal of Integrative Medicine, 2013

Volume transmission (VT) is a widespread mode of intercellular communication that occurs in the e... more Volume transmission (VT) is a widespread mode of intercellular communication that occurs in the extracellular fluid (ECF) and in the cerebrospinal fluid (CSF) of the brain with VT signals moving from source to target cells via energy gradients leading to diffusion and convection (fl ow). The VT channels are diffuse forming a plexus in the extracellular space, while in wiring transmission (WT) the channels (axons, terminals) are private. The speed is slow (seconds-minutes) in VT while rapid in the millisecond range in WT. The extracellular space is the substrate for VT, which is modulated by the extracellular matrix. Extrasynaptic VT is linked to synaptic transmission and likely often takes place due to incomplete diffusion barriers with the synaptic transmitter reaching extrasynaptic domains of the pre-and post-synaptic membrane of the synapse, the astroglia, and even adjacent synapses. Indications exist for the existence of striatal D2-like receptor-mediated extrasynaptic form of dopamine (DA) VT at the local circuit level in vivo in the human striatum. Synaptic glutamate via extrasynaptic VT can act on extrasynaptic metabotropic glutamate receptors located on the astroglia leading to Ca 2+ mediated astrocytic glutamate release into the extracellular space (ECS). Long distance peptide VT and CSF VT is the major long distance VT with distances more than 1 mm and fl ow in the CSF. Indications for long distance VT of beta-endorphin and oxytocin are obtained.

Research paper thumbnail of On the role of volume transmission and receptor–receptor interactions in social behaviour: Focus on central catecholamine and oxytocin neurons

Brain Research, 2012

This article is focused on understanding the mechanisms for the interactions between the central ... more This article is focused on understanding the mechanisms for the interactions between the central catecholamine (CA) and oxytocin (OXY) neurons and their relevance for brain function especially social behaviour in the field of pair bonding. Such a topic is analysed under two perspectives namely the intercellular communication modes between CA and OXT neurons and the molecular integrative mechanisms at the plasma membrane level between their respective decoding systems. As a matter of fact, recent observations strongly indicate a major role of volume transmission and receptor-receptor interactions in the CA/OXT neuron interplay in the brain control of social behaviour and pair bonding.

Research paper thumbnail of Overproduction of human M3 muscarinic acetylcholine receptor: An approach toward structural studies

Biotechnology Progress, 2011

Human M 3 muscarinic acetylcholine receptor (M3R), present in both the central and the peripheral... more Human M 3 muscarinic acetylcholine receptor (M3R), present in both the central and the peripheral nervous system, is involved in several neurodegenerative and autoimmune diseases. Recently, M3R overexpression has been suggested to play a role in certain forms of cancer, showing promise as a new potential pharmacological target. However, the lack of structural information hampered to develop a new potent selective and potent antagonist. We describe here different strategies for overexpressing functional M3R on the perspective of future biophysical studies. To achieve this goal, four tagged M3R genes were engineered and codon optimized. Different heterologous expression systems, including mammalian cells and viral transfection, were employed to overexpress M3R. Although codon optimization resulted in only twofold to threefold increase of M3R expression, we found that epitope tagging of the synthetic M3R, especially with hemagglutinin and Flag epitope tags, could improve M3R expression levels. On the other hand, viral transfection led to a yield of 27 pmol/mg protein that is the highest level reported so far for this receptor subtype in mammalian cells. Taking together several of the strategies used can help increasing M3R expression, not only to start purification efforts but also for secondary structural analysis trial and functional analyses.

Research paper thumbnail of Fibroblast Growth Factor Receptor 1– 5-Hydroxytryptamine 1A Heteroreceptor Complexes and Their Enhancement of Hippocampal Plasticity

Biological Psychiatry, 2012

The hippocampus and its 5-hydroxytryptamine transmission plays an important role in depression re... more The hippocampus and its 5-hydroxytryptamine transmission plays an important role in depression related to its involvement in limbic circuit plasticity.

Research paper thumbnail of Dopamine D2 and D4 receptor heteromerization and its allosteric receptor–receptor interactions

Biochemical and Biophysical Research Communications, 2011

Available online xxxx 22 Keywords: 23 Dopamine D 2 R receptor 24 Dopamine D 4 R receptor 25 Heter... more Available online xxxx 22 Keywords: 23 Dopamine D 2 R receptor 24 Dopamine D 4 R receptor 25 Heteromerization 26 G protein-coupled receptors 27 Allosteric modulation 28 Protein-protein interactions. 29

Research paper thumbnail of Agonist-induced formation of FGFR1 homodimers and signaling differ among members of the FGF family

Biochemical and Biophysical Research Communications, 2011

Fibroblast growth factor receptor 1 (FGFR1) is known to be activated by homodimerization in the p... more Fibroblast growth factor receptor 1 (FGFR1) is known to be activated by homodimerization in the presence of both the FGF agonist ligand and heparan sulfate glycosaminoglycan. FGFR1 homodimers in turn trigger a variety of downstream signaling cascades via autophosphorylation of tyrosine residues in the cytoplasmic domain of FGFR1. By means of Bioluminescence Energy Resonance Transfer (BRET) as a sign of FGFR1 homodimerization, we evaluated in HEK293T cells the effects of all known FGF agonist ligands on homodimer formation. A significant correlation between BRET 2 signaling and ERK1/2 phosphorylation was observed, leading to a further characterization of the binding and signaling properties of the FGF subfamilies. FGF agonist ligand-FGFR1 binding interactions appear as the main mechanism for the control of FGFR1 homodimerization and MAPK signaling which demonstrated a high correlation. The bioinformatic analysis demonstrates the interface of the two pro-triplets SSS (Ser-Ser-Ser) and YGS (Tyr-Gly-Ser) located in the extracellular and intracellular domain of the FGFR1. These pro-triplets are postulated participate in the FGFR1 homodimerization interface interaction. The findings also reveal that FGF agonist ligands within the same subfamily of the FGF gene family produced similar increases in FGFR1 homodimer formation and MAPK signaling. Thus, the evolutionary relationship within this gene family appears to have a distinct functional relevance.

Research paper thumbnail of Characterization of the A2AR–D2R interface: Focus on the role of the C-terminal tail and the transmembrane helices

Biochemical and Biophysical Research Communications, 2010

A single serine point mutation (S374A) in the adenosine A 2A receptor (A 2A R) C-terminal tail re... more A single serine point mutation (S374A) in the adenosine A 2A receptor (A 2A R) C-terminal tail reduces A 2A R-D 2 R heteromerization and prevents its allosteric modulation of the dopamine D 2 receptor (D 2 R).

Research paper thumbnail of Dopamine D2 and 5-hydroxytryptamine 5-HT2A receptors assemble into functionally interacting heteromers

Biochemical and Biophysical Research Communications, 2010

In view of the co-distribution of dopamine D 2L R and 5-hydroxytryptamine 5-HT 2A receptors (D 2L... more In view of the co-distribution of dopamine D 2L R and 5-hydroxytryptamine 5-HT 2A receptors (D 2L R and 5-HT 2A R, respectively) within inter alia regions of the dorsal and ventral striatum and their role as a target of antipsychotic drugs; in this study we assessed the potential existence of D 2L R-5-HT 2A R heteromers in living cells and the functional consequences of this interaction. Thus, by means of a proximity-based bioluminescence resonance energy transfer (BRET) approach we demonstrated that the D 2L R and the 5-HT 2A R form stable and specific heteromers when expressed in HEK293T mammalian cells. Furthermore, when the D 2L R-5-HT 2A R heteromeric signaling was analyzed we found that the 5-HT 2A R-mediated phospholipase C (PLC) activation was synergistically enhanced by the concomitant activation of the D 2L R as shown in a NFAT-luciferase reporter gene assay and a specific and significant rise of the intracellular calcium levels were observed when both receptors were simultaneously activated. Conversely, when the D 2L R-mediated adenylyl cyclase (AC) inhibition was assayed we showed that costimulation of D 2L R and 5-HT 2A R within the heteromer led to inhibition of the D 2L R functioning, thus suggesting the existence of a 5-HT 2A R-mediated D 2L R trans-inhibition phenomenon. Finally, a bioinformatics study reveals that the triplet amino acid homologies LLT (Leu-Leu-Thr) and AIS (Ala-Ile-Ser) in TM1 and TM3, respectively of the D 2 R-5-HT 2A R may be involved in the receptor interface. Overall, the presence of the D 2L R-5-HT 2A R heteromer in discrete brain regions is postulated based on the existence of D 2L R-5-HT 2A receptor-receptor interactions in living cells and their codistribution inter alia in striatal regions. Possible novel therapeutic strategies for treatment of schizophrenia should be explored by targeting this heteromer.

Research paper thumbnail of Dopamine D2 heteroreceptor complexes and their receptor-receptor interactions in ventral striatum: Novel targets for antipsychotic drugs

This review is focused on the D2 heteroreceptor complexes within the ventral striatum with their ... more This review is focused on the D2 heteroreceptor complexes within the ventral striatum with their receptor-receptor interactions and relevance for the treatment of schizophrenia. A "guide-andclasp" manner for receptor-receptor interactions is proposed where "adhesive guides" may be amino acid triplet homologies, which were determined for different kinds of D2 heteroreceptor complexes. The first putative D2 heteroreceptor complex to be discovered in relation to schizophrenia was the A2A-D2 heteroreceptor complex where antagonistic A2A-D2 receptorreceptor interactions were demonstrated after A2A agonist treatment in the ventral striatum. The A2A agonist CGS 21680 with atypical antipsychotic properties may at least in part act by increasing b-arrestin2 signaling over the D2 protomer in the A2A-D2 heteroreceptor complex in the ventral striatum. The antagonistic NTS1-D2 interactions in the NTS1-D2 heteroreceptor complex Progress in Brain Research, Volume 211, ISSN 0079-6123, http://dx.

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

Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 2011

Acetylcholine challenge produces M(3) muscarinic acetylcholine receptor activation and accessory/... more 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 Gα(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 M(3)R and ARF1 an...

Research paper thumbnail of Dopamine D-4 receptor oligomerization: contribution to receptor biogenesis

archive.ugent.be

BRET, bioluminescence resonance energy transfer; CHO, Chinese hamster ovary; DAPI, 4¢,6-diamidino... more BRET, bioluminescence resonance energy transfer; CHO, Chinese hamster ovary; DAPI, 4¢,6-diamidino-2-phenylindole; D n R, dopamine D n receptor; ER, endoplasmic reticulum; GPCR, G protein-coupled receptor; HRP, horseradish peroxidase; MP, milk powder; YFP, yellow fluorescent protein.

Research paper thumbnail of Dopamine D2 and D4 receptor heteromerization and its allosteric receptor–receptor interactions

Biochemical and Biophysical Research Communications, 2011

Dopamine D2 and D4 receptors partially codistribute in the dorsal striatum and appear to play a f... more Dopamine D2 and D4 receptors partially codistribute in the dorsal striatum and appear to play a fundamental role in complex behaviors and motor function. The discovery of D2R–D4.xR (D4.2R, D4.4R or D4.7R) heteromers has been made in cellular models using co-immunoprecipitation, in situ Proximity Ligation Assays and BRET1 techniques with the D2R and D4.7R receptors being the least effective in

Research paper thumbnail of Moonlighting Proteins and Protein–Protein Interactions as Neurotherapeutic Targets in the G Protein-Coupled Receptor Field

Neuropsychopharmacology, 2014

There is serious interest in understanding the dynamics of the receptor-receptor and receptor-pro... more There is serious interest in understanding the dynamics of the receptor-receptor and receptor-protein interactions in space and time and their integration in GPCR heteroreceptor complexes of the CNS. Moonlighting proteins are special multifunctional proteins because they perform multiple autonomous, often unrelated, functions without partitioning into different protein domains. Moonlighting through receptor oligomerization can be operationally defined as an allosteric receptor-receptor interaction, which leads to novel functions of at least one receptor protomer. GPCR-mediated signaling is a more complicated process than previously described as every GPCR and GPCR heteroreceptor complex requires a set of G protein interacting proteins, which interacts with the receptor in an orchestrated spatio-temporal fashion. GPCR heteroreceptor complexes with allosteric receptor-receptor interactions operating through the receptor interface have become major integrative centers at the molecular level and their receptor protomers act as moonlighting proteins. The GPCR heteroreceptor complexes in the CNS have become exciting new targets for neurotherapeutics in Parkinson's disease, schizophrenia, drug addiction, and anxiety and depression opening a new field in neuropsychopharmacology.

Research paper thumbnail of Muscarinic receptor family interacting proteins: Role in receptor function

Journal of Neuroscience Methods, 2011

a b s t r a c t G protein-coupled receptors constitute one of the most important families of memb... more a b s t r a c t G protein-coupled receptors constitute one of the most important families of membrane receptors through which cells respond to extracellular stimuli. Receptors of this superfamily likely function as signal transduction complexes. The identification and analysis of their components provide new insights into a better understanding of these receptors' function and regulation. We used tandem-affinity purification and mass spectrometry as a systematic approach to characterize multiprotein complexes in the acetylcholine muscarinic receptor subfamily. To overcome the limitations associated with membrane protein receptor solubilization with detergents, we developed a strategy in which receptors are co-expressed with a cytoplasmic minigene construct, encoding the third intracellular loop and the C-terminal tail tagged to the tandem-affinity-cassette of each receptor subtype. Numerous protein complexes were identified, including many new interactions in various signalling pathways. Systematic identification data set together with protein interactions reported in the literature revealed a high degree of connectivity. These allow the proposal, for the first time, of an outline of the muscarinic interactome as a network of protein complexes and a context for a more reasoned and informed approach to drug discovery and muscarinic receptor subtype specificities.

Research paper thumbnail of Differential expression of muscarinic acetylcholine receptor subtypes in Jurkat cells and their signaling

Journal of Neuroimmunology, 2011

Muscarinic acetylcholine receptors expression and signaling in the human Jurkat T cell line were ... more Muscarinic acetylcholine receptors expression and signaling in the human Jurkat T cell line were investigated. Semiquantitative real-time PCR and radioligand binding studies, using a wide set of antagonist compounds, showed the co-existence of M 3 , M 4 , and M 5 subtypes. Stimulation of these subpopulations caused a concentration and time-dependent activation of second messengers and ERK signaling pathways, with a major contribution of the M 3 subtype in a G q/11 -mediated response. In addition, we found that T-cell stimulation leads to increased expression of M 3 and M 5 both at transcriptional and protein levels in a PLC/ PKCθ dependent manner. Our data clarifies the functional role of AChR subtypes in Jurkat cells and pave the way to future studies on the potential cross-talk among these subpopulations and their regulation of T lymphocytes immune function.

Research paper thumbnail of On the Existence of a Possible A2A–D2–β-Arrestin2 Complex: A2A Agonist Modulation of D2 Agonist-Induced β-Arrestin2 Recruitment

Journal of Molecular Biology, 2011

Given that coactivation of adenosine A 2A (A 2A R) and dopamine D 2 (D 2 R) receptors results in ... more Given that coactivation of adenosine A 2A (A 2A R) and dopamine D 2 (D 2 R) receptors results in the coaggregation, cointernalization, and codesensitization of the A 2A R and D 2 R and the role of scaffolding protein β-arrestin2 in the desensitization, internalization, and signaling of G-protein-coupled receptors, in this study we explored the ability of the A 2A R agonist CGS21680 in A 2A R-D 2 R-coexpressing cells to modulate the D 2 R agonistinduced recruitment of β-arrestin2 to the D 2 R by means of proximity-based bioluminescence resonance energy transfer (BRET 2 ) and co-trafficking analysis. We found evidence that CGS21680 can increase the maximal BRET 2 signal between β-arrestin2 RLuc and D 2L R GFP2 upon D 2 R activation, by increasing the potency of the D 2 R agonist to exert this action. In addition, this change was associated with an increased formation of cytoplasmic clusters containing β-arrestin2 GFP2 and D 2L R YFP as seen from the cotrafficking analysis. Furthermore, the A 2A R agonist advanced the time for the increase in Akt phosphorylation obtained with the D 2 R agonist. Finally, using a novel bioinformatics approach to predict the protein-protein interface, we have also found that amino acid pro-triplets TNY, LLS, RAF, and VSR may be crucial for the -induced β-arrestin2 recruitment by A 2A R-D 2 R heteromers. Taken together, the results indicate that the antagonistic A 2A R-D 2 R allosteric receptor-receptor interaction in A 2A R-D 2 R heteromers favors β-arrestin2 recruitment to the D 2L R protomer with subsequent cointernalization associated with a reduced time onset of Akt phosphorylation followed by a rapid dephosphorylation. Thus, β-arrestin2 action becomes more rapid and short-lasting and, in this way, mimics G-proteinmediated signaling.

Research paper thumbnail of Altered trafficking and unfolded protein response induction as a result of M3 muscarinic receptor impaired N-glycosylation

Glycobiology, 2011

The human M 3 muscarinic acetylcholine receptor is present in both the central and peripheral ner... more The human M 3 muscarinic acetylcholine receptor is present in both the central and peripheral nervous system, and it is involved in the pathophysiology of several neurodegenerative and autoimmune diseases. We suggested a possible N-glycosylation map for the M 3 muscarinic receptor expressed in COS-7 cells. Here, we examined the role that N-linked glycans play in the folding and in the cell surface trafficking of this receptor. The five potential asparagine-linked glycosylation sites in the muscarinic receptor were mutated and transiently expressed in COS-7 cells. The elimination of N-glycan attachment sites did not affect the cellular expression levels of the receptor. However, proper receptor localization to the plasma membrane was affected as suggested by reduced [ 3 H]-N-methylscopolamine binding. Confocal microscopy confirmed this observation and showed that the nonglycosylated receptor was primarily localized in the intracellular compartments. The mutant variant showed an increase in phosphorylation of the α-subunit of eukaryote initiation factor 2, and other well-known endoplasmic reticulum stress markers of the unfolded protein response pathway, which further supports the proposal of the improper intracellular accumulation of the nonglycosylated receptor. The receptor devoid of glycans showed more susceptibility to events that culminate in apoptosis reducing cell viability. Our findings suggest up-regulation of pro-apoptotic Bax protein, down-regulation of anti-apoptotic Bcl-2, and cleavage of caspase-3 effectors. Collectively, our data provide experimental evidence of the critical role that N-glycan chains play in determining muscarinic receptor distribution, localization, as well as cell integrity.

Research paper thumbnail of Volume transmission and its different forms in the central nervous system

Chinese Journal of Integrative Medicine, 2013

Volume transmission (VT) is a widespread mode of intercellular communication that occurs in the e... more Volume transmission (VT) is a widespread mode of intercellular communication that occurs in the extracellular fluid (ECF) and in the cerebrospinal fluid (CSF) of the brain with VT signals moving from source to target cells via energy gradients leading to diffusion and convection (fl ow). The VT channels are diffuse forming a plexus in the extracellular space, while in wiring transmission (WT) the channels (axons, terminals) are private. The speed is slow (seconds-minutes) in VT while rapid in the millisecond range in WT. The extracellular space is the substrate for VT, which is modulated by the extracellular matrix. Extrasynaptic VT is linked to synaptic transmission and likely often takes place due to incomplete diffusion barriers with the synaptic transmitter reaching extrasynaptic domains of the pre-and post-synaptic membrane of the synapse, the astroglia, and even adjacent synapses. Indications exist for the existence of striatal D2-like receptor-mediated extrasynaptic form of dopamine (DA) VT at the local circuit level in vivo in the human striatum. Synaptic glutamate via extrasynaptic VT can act on extrasynaptic metabotropic glutamate receptors located on the astroglia leading to Ca 2+ mediated astrocytic glutamate release into the extracellular space (ECS). Long distance peptide VT and CSF VT is the major long distance VT with distances more than 1 mm and fl ow in the CSF. Indications for long distance VT of beta-endorphin and oxytocin are obtained.

Research paper thumbnail of On the role of volume transmission and receptor–receptor interactions in social behaviour: Focus on central catecholamine and oxytocin neurons

Brain Research, 2012

This article is focused on understanding the mechanisms for the interactions between the central ... more This article is focused on understanding the mechanisms for the interactions between the central catecholamine (CA) and oxytocin (OXY) neurons and their relevance for brain function especially social behaviour in the field of pair bonding. Such a topic is analysed under two perspectives namely the intercellular communication modes between CA and OXT neurons and the molecular integrative mechanisms at the plasma membrane level between their respective decoding systems. As a matter of fact, recent observations strongly indicate a major role of volume transmission and receptor-receptor interactions in the CA/OXT neuron interplay in the brain control of social behaviour and pair bonding.

Research paper thumbnail of Overproduction of human M3 muscarinic acetylcholine receptor: An approach toward structural studies

Biotechnology Progress, 2011

Human M 3 muscarinic acetylcholine receptor (M3R), present in both the central and the peripheral... more Human M 3 muscarinic acetylcholine receptor (M3R), present in both the central and the peripheral nervous system, is involved in several neurodegenerative and autoimmune diseases. Recently, M3R overexpression has been suggested to play a role in certain forms of cancer, showing promise as a new potential pharmacological target. However, the lack of structural information hampered to develop a new potent selective and potent antagonist. We describe here different strategies for overexpressing functional M3R on the perspective of future biophysical studies. To achieve this goal, four tagged M3R genes were engineered and codon optimized. Different heterologous expression systems, including mammalian cells and viral transfection, were employed to overexpress M3R. Although codon optimization resulted in only twofold to threefold increase of M3R expression, we found that epitope tagging of the synthetic M3R, especially with hemagglutinin and Flag epitope tags, could improve M3R expression levels. On the other hand, viral transfection led to a yield of 27 pmol/mg protein that is the highest level reported so far for this receptor subtype in mammalian cells. Taking together several of the strategies used can help increasing M3R expression, not only to start purification efforts but also for secondary structural analysis trial and functional analyses.

Research paper thumbnail of Fibroblast Growth Factor Receptor 1– 5-Hydroxytryptamine 1A Heteroreceptor Complexes and Their Enhancement of Hippocampal Plasticity

Biological Psychiatry, 2012

The hippocampus and its 5-hydroxytryptamine transmission plays an important role in depression re... more The hippocampus and its 5-hydroxytryptamine transmission plays an important role in depression related to its involvement in limbic circuit plasticity.

Research paper thumbnail of Dopamine D2 and D4 receptor heteromerization and its allosteric receptor–receptor interactions

Biochemical and Biophysical Research Communications, 2011

Available online xxxx 22 Keywords: 23 Dopamine D 2 R receptor 24 Dopamine D 4 R receptor 25 Heter... more Available online xxxx 22 Keywords: 23 Dopamine D 2 R receptor 24 Dopamine D 4 R receptor 25 Heteromerization 26 G protein-coupled receptors 27 Allosteric modulation 28 Protein-protein interactions. 29

Research paper thumbnail of Agonist-induced formation of FGFR1 homodimers and signaling differ among members of the FGF family

Biochemical and Biophysical Research Communications, 2011

Fibroblast growth factor receptor 1 (FGFR1) is known to be activated by homodimerization in the p... more Fibroblast growth factor receptor 1 (FGFR1) is known to be activated by homodimerization in the presence of both the FGF agonist ligand and heparan sulfate glycosaminoglycan. FGFR1 homodimers in turn trigger a variety of downstream signaling cascades via autophosphorylation of tyrosine residues in the cytoplasmic domain of FGFR1. By means of Bioluminescence Energy Resonance Transfer (BRET) as a sign of FGFR1 homodimerization, we evaluated in HEK293T cells the effects of all known FGF agonist ligands on homodimer formation. A significant correlation between BRET 2 signaling and ERK1/2 phosphorylation was observed, leading to a further characterization of the binding and signaling properties of the FGF subfamilies. FGF agonist ligand-FGFR1 binding interactions appear as the main mechanism for the control of FGFR1 homodimerization and MAPK signaling which demonstrated a high correlation. The bioinformatic analysis demonstrates the interface of the two pro-triplets SSS (Ser-Ser-Ser) and YGS (Tyr-Gly-Ser) located in the extracellular and intracellular domain of the FGFR1. These pro-triplets are postulated participate in the FGFR1 homodimerization interface interaction. The findings also reveal that FGF agonist ligands within the same subfamily of the FGF gene family produced similar increases in FGFR1 homodimer formation and MAPK signaling. Thus, the evolutionary relationship within this gene family appears to have a distinct functional relevance.

Research paper thumbnail of Characterization of the A2AR–D2R interface: Focus on the role of the C-terminal tail and the transmembrane helices

Biochemical and Biophysical Research Communications, 2010

A single serine point mutation (S374A) in the adenosine A 2A receptor (A 2A R) C-terminal tail re... more A single serine point mutation (S374A) in the adenosine A 2A receptor (A 2A R) C-terminal tail reduces A 2A R-D 2 R heteromerization and prevents its allosteric modulation of the dopamine D 2 receptor (D 2 R).

Research paper thumbnail of Dopamine D2 and 5-hydroxytryptamine 5-HT2A receptors assemble into functionally interacting heteromers

Biochemical and Biophysical Research Communications, 2010

In view of the co-distribution of dopamine D 2L R and 5-hydroxytryptamine 5-HT 2A receptors (D 2L... more In view of the co-distribution of dopamine D 2L R and 5-hydroxytryptamine 5-HT 2A receptors (D 2L R and 5-HT 2A R, respectively) within inter alia regions of the dorsal and ventral striatum and their role as a target of antipsychotic drugs; in this study we assessed the potential existence of D 2L R-5-HT 2A R heteromers in living cells and the functional consequences of this interaction. Thus, by means of a proximity-based bioluminescence resonance energy transfer (BRET) approach we demonstrated that the D 2L R and the 5-HT 2A R form stable and specific heteromers when expressed in HEK293T mammalian cells. Furthermore, when the D 2L R-5-HT 2A R heteromeric signaling was analyzed we found that the 5-HT 2A R-mediated phospholipase C (PLC) activation was synergistically enhanced by the concomitant activation of the D 2L R as shown in a NFAT-luciferase reporter gene assay and a specific and significant rise of the intracellular calcium levels were observed when both receptors were simultaneously activated. Conversely, when the D 2L R-mediated adenylyl cyclase (AC) inhibition was assayed we showed that costimulation of D 2L R and 5-HT 2A R within the heteromer led to inhibition of the D 2L R functioning, thus suggesting the existence of a 5-HT 2A R-mediated D 2L R trans-inhibition phenomenon. Finally, a bioinformatics study reveals that the triplet amino acid homologies LLT (Leu-Leu-Thr) and AIS (Ala-Ile-Ser) in TM1 and TM3, respectively of the D 2 R-5-HT 2A R may be involved in the receptor interface. Overall, the presence of the D 2L R-5-HT 2A R heteromer in discrete brain regions is postulated based on the existence of D 2L R-5-HT 2A receptor-receptor interactions in living cells and their codistribution inter alia in striatal regions. Possible novel therapeutic strategies for treatment of schizophrenia should be explored by targeting this heteromer.

Research paper thumbnail of Dopamine D2 heteroreceptor complexes and their receptor-receptor interactions in ventral striatum: Novel targets for antipsychotic drugs

This review is focused on the D2 heteroreceptor complexes within the ventral striatum with their ... more This review is focused on the D2 heteroreceptor complexes within the ventral striatum with their receptor-receptor interactions and relevance for the treatment of schizophrenia. A "guide-andclasp" manner for receptor-receptor interactions is proposed where "adhesive guides" may be amino acid triplet homologies, which were determined for different kinds of D2 heteroreceptor complexes. The first putative D2 heteroreceptor complex to be discovered in relation to schizophrenia was the A2A-D2 heteroreceptor complex where antagonistic A2A-D2 receptorreceptor interactions were demonstrated after A2A agonist treatment in the ventral striatum. The A2A agonist CGS 21680 with atypical antipsychotic properties may at least in part act by increasing b-arrestin2 signaling over the D2 protomer in the A2A-D2 heteroreceptor complex in the ventral striatum. The antagonistic NTS1-D2 interactions in the NTS1-D2 heteroreceptor complex Progress in Brain Research, Volume 211, ISSN 0079-6123, http://dx.