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.

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.

Synthesis toward Bivalent Ligands for the Dopamine D 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 D receptor (DR). Bivalent ligand 22a with a linker consisting of 20 atoms showed 4-fold increase in affinity for cells coexpressing DR and mGluR5 compared to cells solely expressing DR. Likewise, the affinity of 22a for mGluR5 increased 2-fold in the coexpressing cells.

Design, Synthesis, and Biological Evaluation of Bivalent Ligands Targeting Dopamine D -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 D -like receptors (D -likeR) and the μ-opioid receptor (μOR) and their evaluation using ligand binding and functional assays.

Molecular dissection of the human A adenosine receptor coupling with β-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 β-arrestin2 (βarr2). The elucidation of the molecular mechanism of the GPCR-βarr2 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.

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.

Dopamine D receptor stimulation prevents nigrostriatal dopamine pathway activation by morphine: relevance for drug addiction.

Morphine is one of the most effective drugs used for pain management, but it is also highly addictive. Morphine elicits acute and long-term adaptive changes at cellular and molecular level in the brain, which play a critical role in the development of tolerance, dependence and addiction. Previous studies indicated that the dopamine D receptor (D R) activation counteracts morphine-induced adaptive changes of the μ opioid receptor (MOR) signaling in the striosomes of the caudate putamen (CPu), as well as the induction of several Fos family transcription factors.

Characterization of the interaction between the dopamine D4 receptor, KLHL12 and β-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 β-arrestins which can control receptor desensitization, ubiquitination and signaling. Previously, we have reported that β-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.

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.

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.

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.

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.

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.

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 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.

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.

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.

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.

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.

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.

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.

Biochemical and pharmacological study of N-linked glycosylation of the human serotonin 5-HT₇a receptor.

The 5-hydroxytryptamine (5-HT)(7(a)) receptor is a G-protein-coupled receptor critically involved in human psychiatric and neurological disorders. In the present study, we evaluate the presence and the functional role of N-glycosylation of the human 5-HT(7) receptor. Western blot analysis of HEK293T cells transiently expressing the 5-HT(7(a)) receptor in the presence of tunicamycin gave rise to a band shift, indicating the existence of an N-glycosylated form of the 5-HT(7(a)) receptor.

RhoBTB3 interacts with the 5-HT7a receptor and inhibits its proteasomal degradation.

The 5-hydroxytryptamine (5-HT)7 receptor is the most recently identified serotonin receptor and is involved in a wide variety of central nervous system (CNS) functions, namely circadian rhythm, REM sleep, depression, thermoregulation, obsessive-compulsive disorder (OCD), anxiety, schizophrenia, epilepsy, nociception, migraine, sensation-seeking behavior, impulsivity, learning and memory. These numerous (patho)physiological processes of the CNS, in which the 5-HT7 receptor is involved, most likely reflect a diverse set of signaling pathways arising from this receptor. In order to reveal new interaction partners and possibly new signaling and/or trafficking pathways, we performed a yeast two-hybrid screening, using the C-terminal tail of the 5-HT7a receptor as bait and an adult-human brain cDNA library as prey.

Extracellular complexes of the hematopoietic human and mouse CSF-1 receptor are driven by common assembly principles.

The hematopoietic colony stimulating factor-1 receptor (CSF-1R or FMS) is essential for the cellular repertoire of the mammalian immune system. Here, we report a structural and mechanistic consensus for the assembly of human and mouse CSF-1:CSF-1R complexes. The EM structure of the complete extracellular assembly of the human CSF-1:CSF-1R complex reveals how receptor dimerization by CSF-1 invokes a ternary complex featuring extensive homotypic receptor contacts and striking structural plasticity at the extremities of the complex.

Role of the 5-HT7 receptor in the central nervous system: from current status to future perspectives.

Pharmacological and genetic tools targeting the 5-hydroxytryptamine (5-HT)7 receptor in preclinical animal models have implicated this receptor in diverse (patho)physiological processes of the central nervous system (CNS). Some data obtained with 5-HT7 receptor knockout mice, selective antagonists, and, to a lesser extent, agonists, however, are quite contradictory. In this review, we not only discuss in detail the role of the 5-HT7 receptor in the CNS but also propose some hypothetical models, which could explain the observed inconsistencies.