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The adenosine A 2A receptor (A 2A R), dopamine D 2 receptor (D 2 R) and metabotropic glutamate receptor type 5 (mGluR 5 ) form A 2A R-D 2 R-mGluR 5 heteroreceptor complexes in living cells and in rat striatal neurons. In the current study, we present experimental data supporting the view that the A 2A R protomer plays a major role in the inhibitory modulation of the density and the allosteric receptor-receptor interaction within the D 2 R-mGluR 5 heteromeric component of the A 2A R-D 2 R-mGluR 5 complex in vitro and in vivo. The A 2A R and mGluR 5 protomers interact and modulate D 2 R protomer recognition and signalling upon forming a trimeric complex from these receptors. Expression of A 2A R in HEK293T cells co-expressing D 2 R and mGluR 5 resulted in a significant and marked increase in the formation of the D 2 R-mGluR 5 heteromeric component in both bioluminescence resonance energy transfer and proximity ligation assays. A highly significant increase of the the high-affinity component of D 2 R (D2R Ki High) values was found upon cotreatment with the mGluR 5 and A 2A R agonists in the cells expressing A 2A R, D 2 R and mGluR 5 with a significant effect observed also with the mGluR 5 agonist alone compared to cells expressing only D 2 R and mGluR 5 . In cells co-expressing A 2A R, D 2 R and mGluR 5 , stimulation of the cells with an mGluR 5 agonist like or D 2 R antagonist fully counteracted the D 2 R agonist-induced inhibition of the cAMP levels which was not true in cells only expressing mGluR 5 and D 2 R. In agreement, the mGluR 5 -negative allosteric modulator raseglurant significantly reduced the haloperidol-induced catalepsy in mice, and in A 2A R knockout mice, the haloperidol action had almost disappeared, supporting a functional role for mGluR 5 and A 2A R in enhancing D 2 R blockade resulting in catalepsy. The results represent a relevant example of integrative activity within higher-order heteroreceptor complexes., (© 2022. The Author(s).)

The current study was performed to establish the actions of nanomolar concentrations of cocaine, not blocking the dopamine transporter, on dopamine D2 receptor (D 2 R)-sigma 1 receptor (δ1R) heteroreceptor complexes and the D 2 R protomer recognition, signaling and internalization in cellular models. We report the existence of D 2 R-δ1R heteroreceptor complexes in subcortical limbic areas as well as the dorsal striatum, with different distribution patterns using the in situ proximity ligation assay. Also, through BRET, these heteromers were demonstrated in HEK293 cells. Furthermore, saturation binding assay demonstrated that in membrane preparations of HEK293 cells coexpressing D 2 R and δ1R, cocaine (1 nM) significantly increased the D 2 R B max values over cells singly expressing D 2 R. CREB reporter luc-gene assay indicated that coexpressed δ1R significantly reduced the potency of the D 2 R-like agonist quinpirole to inhibit via D 2 R activation the forskolin induced increase of the CREB signal. In contrast, the addition of 100 nM cocaine was found to markedly increase the quinpirole potency to inhibit the forskolin-induced increase of the CREB signal in the D 2 R-δ1R cells. These events were associated with a marked reduction of cocaine-induced internalization of D 2 R protomers in D 2 R-δ1R heteromer-containing cells vs D 2 R singly expressing cells as studied by means of confocal analysis of D 2 R-δ1R trafficking and internalization. Overall, the formation of D 2 R-δ1R heteromers enhanced the ability of cocaine to increase the D 2 R protomer function associated with a marked reduction of its internalization. The existence of D 2 R-δ1R heteromers opens up a new understanding of the acute actions of cocaine.

Antagonistic allosteric A2AR-D2R receptor-receptor interactions in heteroreceptor complexes counteract cocaine self-administration and cocaine seeking in rats as seen in biochemical and behavioral experiments. It was shown that the human A2AR transmembrane five (TM5) was part of the interface of the human A2AR-D2R receptor heteromer. In the current paper, the rat A2AR synthetic TM5 (synthTM5) peptide disrupts the A2AR-D2R heteroreceptor complex in HEK293 cells as shown by the bioluminescence resonance energy transfer method. Rat A2AR synthTM5 peptide, microinjected into the nucleus accumbens, produced a complete counteraction of the inhibitory effects of the A2AR agonist CGS21680 on cocaine self-administration. It was linked to a disappearance of the accumbal A2AR-D2R heteroreceptor complexes and the A2AR agonist induced inhibition of D2R recognition using proximity ligation assay and biochemical binding techniques. However, possible effects of the A2AR synthTM5 peptide on accumbal A2AR-D3R and A2AR-D4R heteroreceptor complexes remain to be excluded. Evidence is provided that accumbal A2AR-D2R-like heteroreceptor complexes with their antagonistic receptor-receptor interactions can be major targets for treatment of cocaine use disorder.

The effects of cocaine (150 nM, 300 nM, and 150 μM) on human glioblastoma cell cultures were studied on tunneling nanotube formation (1-h cocaine treatment) and extracellular vesicle release (1-, 3-, and 8-h cocaine treatment). Cocaine significantly increased the number of tunneling nanotubes only at the lowest concentration used. The release of extracellular vesicles (mainly exosomes) into the medium was stimulated by cocaine at each concentration used with a maximum effect at the highest concentration tested (150 μM). Moreover, cocaine (150 nM) significantly increased the number of vesicles with 61-80 nm diameter while at concentrations of 300 nM and 150 μM, and the smaller vesicles (30-40 nm diameter) were significantly increased with a reduction of the larger vesicles (41-60 nm diameter). A time dependence in the release of extracellular vesicles was observed. In view of the proposed role of these novel intercellular communication modes in the glial-neuronal plasticity, it seems possible that they can participate in the processes leading to cocaine addiction. The molecular target/s involved in these cocaine effects could be specific molecular components of plasma membrane lipid rafts and/or cocaine-induced modifications in cytoplasmic lipid composition.

Based on our theory, we have discovered main triplets of amino acid residues in cell-adhesion receptors of marine sponges, which appear also as homologies in several receptor heteromers of human brain. The obtained results strengthen our hypothesis that these triplets may "guide-and-clasp" receptor-receptor interactions.

Based on our theory, we have discovered main triplets of amino acid residues in the GABAB1 receptor and several other neural receptors which seem to come from immunoglobulin chains and appear also as homologies in receptor heteromers. The obtained results strengthen our hypothesis that these triplets may "guide-and-clasp" receptor-receptor interactions playing a role, e.g., in neuroinflammation disorders.

Based on our theory, we point out main triplets of amino acid residues in the GABAB1 receptor, found in the central and peripheral nervous system, which seem to be critical for both receptor heterodimerization and chemokine binding. The obtained results suggest that these triplets may "guide-and-clasp" protein-protein interactions playing a role, e.g., in neuroinflammation disorders.

Patients affected by recurrent seizures frequently present increased homocysteine plasma levels in consequence of treatment with antiepileptic drugs. Homocysteine is proconvulsant and can affect the response to antiepileptic drugs. In addition, high homocysteine plasma levels represent a risk factor for cardiovascular and neurodegenerative diseases. To better define the role of increased homocysteine in epilepsy, we analyzed the effects of homocysteine pretreatment in the pilocarpine model of status epilepticus (SE), which is used to mimic temporal lobe epilepsy (TLE) in rodents. Precisely, we investigated whether a moderate hyperhomocysteinemia, unable to cause seizures, could sensitize rats to pilocarpine and cooperate in inducing brain lesions. We found that a subthreshold dose of pilocarpine (200 mg/kg) is sufficient to induce SE in the majority (approximately 90%) of rats pretreated with homocysteine for 2 weeks, whereas only 40% of saline-treated controls developed SE following the same pilocarpine dose. Furthermore, homocysteine pretreatment led to a significant increase in neuronal cell loss evaluated by counting toluidine blue-stained or Fluoro-Jade-positive cells in hippocampal and parahippocampal regions. Pilocarpine augmented amyloid beta expression in both animal groups. However, pretreatment with homocysteine favored the intraneuronal fibrillar conformation of amyloid beta, thus promoting neurodegeneration. These findings indicate that increased homocysteine levels enhance seizure activity and neurodegeneration in pilocarpine-treated rats and suggest that similar detrimental effects may occur in patients affected by TLE.

The effects of low and high concentrations of cocaine have been studied in vitro on the trafficking of plasma membrane A(2A) and D(2) immunoreactivities in previously characterized A(2A)-D(2) CHO cell lines. Receptor double immunofluorescence staining was performed with D(2) and A(2A) antibodies, planar lipid rafts immunolabeling with biotinylated cholera toxin subunit B and membrane invaginations with an anti-caveolin-1 antibody. A computer-assisted image analysis demonstrated a substantial and highly significant rise of membrane-associated D(2) immunoreactivity (IR) after 8 h of exposure to a low concentration of cocaine (150 nM). At this low concentration of cocaine, there was also an increase of membrane associated A(2A) immunoreactivity but smaller and less significant. However, this increase became considerably larger and highly significant at 150 microM at which concentration the rise of D(2) immunoreactivity had begun to disappear. It may be suggested that an allosteric action of cocaine at 150 nM on the D(2) receptors may primarily increase the insertion of D(2) monomers, homomers and also of a subpopulation of A(2A)-D(2) heteromers from the cytoplasm into the plasma membrane due to the conformational change induced by cocaine in the D(2) receptor. The planar lipid rafts and the caveolae are only affected by the higher concentrations of cocaine. It is proposed that changes in D(2) and A(2A)-D(2) trafficking induced by allosteric actions of cocaine at D(2) receptors may contribute to the alterations of D(2) signaling found in cocaine abusers.

Based on a mathematical approach, we deduce a set of triplet homologies that may be responsible for receptor-receptor interactions. We show how such triplets of amino acid residues and their 'teams' may be utilized to construct a kind of code that determines (and/or predicts) which receptors should or should not form heterodimers. Based on the obtained results, we propose a 'guide-and-clasp' manner for receptor-receptor interactions where 'adhesive guides' might be the triplet homologies. We also demonstrate their relevance to protein-protein interactions and mention possible implications for novel pharmacological targets and strategies for treatment of diseases, e.g. neuroinflammatory diseases.