Evaluating the applicability of mouse SINEs as an alternative normalization approach for RT-qPCR in brain tissue of the APP23 model for Alzheimer's disease.

BACKGROUND: The choice of appropriate reference genes (RGs) for use in reverse transcription quantitative polymerase chain reaction (RT-qPCR) has been thoroughly investigated, since the inclusion of unstable RGs might cause inaccurate gene expression results. NEW METHOD: Short interspersed nuclear elements (SINEs) such as B elements, might represent an alternative solution given the high occurrence of these repetitive elements in the rodent genome and transcriptome. We performed RT-qPCR to investigate the stability of nine commonly used RGs and two B elements, B1 and B2, across different age- and genotype-related experimental conditions in the hippocampus and cortex of the APP23 amyloidosis mouse model for Alzheimer's disease. Gene stability was assessed using geNorm, NormFinder and BestKeeper. Human amyloid precursor protein (APP) levels in transgenic versus wild-type animals were also determined to validate the use of B elements as an alternative normalization approach. RESULTS: Whereas B elements were stably expressed in the hippocampus, they were ranked as least stable in the cortex. The optimal normalization factor (NF) in hippocampus was a combination of Gapdh and Rpl13a, whereas in cortex, Actb and Tbp constituted the ideal NF. COMPARISON WITH EXISTING METHOD: When comparing B1 and B2 as NFs for APP with the optimal panel of RGs in hippocampus, we found that B1 and B2 performed similarly to the optimal NF, while these SINEs performed less well in cortex. CONCLUSIONS: Although B elements are suitable as an alternative normalization strategy in the hippocampus, they do not represent a universal normalization approach in the APP23 model.

The validation of Short Interspersed Nuclear Elements (SINEs) as a RT-qPCR normalization strategy in a rodent model for temporal lobe epilepsy.

BACKGROUND: In gene expression studies via RT-qPCR many conclusions are inferred by using reference genes. However, it is generally known that also reference genes could be differentially expressed between various tissue types, experimental conditions and animal models. An increasing amount of studies have been performed to validate the stability of reference genes. In this study, two rodent-specific Short Interspersed Nuclear Elements (SINEs), which are located throughout the transcriptome, were validated and assessed against nine reference genes in a model of Temporal Lobe Epilepsy (TLE). Two different brain regions (i.e. hippocampus and cortex) and two different disease stages (i.e. acute phase and chronic phase) of the systemic kainic acid rat model for TLE were analyzed by performing expression analyses with the geNorm and NormFinder algorithms. Finally, we performed a rank aggregation analysis and validated the reference genes and the rodent-specific SINEs (i.e. B elements) individually via Gfap gene expression. RESULTS: GeNorm ranked Hprt1, Pgk1 and Ywhaz as the most stable genes in the acute phase, while Gusb and B2m were ranked as the most unstable, being significantly upregulated. The two B elements were ranked as most stable for both brain regions in the chronic phase by geNorm. In contrast, NormFinder ranked the B1 element only once as second best in cortical tissue for the chronic phase. Interestingly, using only one of the two algorithms would have led to skewed conclusions. Finally, the rank aggregation method indicated the use of the B1 element as the best option to normalize target genes, independent of the disease progression and brain region. This result was supported by the expression profile of Gfap. CONCLUSION: In this study, we demonstrate the potential of implementing SINEs -notably the B1 element- as a stable normalization factor in a rodent model of TLE, independent of brain region or disease progression.

Synthesis toward Bivalent Ligands for the Dopamine D2 and Metabotropic Glutamate 5 Receptors.

In this study, we designed and synthesized heterobivalent ligands targeting heteromers consisting of the metabotropic glutamate 5 receptor (mGluR5) and the dopamine D2 receptor (D2R). Bivalent ligand 22a with a linker consisting of 20 atoms showed 4-fold increase in affinity for cells coexpressing D2R and mGluR5 compared to cells solely expressing D2R. Likewise, the affinity of 22a for mGluR5 increased 2-fold in the coexpressing cells. Additionally, 22a exhibited a 5-fold higher mGluR5 affinity than its monovalent precursor 21a in cells coexpressing D2R and mGluR5. These results indicate that 22a is able to bridge binding sites on both receptors constituting the heterodimer. Likewise, cAMP assays revealed that 22a had a 4-fold higher potency in stable D2R and mGluR5 coexpressing cell lines than 1. Furthermore, molecular modeling reveals that 22a is able to simultaneously bind both receptors by passing between the TM5-TM6 interface and establishing six protein-ligand H-bonds.

Design, Synthesis, and Biological Evaluation of Bivalent Ligands Targeting Dopamine D2 -Like Receptors and the µ-Opioid Receptor.

Currently, there is mounting evidence that intermolecular receptor-receptor interactions may result in altered receptor recognition, pharmacology and signaling. Heterobivalent ligands have been proven useful as molecular probes for confirming and targeting heteromeric receptors. This report describes the design and synthesis of novel heterobivalent ligands for dopamine D2 -like receptors (D2 -likeR) and the µ-opioid receptor (µOR) and their evaluation using ligand binding and functional assays. Interestingly, we identified a potent bivalent ligand that contains a short 18-atom linker and combines good potency with high efficacy both in ß-arrestin 2 recruitment for µOR and MAPK-P for D4 R. Furthermore, this compound was characterized by a biphasic competition binding curve for the D4 R-µOR heterodimer, indicative of a bivalent binding mode. As this compound possibly bridges the D4 R-µOR heterodimer, it could be used as a pharmacological tool to further investigate the interactions of D4 R and µOR.

Molecular dissection of the human A3 adenosine receptor coupling with ß-arrestin2.

Besides classical G protein coupling, G protein-coupled receptors (GPCRs) are nowadays well known to show significant signalling via other adaptor proteins, such as ß-arrestin2 (ßarr2). The elucidation of the molecular mechanism of the GPCR-ßarr2 interaction is a prerequisite for the structure-activity based design of biased ligands, which introduces a new chapter in drug discovery. The general mechanism of the interaction is believed to rely on phosphorylation sites, exposed upon agonist binding. However, it is not known whether this mechanism is universal throughout the GPCR family or if GPCR-specific patterns are involved. In recent years, promising orally active agonists for the human A3 adenosine receptor (A3AR), a GPCR highly expressed in inflammatory and cancer cells, have been evaluated in clinical trials for the treatment of rheumatoid arthritis, psoriasis, and hepatocellular carcinoma. In this study, the effect of cytoplasmic modifications of the A3AR on ßarr2 recruitment was evaluated in transiently transfected HEK293T cells, using a live-cell split-reporter system (NanoBit®, Promega), based on the structural complementation of NanoLuc luciferase, allowing real-time ßarr2 monitoring. The A3AR-selective reference agonist 2-Cl-IB-MECA yielded a robust, concentration dependent (5 nM-1 µM) recruitment of ßarr2 (logEC50: -7.798 ±â€¯0.076). The role of putative phosphorylation sites, located in the C-terminal part and cytoplasmic loops, and the role of the 'DRY' motif was evaluated. It was shown that the A3AR C-terminus was dispensable for ßarr2 recruitment. This contrasts with studies in the past for the rat A3AR, which pointed at crucial C-terminal phosphorylation sites. When combining truncation of the A3AR with modification of the 'DRY' motif to 'AAY', the ßarr2 recruitment was drastically reduced. Recruitment could be partly rescued by back-mutation to 'NQY', or by extending the C-terminus again. In conclusion, other parts of the human A3AR, either cytosolic or exposed upon receptor activation, rather than the C-terminus alone, are responsible for ßarr2 recruitment in a complementary or synergistic way.

Regulation of G Protein-Coupled Receptors by Ubiquitination.

G protein-coupled receptors (GPCRs) comprise the largest family of membrane receptors that control many cellular processes and consequently often serve as drug targets. These receptors undergo a strict regulation by mechanisms such as internalization and desensitization, which are strongly influenced by posttranslational modifications. Ubiquitination is a posttranslational modification with a broad range of functions that is currently gaining increased appreciation as a regulator of GPCR activity. The role of ubiquitination in directing GPCRs for lysosomal degradation has already been well-established. Furthermore, this modification can also play a role in targeting membrane and endoplasmic reticulum-associated receptors to the proteasome. Most recently, ubiquitination was also shown to be involved in GPCR signaling. In this review, we present current knowledge on the molecular basis of GPCR regulation by ubiquitination, and highlight the importance of E3 ubiquitin ligases, deubiquitinating enzymes and ß-arrestins. Finally, we discuss classical and newly-discovered functions of ubiquitination in controlling GPCR activity.

Characterization of the interaction between the dopamine D4 receptor, KLHL12 and ß-arrestins.

Dopamine receptors are G protein-coupled receptors involved in regulation of cognition, learning, movement and endocrine signaling. The action of G protein-coupled receptors is highly regulated by multifunctional proteins, such as ß-arrestins which can control receptor desensitization, ubiquitination and signaling. Previously, we have reported that ß-arrestin 2 interacts with KLHL12, a BTB-Kelch protein which functions as an adaptor in a Cullin3-based E3 ligase complex and promotes ubiquitination of the dopamine D4 receptor. Here, we have investigated the molecular basis of the interaction between KLHL12 and ß-arrestins and questioned its functional relevance. Our data demonstrate that ß-arrestin 1 and ß-arrestin 2 bind constitutively to the most common dopamine D4 receptor polymorphic variants and to KLHL12 and that all three proteins can interact within a single macromolecular complex. Surprisingly, stimulation of the receptor has no influence on the association between these proteins or their cellular distribution. We found that Cullin3 also interacts with both ß-arrestins but has no influence on their ubiquitination. Knockout of one of the two ß-arrestins hampers neither interaction between the dopamine D4 receptor and KLHL12, nor ubiquitination of the receptor. Finally, our results indicate that p44/42 MAPK phosphorylation, the signaling pathway which is often regulated by ß-arrestins is not influenced by KLHL12, but seems to be exclusively mediated by Gαi protein upon dopamine D4 receptor stimulation.

Dopamine D4 receptor ubiquitination.

Ubiquitination is a post-translational modification that targets proteins for degradation but can also regulate other cellular processes such as endocytosis, trafficking and DNA repair. We investigate ubiquitination of the dopamine D4receptor (D4R) which belongs to the superfamily of G protein-coupled receptors (GPCR). Several polymorphic variants of the D4R exist, which differ in the number of 16-amino acid repeats in the third intracellular loop (IC3) of the receptor. The functional role of this polymorphic region is not known but persons with the seven-repeat allele show a predisposition to develop attention deficit hyperactivity disorder (ADHD). We identified a protein, KLHL12, which specifically interacts with this polymorphic region and enhances ubiquitination of the D4R. We have tested the influence of KLHL12 on the ubiquitination of the most common D4R polymorphic variants and found that KLHL12 strongly promotes ubiquitination of the two- and four-repeat variant but has hardly any effect on ubiquitination of the seven-repeat D4R. This suggests that differential ubiquitination of the D4R may have functional implications. Moreover, we were able to demonstrate that KLHL12-mediated D4R ubiquitination does not lead to receptor degradation. Next, we aimed to identify specific residues in the sequence of D4R which undergo ubiquitination and observed that the lysine-less receptor mutant is still ubiquitinated. Subsequently, we have tested the hypothesis whether KLHL12 could promote ubiquitination on non-lysine residues of the D4R. The importance of the cysteine and serine/threonine residues in the ubiquitination process of the receptor was examined and the obtained results confirmed that D4R can be ubiquitinated on non-lysine residues. In this review we summarize our data on D4R ubiquitination and put this in the light of other GPCR ubiquitination studies.

Characterization of Antibodies to Identify Cellular Expression of Dopamine Receptor 4.

The dopamine receptor D4 (DRD4) plays an important role in vision. In order to study the DRD4 expression in vivo, it is important to have antibodies that are specific for DRD4 for both immunoblot and immunohistochemical (IHC) applications. In this study, six antibodies raised against DRD4 peptides were tested in vitro, using transfected mammalian cells, and in vivo, using mouse retinas. Three Santa Cruz (SC) antibodies, D-16, N-20, and R-20, were successful in IHC of transfected DRD4; however, N-20 was the only one effective on immunoblot analysis in DRD4 transfected cells and IHC of mouse retinal sections, while R-20, 2B9, and Antibody Verify AAS63631C were non-specific or below detection.

KLHL12 Promotes Non-Lysine Ubiquitination of the Dopamine Receptors D4.2 and D4.4, but Not of the ADHD-Associated D4.7 Variant.

DOPAMINE D4 RECEPTOR POLYMORPHISM: The dopamine D4 receptor has an important polymorphism in its third intracellular loop that is intensively studied and has been associated with several abnormal conditions, among others, attention deficit hyperactivity disorder. KLHL12 PROMOTES UBIQUITINATION OF THE DOPAMINE D4 RECEPTOR ON NON-LYSINE RESIDUES: In previous studies we have shown that KLHL12, a BTB-Kelch protein, specifically interacts with the polymorphic repeats of the dopamine D4 receptor and enhances its ubiquitination, which, however, has no influence on receptor degradation. In this study we provide evidence that KLHL12 promotes ubiquitination of the dopamine D4 receptor on non-lysine residues. By using lysine-deficient receptor mutants and chemical approaches we concluded that ubiquitination on cysteine, serine and/or threonine is possible. DIFFERENTIAL UBIQUITINATION OF THE DOPAMINE D4 RECEPTOR POLYMORPHIC VARIANTS: Additionally, we show that the dopamine D4.7 receptor variant, which is associated with a predisposition to develop attention deficient hyperactivity disorder, is differentially ubiquitinated compared to the other common receptor variants D4.2 and D4.4. Together, our study suggests that GPCR ubiquitination is a complex and variable process.

Dopamine receptor D4 internalization requires a beta-arrestin and a visual arrestin.

PURPOSE: The G-protein coupled receptor (GPCR) Dopamine Receptor D4 (DRD4) plays an essential role in cAMP regulation and gap junctional coupling in the photoreceptors, where DRD4 expression is under circadian control. Previous in vitro transfection studies of human DRD4 desensitization have reported that DRD4 is not internalized upon dopamine stimulation when beta-arrestin is co-transfected with DRD4. We hypothesized that the visual arrestins, ARR1 and ARR4, play a modulatory role in DRD4 desensitization in the photoreceptors. METHODS: To test this hypothesis, immunohistochemistry analysis of mouse retinas was used to determine the cellular localization of beta-arrestins and DRD4 in photoreceptors. In vitro studies were performed in HEK293T cells transiently transfected with human DRD4 and arrestins. First, co-immunoprecipitation experiments were executed to test protein-protein interactions and to investigate the effect of dopamine stimulation. Second, immunohistochemistry analysis was implemented to study DRD4 internalization and translocation of ARR4. RESULTS: Immunohistochemistry studies of mouse retinas confirmed the expression of beta-arrestin 2, ARR1 and ARR4, as well as DRD4 in mouse cone photoreceptor inner segments. Co-immunoprecipitation experiments revealed a dopamine-dependent protein-protein interaction between human DRD4 and ARR4. In vitro internalization experiments showed that no detectable internalization of DRD4 was observed with any single arrestin co-transfected. However, a dopamine-dependent internalization of DRD4 was observed with three out of six sets of two arrestins co-transfected with DRD4. Each of these pairs of arrestins contained one visual arrestin and one beta-arrestin, and no internalization was observed with either two visual arrestins or two beta-arrestins. Additional time-course experiments revealed that in vitro, ARR4 translocates to co-localize with DRD4 at the plasma membrane in response to 30min of dopamine stimulation. CONCLUSIONS: The results have functional implications and we hypothesize that the desensitization and internalization of DRD4 in photoreceptors are synergistically mediated by both visual and beta-arrestins. These results are additionally unique because they demonstrate for the first time that at least one G-protein coupled receptor, DRD4, requires two arrestins for desensitization and internalization, and opens up the possibility that other G-protein coupled receptors may require more than one arrestin for desensitization and/or internalization.

Downregulation of 5-HT7 Serotonin Receptors by the Atypical Antipsychotics Clozapine and Olanzapine. Role of Motifs in the C-Terminal Domain and Interaction with GASP-1.

The human 5-HT7 serotonin receptor, a G-protein-coupled receptor (GPCR), activates adenylyl cyclase constitutively and upon agonist activation. Biased ligands differentially activate 5-HT7 serotonin receptor desensitization, internalization and degradation in addition to G protein activation. We have previously found that the atypical antipsychotics clozapine and olanzapine inhibited G protein activation and, surprisingly, induced both internalization and lysosomal degradation of 5-HT7 receptors. Here, we aimed to determine the mechanism of clozapine- and olanzapine-mediated degradation of 5-HT7 receptors. In the C-terminus of the 5-HT7 receptor, we identified two YXXΦ motifs, LR residues, and a palmitoylated cysteine anchor as potential sites involved in receptor trafficking to lysosomes followed by receptor degradation. Mutating either of these sites inhibited clozapine- and olanzapine-mediated degradation of 5-HT7 receptors and also interfered with G protein activation. In addition, we tested whether receptor degradation was mediated by the GPCR-associated sorting protein-1 (GASP-1). We show that GASP-1 binds the 5-HT7 receptor and regulates the clozapine-mediated degradation. Mutations of the identified motifs and residues, located in or close to Helix-VIII of the 5-HT7 receptor, modified antipsychotic-stimulated binding of proteins (such as GASP-1), possibly by altering the flexibility of Helix-VIII, and also interfered with G protein activation. Taken together, our data demonstrate that binding of clozapine or olanzapine to the 5-HT7 receptor leads to antagonist-mediated lysosomal degradation by exposing key residues in the C-terminal tail that interact with GASP-1.

Role of dimerization in dopamine D(4) receptor biogenesis.

Dopamine receptors are G protein-coupled receptors critically involved in locomotion, reward, and cognitive processes. Export of dopamine receptors to the plasma membrane is thought to follow the default secretory pathway, whereby proteins travel from the endoplasmatic reticulum (ER), through the Golgi apparatus, to arrive at the cell surface. Several observations indicate that trafficking from the ER to the plasma membrane is tightly regulated, and that correct folding in the ER acts as a bottle neck to the maturation of the dopamine D4 receptors. The dopamine D(4) receptor is an interesting receptor since it has a polymorphic region in its third intracellular loop, resulting in receptor isoforms of varying length and amino acid composition. Correct folding is enhanced by: (1) interaction with specific proteins, such as ER resident chaperones, (2) interaction with pharmacological chaperones, for example, ligands that are membrane permeable and can bind to the receptor in the ER, and (3) receptor dimerization; the assembly of multisubunit proteins into a quaternary structure is started in the ER before cell surface delivery, which helps in correct folding and subsequent expression. These interactions help the process of GPCR folding, but more importantly they ensure that only properly folded proteins proceed from the ER to the trans-Golgi network. In this review we will mainly focus on the role of receptor dimerization in dopamine D(4) receptor maturation.

The G protein-coupled receptor heterodimer network (GPCR-HetNet) and its hub components.

G protein-coupled receptors (GPCRs) oligomerization has emerged as a vital characteristic of receptor structure. Substantial experimental evidence supports the existence of GPCR-GPCR interactions in a coordinated and cooperative manner. However, despite the current development of experimental techniques for large-scale detection of GPCR heteromers, in order to understand their connectivity it is necessary to develop novel tools to study the global heteroreceptor networks. To provide insight into the overall topology of the GPCR heteromers and identify key players, a collective interaction network was constructed. Experimental interaction data for each of the individual human GPCR protomers was obtained manually from the STRING and SCOPUS databases. The interaction data were used to build and analyze the network using Cytoscape software. The network was treated as undirected throughout the study. It is comprised of 156 nodes, 260 edges and has a scale-free topology. Connectivity analysis reveals a significant dominance of intrafamily versus interfamily connections. Most of the receptors within the network are linked to each other by a small number of edges. DRD2, OPRM, ADRB2, AA2AR, AA1R, OPRK, OPRD and GHSR are identified as hubs. In a network representation 10 modules/clusters also appear as a highly interconnected group of nodes. Information on this GPCR network can improve our understanding of molecular integration. GPCR-HetNet has been implemented in Java and is freely available at http://www.iiia.csic.es/~ismel/GPCR-Nets/index.html.

How the venom from the ectoparasitoid Wasp nasonia vitripennis exhibits anti-inflammatory properties on mammalian cell lines.

With more than 150,000 species, parasitoids are a large group of hymenopteran insects that inject venom into and then lay their eggs in or on other insects, eventually killing the hosts. Their venoms have evolved into different mechanisms for manipulating host immunity, physiology and behavior in such a way that enhance development of the parasitoid young. The venom from the ectoparasitoid Nasonia vitripennis inhibits the immune system in its host organism in order to protect their offspring from elimination. Since the major innate immune pathways in insects, the Toll and Imd pathways, are homologous to the NF-κB pathway in mammals, we were interested in whether a similar immune suppression seen in insects could be elicited in a mammalian cell system. A well characterized NF-κB reporter gene assay in fibrosarcoma cells showed a dose-dependent inhibition of NF-κB signaling caused by the venom. In line with this NF-κB inhibitory action, N. vitripennis venom dampened the expression of IL-6, a prototypical proinflammatory cytokine, from LPS-treated macrophages. The venom also inhibited the expression of two NF-κB target genes, IκBα and A20, that act in a negative feedback loop to prevent excessive NF-κB activity. Surprisingly, we did not detect any effect of the venom on the early events in the canonical NF-κB activation pathway, leading to NF-κB nuclear translocation, which was unaltered in venom-treated cells. The MAP kinases ERK, p38 and JNK are other crucial regulators of immune responses. We observed that venom treatment did not affect p38 and ERK activation, but induced a prolonged JNK activation. In summary, our data indicate that venom from N. vitripennis inhibits NF-κB signaling in mammalian cells. We identify venom-induced up regulation of the glucocorticoid receptor-regulated GILZ as a most likely molecular mediator for this inhibition.

Dopamine D4 receptor counteracts morphine-induced changes in µ opioid receptor signaling in the striosomes of the rat caudate putamen.

The mu opioid receptor (MOR) is critical in mediating morphine analgesia. However, prolonged exposure to morphine induces adaptive changes in this receptor leading to the development of tolerance and addiction. In the present work we have studied whether the continuous administration of morphine induces changes in MOR protein levels, its pharmacological profile, and MOR-mediated G-protein activation in the striosomal compartment of the rat CPu, by using immunohistochemistry and receptor and DAMGO-stimulated [35S]GTPγS autoradiography. MOR immunoreactivity, agonist binding density and its coupling to G proteins are up-regulated in the striosomes by continuous morphine treatment in the absence of changes in enkephalin and dynorphin mRNA levels. In addition, co-treatment of morphine with the dopamine D4 receptor (D4R) agonist PD168,077 fully counteracts these adaptive changes in MOR, in spite of the fact that continuous PD168,077 treatment increases the [3H]DAMGO Bmax values to the same degree as seen after continuous morphine treatment. Thus, in spite of the fact that both receptors can be coupled to Gi/0 protein, the present results give support for the existence of antagonistic functional D4R-MOR receptor-receptor interactions in the adaptive changes occurring in MOR of striosomes on continuous administration of morphine.

Detection of G protein-coupled receptor (GPCR) dimerization by coimmunoprecipitation.

With 356 members in the human genome, G protein-coupled receptors (GPCRs) constitute the largest family of proteins involved in signal transduction across biological membranes. GPCRs are integral membrane proteins featuring a conserved structural topology with seven transmembrane domains. By recognizing a large diversity of hormones and neurotransmitters, GPCRs mediate signal transduction pathways through their interactions with both extracellular small-molecule ligands and intracellular G proteins to initiate appropriate cellular signaling cascades. As there is a clear link between GPCRs and several disorders, GPCRs currently constitute the largest family of proteins targeted by marketed pharmaceuticals. Therefore, a detailed understanding of the biogenesis of these receptors and of GPCR-protein complex assembly can help to answer some important questions. In this chapter, we will discuss several methods to isolate GPCRs and to study, via coimmunoprecipitation, protein-protein interactions. Special attention will be given to GPCR dimerization, which often starts already in the endoplasmic reticulum and influences the maturation of the receptor. Next, we will also explain an elegant tool to study GPCR biogenesis based on the glycosylation pattern of the receptor of interest.

The serotonin 5-HT7 receptors: two decades of research.

Like most neurotransmitters, serotonin possesses a simple structure. However, the pharmacological consequences are more complex and diverse. Serotonin is involved in numerous functions in the human body including the control of appetite, sleep, memory and learning, temperature regulation, mood, behavior, cardiovascular function, muscle contraction, endocrine regulation, and depression. Low levels of serotonin may be associated with several disorders, namely increase in aggressive and angry behaviors, clinical depression, Parkinson's disease, obsessive-compulsive disorder, eating disorders, migraine, irritable bowel syndrome, tinnitus, and bipolar disease. These effects are mediated via different serotonin (5-HT) receptors. In this review, we will focus on the last discovered member of this serotonin receptor family, the 5-HT7 receptor. This receptor belongs to the G protein-coupled receptor superfamily and was cloned two decades ago. Later, different splice variants were described but no major functional differences have been described so far. All 5-HT7 receptor variants are coupled to Gαs proteins and stimulate cAMP formation. Recently, several interacting proteins have been reported, which can influence receptor signaling and trafficking.

On the g-protein-coupled receptor heteromers and their allosteric receptor-receptor interactions in the central nervous system: focus on their role in pain modulation.

The modulatory role of allosteric receptor-receptor interactions in the pain pathways of the Central Nervous System and the peripheral nociceptors has become of increasing interest. As integrators of nociceptive and antinociceptive wiring and volume transmission signals, with a major role for the opioid receptor heteromers, they likely have an important role in the pain circuits and may be involved in acupuncture. The delta opioid receptor (DOR) exerts an antagonistic allosteric influence on the mu opioid receptor (MOR) function in a MOR-DOR heteromer. This heteromer contributes to morphine-induced tolerance and dependence, since it becomes abundant and develops a reduced G-protein-coupling with reduced signaling mainly operating via ß -arrestin2 upon chronic morphine treatment. A DOR antagonist causes a return of the Gi/o binding and coupling to the heteromer and the biological actions of morphine. The gender- and ovarian steroid-dependent recruitment of spinal cord MOR/kappa opioid receptor (KOR) heterodimers enhances antinociceptive functions and if impaired could contribute to chronic pain states in women. MOR1D heterodimerizes with gastrin-releasing peptide receptor (GRPR) in the spinal cord, mediating morphine induced itch. Other mechanism for the antinociceptive actions of acupuncture along meridians may be that it enhances the cross-desensitization of the TRPA1 (chemical nociceptor)-TRPV1 (capsaicin receptor) heteromeric channel complexes within the nociceptor terminals located along these meridians. Selective ionotropic cannabinoids may also produce cross-desensitization of the TRPA1-TRPV1 heteromeric nociceptor channels by being negative allosteric modulators of these channels leading to antinociception and antihyperalgesia.

GPCR oligomerization: contribution to receptor biogenesis.

G protein-coupled receptor (GPCR) export to the plasma membrane is considered to follow the default secretory pathway. Several observations indicate that trafficking from the endoplasmic reticulum to the plasma membrane is strictly regulated and involves interactions with specific proteins, such as resident ER chaperones. These interactions help with GPCR folding, but more importantly, they ensure that only properly folded proteins proceed from the ER to the trans-golgi network. The assembly of several GPCRs into a quaternary structure is started in the ER, before cell surface delivery, and helps in the correct expression of the GPCRs. This review will mainly focus on the role of GPCR oligomerization in receptor biogenesis.