Dopamine D2 and Serotonin 5-HT1A Dimeric Receptor-Binding Monomeric Antibody scFv as a Potential Ligand for Carrying Drugs Targeting Selected Areas of the Brain.

Targeted therapy uses multiple ways of ensuring that the drug will be delivered to the desired site. One of these ways is an encapsulation of the drug and functionalization of the surface. Among the many molecules that can perform such a task, the present work focused on the antibodies of single-chain variable fragments (scFvs format). We studied scFv, which specifically recognizes the dopamine D2 and serotonin 5-HT1A receptor heteromers. The scFvD2-5-HT1A protein was analyzed biochemically and biologically, and the obtained results indicated that the antibody is properly folded and non-toxic and can be described as low-immunogenic. It is not only able to bind to the D2-5-HT1A receptor heteromer, but it also influences the cAMP signaling pathway and-when surfaced on nanogold particles-it can cross the blood-brain barrier in in vitro models. When administered to mice, it decreased locomotor activity, matching the effect induced by clozapine. Thus, we are strongly convinced that scFvD2-5-HT1A, which was a subject of the present investigation, is a promising targeting ligand with the potential for the functionalization of nanocarriers targeting selected areas of the brain.

Development of a New Polymeric Nanocarrier Dedicated to Controlled Clozapine Delivery at the Dopamine D2-Serotonin 5-HT1A Heteromers.

Clozapine, the second generation antipsychotic drug, is one of the prominent compounds used for treatment of schizophrenia. Unfortunately, use of this drug is still limited due to serious side effects connected to its unspecific and non-selective action. Nevertheless, clozapine still remains the first-choice drug for the situation of drug-resistance schizophrenia. Development of the new strategy of clozapine delivery into well-defined parts of the brain has been a great challenge for modern science. In the present paper we focus on the presentation of a new nanocarrier for clozapine and its use for targeted transport, enabling its interaction with the dopamine D2 and serotonin 5-HT1A heteromers (D2-5-HT1A) in the brain tissue. Clozapine polymeric nanocapsules (CLO-NCs) were prepared using anionic surfactant AOT (sodium docusate) as an emulsifier, and bio-compatible polyelectrolytes such as: poly-l-glutamic acid (PGA) and poly-l-lysine (PLL). Outer layer of the carrier was grafted by polyethylene glycol (PEG). Several variants of nanocarriers containing the antipsychotic varying in physicochemical parameters were tested. This kind of approach may enable the availability and safety of the drug, improve the selectivity of its action, and finally increase effectiveness of schizophrenia therapy. Moreover, the purpose of the manuscript is to cover a wide scope of the issues, which should be considered while designing a novel means for drug delivery. It is important to determine the interactions of a new nanocarrier with many cell components on various cellular levels in order to be sure that the new nanocarrier will be safe and won't cause undesired effects for a patient.

Polycaprolactone Nanoparticles as Promising Candidates for Nanocarriers in Novel Nanomedicines.

An investigation of the interactions between bio-polymeric nanoparticles (NPs) and the RAW 264.7 mouse murine macrophage cell line has been presented. The cell viability, immunological response, and endocytosis efficiency of NPs were studied. Biopolymeric NPs were synthesized from a nanoemulsion using the phase inversion composition (PIC) technique. The two types of biopolymeric NPs that were obtained consisted of a biocompatible polymer, polycaprolactone (PCL), either with or without its copolymer with poly(ethylene glycol) (PCL-b-PEG). Both types of synthesized PCL NPs passed the first in vitro quality assessments as potential drug nanocarriers. Non-pegylated PCL NPs were internalized more effectively and the clathrin-mediated pathway was involved in that process. The investigated NPs did not affect the viability of the cells and did not elicit an immune response in the RAW 264.7 cells (neither a significant increase in the expression of genes encoding pro-inflammatory cytokines nor NO (nitric oxide) production were observed). It may be concluded that the synthesized NPs are promising candidates as nanocarriers of therapeutic compounds.

Towards plant-mediated chemistry - Au nanoparticles obtained using aqueous extract of Rosa damascena and their biological activity in vitro.

An eco-friendly, efficient, and controlled synthesis of gold nanoparticles with application of the aqueous extract of Rosa damascena (Au@RD NPs) without using any other reducing agents was studied. Au@RD NPs of narrow size distribution were characterized by UV-vis and FT-IR spectroscopies, transmission electron microscopy, X-ray powder diffraction, X-ray photoelectron spectroscopy, particle size analysis, and zeta potential measurements. In vitro stability experiments revealed that the Au@RD NPs were stable for over a year (pH ~ 3.5), proving a significant stabilizing potential of the aqueous RD extract. The high total content of polyphenols, flavonoids, and reducing sugars along with the powerful antioxidant activity of the RD extract was determined by spectroscopic and analytical methods. Colloids prepared from the purified and lyophilized Au@RD NPs (electrokinetic potential of ca. -33 mV) were stable for at least 24 h under terms similar to physiological conditions (pH = 7.4, PBS). The in vitro cytotoxicity of Au@RD NPs was investigated against peripheral blood mononuclear lymphocytes (PBML), acute promyelocytic leukemia (HL60), and human lung adenocarcinoma (A549). Selective cytotoxicity of Au@RD NPs towards cancer cells (HL60, A549) over normal cells (PBML) in vitro was explicitly demonstrated by viability assays. Comet assay revealed a higher level of DNA damages in cancer cells when compared with normal ones. Apoptotic death in cancer cells was proved by measuring caspases activity. Thus, the developed Au@RD NPs, obtained by the plant-mediated green synthesis, are attractive hybrid materials for the medical applications combining two active components - metal nanoparticles platform and plant-derived metabolites.

Paroxetine and Low-dose Risperidone Induce Serotonin 5-HT1A and Dopamine D2 Receptor Heteromerization in the Mouse Prefrontal Cortex.

Recently, it has been shown that serotonin 5-HT1A receptor interacts with dopamine D2 receptor in vitro. However, the existence of 5-HT1A-D2 heteromers in native tissue remains unexplored. In the present study, we investigated 5-HT1A-D2 receptor heteromerization in mice treated acutely or chronically with paroxetine (10 mg/kg) or risperidone (0.05 mg/kg). Receptor heteromerization was visualized and quantified in the mouse brain by in situ proximity ligation assay (PLA). Additionally, we aimed to determine the cellular localization of 5-HT1A-D2 receptor heteromers in mouse adult primary neuronal cells by immunofluorescent staining with markers for astrocytes (GFAP) and neurons (NeuN and MAP2). The results from the current study demonstrated that 5-HT1A and D2 receptor co-localization and heteromerization occurred in the mouse prefrontal cortex. Counterstaining after PLA confirmed neuronal (pyramidal and GABAergic) as well as astrocytal localization of 5-HT1A-D2 receptor heteromers. Chronic administration of paroxetine or risperidone increased the level of 5-HT1A-D2 receptor heteromers in the prefrontal cortex. These changes were not accompanied by any changes in the expression of mRNAs (measured by in situ hybridization) or densities of 5-HT1A and D2 receptors (quantified by receptor autoradiography with [3H]8-OH-DPAT and [3H]domperidone, respectively), what all indicated that paroxetine and risperidone facilitated 5-HT1A-D2 heteromer formation independently of the receptor expression. In vitro homogenous time-resolved FRET (HTRF) study confirmed the ability of tested drugs to influence the human 5-HT1A-D2 heteromer formation. The obtained data indicate that the increase in 5-HT1A-D2 receptor heteromerization is a common molecular characteristic of paroxetine and low-dose risperidone treatment.

The interaction of clozapine loaded nanocapsules with the hCMEC/D3 cells - In vitro model of blood brain barrier.

Despite progress in the development of novel pharmacological compounds, their efficacy in the treatment of neuropathologies is not satisfactory. One strategy to achieve safe and efficient brain targeting therapy is to design nanocarriers capable of transporting antipsychotic drugs through the BBB (without affecting the normal functions of the barrier) in a defined part of the brain. Here we investigate the interaction of clozapine-loaded polymeric Nano capsules (CLO-NCs) with hCMEC/D3 (human cerebral microvascular endothelial cells, D3 clone) cells that constitutes an in vitro model of the blood brain barrier (BBB). CLO-NCs (average size of 100nm) were constructed by the technique of sequential adsorption of polyelectrolytes (LbL), using biocompatible polyanion PGA (Poly-l-glutamic acid sodium salt) and polycation PLL (poly L-lysine) on clozapine-loaded nano-emulsion cores. Pegylated external layers were prepared using PGA-g(39)-PEG (PGA grafted by PEG poly(ethylene glycol)). The influence of the physicochemical properties of the CLO-NCs (charge, size, surface modification) on cell viability was determined. Advanced studies of CLO-NCs internalization (including endocytosis and transcytosis experiments) using confocal microscopy, flow cytometry and fluorescence spectroscopy are presented. Our results indicate that among the studied NCs, the pegylated clozapine-loaded NCs were the most protected from their uptake by macrophages, and they were the least toxic to hCMEC/D3 cells. They were also the most efficient in the transcytosis experiment, which serves as an indicator of their ability to cross a model BBB.

Genetic variants of dopamine D2 receptor impact heterodimerization with dopamine D1 receptor.

BACKGROUND: The human dopamine D2 receptor gene has three polymorphic variants that alter its amino acid sequence: alanine substitution by valine in position 96 (V96A), proline substitution by serine in position 310 (P310S) and serine substitution by cysteine in position 311 (S311C). Their functional role has never been the object of extensive studies, even though there is some evidence that their occurrence correlates with schizophrenia. METHODS: The HEK293 cell line was transfected with dopamine D1 and D2 receptors (or genetic variants of the D2 receptor), coupled to fluorescent proteins which allowed us to measure the extent of dimerization of these receptors, using a highly advanced biophysical approach (FLIM-FRET). Additionally, Fluoro-4 AM was used to examine changes in the level of calcium release after ligand stimulation of cells expressing different combinations of dopamine receptors. RESULTS: Using FLIM-FRET experiments we have shown that in HEK 293 expressing dopamine receptors, polymorphic mutations in the D2 receptor play a role in dimmer formation with the dopamine D1 receptor. The association level of dopamine receptors is affected by ligand administration, with variable effects depending on polymorphic variant of the D2 dopamine receptor. We have found that the level of heteromer formation is reflected by calcium ion release after ligand stimulation and have observed variations of this effect dependent on the polymorphic variant and the ligand. CONCLUSION: The data presented in this paper support the hypothesis on the role of calcium signaling regulated by the D1-D2 heteromer which may be of relevance for schizophrenia etiology.

Dopamine D2 and serotonin 5-HT1A receptor interaction in the context of the effects of antipsychotics - in vitro studies.

The serotonin 5-HT1A receptor (5-HT1 A R) and dopamine D2 receptor (D2 R) have been implicated as important sites of action in antipsychotics. Several lines of evidence indicate the key role of G protein-coupled receptors (GPCRs) heteromers in pathophysiology of schizophrenia and highlight these complexes as novel drug targets. Because heterodimers can form only on those cells co-expressing constituent receptors, they present a target of high pharmacological specificity in the context of biochemical effects induced by antipsychotic drugs. In studies conducted in the HEK 293 cell line, we demonstrated that 5-HT1 A R and D2 R are able to form constitutive heterodimers, and antipsychotic drugs (clozapine, olanzapine, aripiprazole, and lurasidone) enhanced this process, with clozapine being most effective. Various functional tests (cAMP and IP1 as well as ERK activation) indicated that the drugs had different effects on signal transduction by the heteromer. Interestingly, co-incubation of heterodimer-expressing HEK 293 cells with clozapine and the 5-HT1 A R agonist 8-OH DPAT potentiated post-synaptic effects, especially with respect to ERK activation. Our results indicate that the D2 -5-HT1A complex possesses biochemical, pharmacological, and functional properties distinct from those of mono- and homomers. This result has implications for the development of improved pharmacotherapy for schizophrenia or other disorders (activating the heteromer might be cognitive enhancing, since it is expressed in frontal cortex) through the specific targeting of heterodimers. We reported the constitutive formation of D2 -5-HT1A heteromers, which possess biochemical, pharmacological, and functional properties distinct from those of mono- and homomers, as revealed by antipsychotics action. We also showed that these two receptors are co-expressed in mouse cortical neurons; therefore their potential to heterodimerize may comprise an essential target for the development of novel strategies for schizophrenia treatment.

Encapsulation of clozapine in polymeric nanocapsules and its biological effects.

Clozapine is an effective atypical antipsychotic drug that unfortunately exhibits poor oral bioavailability. Moreover, the clinical use of the compound is limited because of its numerous unfavorable and unsafe side effects. Therefore, the aim of the present study was the development of a new nanocarrier for a more effective clozapine delivery. Here, clozapine was encapsulated into polymeric nanocapsules (NCs). Polyelectrolyte multilayer shells were constructed by the technique of sequential adsorption of polyelectrolytes (LbL) using biocompatible polyanion PGA (Poly-L-glutamic acid, sodium salt) and polycation PLL (poly-L-lysine) on clozapine-loaded nanoemulsion cores. Pegylated external layers were prepared using PGA-g-PEG (PGA grafted by PEG (polyethylene glycol)). Clozapine was successfully loaded into the PLL-PGA nanocarriers (CLO-NCs) with an average size of 100 nm. In vitro analysis of the interactions of the CLO-NCs with the cells of the mononuclear phagocytic system (MPS) was conducted. Cell biocompatibility, phagocytosis potential, and cellular uptake were studied. Additionally, the biodistribution and behavioral effects of the encapsulated clozapine were also studied. The results indicate that surface modified (by PEG grafting) polymeric PLL-PGA CLO-NCs are very promising nanovehicles for improving clozapine delivery.

Proteomic and bioinformatic analysis of a nuclear intrinsically disordered proteome.

UNLABELLED: Intrinsically disordered proteins (IDPs) are biologically active and crucial for cell function although they do not possess defined three-dimensional architecture. IDPs are especially prevalent in eukaryotic proteomes, and large-scale experiments have shown that many IDPs are nuclear proteins. Bioinformatic analyses have also demonstrated that the vast majority of transcription factors contain extended regions of intrinsic disorder. In the current study, we isolated and functionally analyzed IDPs expressed in the nuclei of HEK293 human cells. According to the results of MS analysis followed by subsequent analysis with the bioinformatic tools IUPred and RAPID (regression-based accurate predictor of intrinsic disorder), a heat-treatment method was able to enrich the nuclear lysate in IDPs. For approximately 85% of the proteins obtained, IUPred predicted a sequence of 30 or more consecutive disordered residues (DRs), and for approximately 83% of the proteins RAPID reported a content of at least 25% DRs (compared to ~66% and 49%, respectively, for the nuclear lysate). Gene Ontology analysis in terms of molecular function revealed that the obtained fraction was generally enriched in proteins involved in the process of transcription and especially in transcription factors. We also showed experimentally that IDPs are overrepresented in the cell nucleus. SIGNIFICANCE: Intrinsically disordered proteins (IDPs) are crucial cellular molecules and are especially numerous in eukaryotes. In particular, IDPs act as signaling and regulatory proteins, and impairment in their functioning may lead to serious diseases. Large-scale bioinformatic studies of IDPs have provided essential knowledge about this group of proteins. However, experimental data reflect the actual situation in living cells. Our study is the first large-scale proteomic analysis of nuclear IDPs. We showed experimentally that IDPs are overrepresented in the nucleus in comparison to the whole cell. Analysis of molecular function indicated that the nuclear intrinsically disordered proteome (IDP-ome) is enriched in proteins involved in transcription regulation and especially in transcription factors. The IDP isolation method from human cell nuclei presented in this article could be further applied in differential proteomic studies.

Polyelectrolyte-coated nanocapsules containing undecylenic acid: Synthesis, biocompatibility and neuroprotective properties.

The main objectives of the present study were to investigate the biocompatibility of polyelectrolyte-coated nanocapsules and to evaluate the neuroprotective action of the nanoencapsulated water-insoluble neuroprotective drug-undecylenic acid (UDA), in vitro. Core-shell nanocapsules were synthesized using nanoemulsification and the layer-by-layer (LbL) technique (by saturation method). The average size of synthesized nanocapsules was around 80 nm and the concentration was 2.5 × 10(10) particles/ml. Their zeta potential values ranged from less than -30 mV for the ones with external polyanion layers through -4 mV for the PEG-ylated layers to more than 30 mV for the polycation layers. Biocompatibility of synthesized nanocarriers was evaluated in the SH-SY5Y human neuroblastoma cell line using cell viability/toxicity assays (MTT reduction, LDH release). The results obtained showed that synthesized nanocapsules coated with PLL and PGA (also PEG-ylated) were non-toxic to SH-SY5Y cells, therefore, they were used as nanocarriers for UDA. Moreover, studies with ROD/FITC-labeled polyelectrolytes demonstrated approximately 20% cellular uptake of synthetized nanocapsules. Further studies showed that nanoencapsulated form of UDA was biocompatible and protected SH-SY5Y cells against the staurosporine-induced damage in lower concentrations than those of the same drug added directly to the culture medium. These data suggest that designed nanocapsules might serve as novel, promising delivery systems for neuroprotective agents.

Biocompatible Polymeric Nanoparticles as Promising Candidates for Drug Delivery.

The use of polymeric nanoparticles (NPs) in pharmacology provides many benefits because this approach can increase the efficacy and selectivity of active compounds. However, development of new nanocarriers requires better understanding of the interactions between NPs and the immune system, allowing for the optimization of NP properties for effective drug delivery. Therefore, in the present study, we focused on the investigation of the interactions between biocompatible polymeric NPs and a murine macrophage cell line (RAW 264.7) and a human monocytic leukemia cell line (THP-1). NPs based on a liquid core with polyelectrolyte shells were prepared by sequential adsorption of polyelectrolytes (LbL) using AOT (docusate sodium salt) as the emulsifier and the biocompatible polyelectrolytes polyanion PGA (poly-l-glutamic acid sodium salt) and polycation PLL (poly l-lysine). The average size of the obtained NPs was 80 nm. Pegylated external layers were prepared using PGA-g-PEG (PGA grafted by PEG poly(ethylene glycol)). The influence of the physicochemical properties of the NPs (charge, size, surface modification) on viability, phagocytosis potential, and endocytosis was studied. Internalization of NPs was determined by flow cytometry and confocal microscopy. Moreover, we evaluated whether addition of PEG chains downregulates particle uptake by phagocytic cells. The presented results confirm that the obtained PEG-grafted NPs are promising candidates for drug delivery.

In vitro interaction of polyelectrolyte nanocapsules with model cells.

The nanocapsules based on a liquid core with polyelectrolyte shells prepared by the technique of sequential adsorption of polyelectrolytes (LbL) were investigated to verify capsules bioacceptance. Using AOT (docusate sodium salt) as emulsifier, we obtained liquid cores, stabilized by the interfacial complex AOT/PLL (poly-l-lysine hydrobromide). These liquid cores were encapsulated by sequential adsorption of polyelectrolytes using biocompatible polyanion PGA (poly-l-glutamic acid sodium salt) and biocompatible polycation PLL. The average size of the formed capsules was 60-80 nm. The influence of a number of polyelectrolytes layer in the shell (thickness of polyelectrolytes shell), surface charge, and capsule doses on cell viability was studied in a cellular coculture assay. In order to improve nanocapsules biocompatibility, the PEG-ylated external layers were prepared using PGA-g-PEG (PGA grafted by PEG poly(ethylene glycol)). For the most toxic nanocapsules (with only one polycation layer) about 90% of cells could survive when the concentration of nanocapsules was below 0.2 × 10(6) per one cell. That suggests that they use as a delivery vehicles is quite safe for living cells. Analysis of internalization of AOT(PLL/PGA)4-g-PEG in HEK 293 cells indicates that tested nanocapsules can easily penetrate cells membrane.

A biophysical approach for the study of dopamine receptor oligomerization.

The ability of certain neurotransmitter receptors to form oligomers provides an additional level of fine-tuning of intracellular signaling. Among the techniques allowing study of receptor oligomerization as well as influence of specific ligands on these processes, a biophysical approach with the use of fluorescently tagged receptors is the most sensitive. Measurement of the fluorescence resonance energy transfer (FRET) phenomenon between two fluorescently tagged receptors is considered a very useful and measurable tool to study the physical interactions between receptors either in a single cell or in a population of living cells. Here we describe the use of FRET measurement specifically to monitor protein oligomer formation between dopamine D(1)R and D(2)R, but the same methodology can be used to study other receptor proteins as well as their mutants.

Antidepressant drugs promote the heterodimerization of the dopamine D2 and somatostatin Sst5 receptors--fluorescence in vitro studies.

BACKGROUND: The interaction between the dopaminergic and somatostatinergic systems and their role in mood regulation have been well-documented. Therefore, we decided to investigate the effect of antidepressant drugs on the heterodimerization of the dopamine D2 and somatostatin Sst5 receptors. METHODS: The human receptor proteins were tagged with fluorescent proteins, expressed in the HEK 293 cells and incubated with antidepressant drugs: desipramine and citalopram. To determine the FRET efficiency, the fluorescence resonance energy transfer (FRET) and photobleaching confocal microscopy techniques were used. RESULTS: We found that the efficiency of FRET is markedly increased in cells coexpressing the somatostatin Sst5 and dopamine D2 receptors after 48 h of incubation with desipramine and citalopram. CONCLUSIONS: In the present study we provide physical evidence, based on FRET analysis, that antidepressants increase Sst5 and D2 receptors heterodimerization. The effect is specific because desipramine in the incubation medium uncouples other pairs of receptors, such as the dopamine D1-D2 receptors.

Effect of clozapine on the dimerization of serotonin 5-HT(2A) receptor and its genetic variant 5-HT(2A)H425Y with dopamine D(2) receptor.

Oligomerization of G protein-coupled receptors has become a very important issue in a present molecular pharmacology. In the present study the level of the serotonin 5-HT(2A) and the dopamine D(2) receptor interactions have been studied since it may have a key significance in understanding the mechanism of action of drugs used to treat schizophrenia. With the use of fluorescence resonance energy transfer we demonstrated that the serotonin 5-HT(2A) receptors form homo- and hetero-dimers with the dopamine D(2) receptors and polymorphism H452Y within the 5-HT(2A) receptor, implicated as a cause of altered response to antipsychotic treatment, disturbs both processes. Clozapine affected the hetero-dimers (5-HT(2A)H452Y/D(2)) complexes and increased the otherwise weakened dimerization to the value observed for combination of both wild type receptors, and had no effect on the serotonin receptor homo-dimers (5-HT(2A)H452Y/5-HT(2A)), while haloperidol has restored the weakened interaction within homo-complexes and did not effect the hetero-complexes. The obtained data suggest that H452Y polymorphism has an influence not only on the level of constitutive oligomerization of investigated receptors but also it changes their pharmacological properties within both homo- and hetero-complexes.

Hetero-dimerization of serotonin 5-HT(2A) and dopamine D(2) receptors.

In the present study, detailed information is presented on the hetero-dimerization of the serotonin 5-HT(2A) receptor and the dopamine D(2) receptor. Biophysical approaches (fluorescence spectroscopy as well as fluorescence lifetime microscopy) were used to determine the degree of fluorescence resonance energy transfer (FRET) between cyan and yellow fluorescent protein labeled receptor variants co-expressed in human embryonic kidney 293 cells (HEK293). Recorded data demonstrate the existence of energy transfer between the wild-type forms of 5-HT(2A)R and D(2)R, pointing toward the formation of hetero-5-HT(2A)R/D(2)R dimers and homo-5-HT(2A)R/5-HT(2A)R dimers. Moreover, the present study investigates the role of specific motifs (one containing adjacent arginine residues (217RRRRKR222) in the third intracellular loop (ic3) of D(2)R, and the other consisting of acidic glutamate residues (454EE455) in the C-tail of (5-HT(2A)R) in the formation of noncovalent complexes between these receptors. Our results suggest that these regions of 5-HT(2A)R and D(2)R may be involved in the interaction between these two proteins. On the other hand, the above-mentioned motifs do not play an important role in the homo-dimerization of these receptors. Furthermore, we estimated the influence of specific receptor ligands on the dimerization processes. Agonists (DOI and quinpirole) and antagonists (ketanserin and butaclamol) cause different effects on FRET efficiency depending on whether homo- or hetero-complexes are present. These data may have therapeutic implications, since (using the immunofluorescence double labeling protocols) the co-localization of these two receptors was demonstrated in the medial prefrontal cortex and pars reticulate of the substantia nigra of the rat brain.

Studies on the role of the receptor protein motifs possibly involved in electrostatic interactions on the dopamine D1 and D2 receptor oligomerization.

We investigated the influence of an epitope from the third intracellular loop (ic3) of the dopamine D(2) receptor, which contains adjacent arginine residues (217RRRRKR222), and an acidic epitope from the C-terminus of the dopamine D(1) receptor (404EE405) on the receptors' localization and their interaction. We studied receptor dimer formation using fluorescence resonance energy transfer. Receptor proteins were tagged with fluorescence proteins and expressed in HEK293 cells. The degree of D(1)-D(2) receptor heterodimerization strongly depended on the number of Arg residues replaced by Ala in the ic3 of D(2)R, which may suggest that the indicated region of ic3 in D(2)R might be involved in interactions between two dopamine receptors. In addition, the subcellular localization of these receptors in cells expressing both receptors D(1)-cyan fluorescent protein, D(2)-yellow fluorescent protein, and various mutants was examined by confocal microscopy. Genetic manipulations of the Arg-rich epitope induced alterations in the localization of the resulting receptor proteins, leading to the conclusion that this epitope is responsible for the cellular localization of the receptor. The lack of energy transfer between the genetic variants of yellow fluorescent protein-tagged D(2)R and cyan fluorescent protein-tagged D(1)R may result from differing localization of these proteins in the cell rather than from the possible role of the D(2)R basic domain in the mechanism of D(1)-D(2) receptor heterodimerization. However, we find that the acidic epitope from the C-terminus of the dopamine D(1) receptor is engaged in the heterodimerization process.

Role of silent polymorphisms within the dopamine D1 receptor associated with schizophrenia on D1-D2 receptor hetero-dimerization.

Within the coding region of the dopamine D(1) receptor (D(1)R), two synonymous polymorphisms, D(1)R(G198A) and D(1)R(G1263), have been identified and postulated to correlate with the schizophrenia phenotype. Binding studies revealed that the density of these genetic variants was much lower than the density of wild type D(1)R in the human embryonic kidney (HEK) 293 cell line, used as a model system. From the data obtained using MFOLD software it is apparent that the G198A mutation has a greater impact on the secondary structure of the mRNA, which may affect its translation. However, the G1263A mutation is localized within the serine 421 codon of D(1)R, which is predicted to be a potential site of phosphorylation according to the PHOSIDA database. In order to determine whether the studied synonymous polymorphisms influence the process of dopamine D(1)-D(2) receptors heterodimerization, we employed fluorescence resonance energy transfer (FRET) technology. The dopamine D(2) receptor (D(2)R) was tagged with cyan fluorescence protein and the D(1)R and its genetic variants were tagged with yellow fluorescence protein. The degree of D(1)-D(2) receptor hetero-dimerization was significantly decreased when genetic variants of D(1)R were co-expressed with D(2)R. Since the D(1)R mutations affected the expression levels of the proteins in the cell membrane without affecting the cellular localization of the receptor proteins, we postulated that the D(1)R polymorphisms altered the translation rate and protein structure of the receptor. The altered hetero-dimerization that likely results from the lower expression of these genetic variants of D(1)R with D(2)R may be partially responsible for the association of both G198A and G1263A polymorphisms with the schizophrenia phenotype.

[Fluorescence studies of G-protein coupled receptors oligomerization]. / Fluorescencyjne metody badania oligomeryzacji receptorów zwiazanych z bialkami G.

Fluorescence methods based on the resonance energy transfer phenomenon are currently extensively used to study biological processes, particularly interactions of G-protein coupled receptors. The development of molecular engineering has made it feasible to produce fluorescently tagged fusion proteins, which constitute a model system for studies of receptors oligomerization. It is widely accepted, that physical interactions between receptors might be of great importance for development of potential therapeutic factors, which influence signal transduction across the membrane. Here, we report on a variety of fluorescence approaches commonly used to investigate receptors oligomerization. Comparative studies reveal that even a relatively simple method, such as steady-state fluorescence spectroscopy could be useful in verifying receptor interactions. However, more sophisticated techniques as confocal microscopy and fluorescence lifetime microscopy not only provide quantifiable data of high relevance but also allow observations of dynamic processes in complex biological systems.