Actin cytoskeleton-dependent regulation of corticotropin-releasing factor receptor heteromers

Stress responses are highly nuanced and variable, but how this diversity is achieved by modulating receptor function is largely unknown. Corticotropin-releasing factor receptors (CRFRs), class B G protein–coupled receptors, are pivotal in mediating stress responses. Here we show that the two known CRFRs interact to form heteromeric complexes in HEK293 cells coexpressing both CRFRs and in vivo in mouse pancreas. Coimmunoprecipitation and mass spectrometry confirmed the presence of both CRF1R and CRF2βR, along with actin in these heteromeric complexes. Inhibition of actin filament polymerization prevented the transport of CRF2βR to the cell surface but had no effect on CRF1R. Transport of CRF1R when coexpressed with CRF2βR became actin dependent. Simultaneous stimulation of cells coexpressing CRF1R+CRF2βR with their respective high-affinity agonists, CRF+urocortin2, resulted in approximately twofold increases in peak Ca2+responses, whereas stimulation with urocortin1 that binds both receptors with 10-fold higher affinity did not. The ability of CRFRs to form heteromeric complexes in association with regulatory proteins is one mechanism to achieve diverse and nuanced function

KLHL12 promotes non-lysine ubiquitination of the dopamine receptors D-4.2 and D-4.4, but not of the ADHD-associated D-4.7 variant

Dopamine D-4 Receptor Polymorphism : The dopamine D-4 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 D-4 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 D-4 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 D-4 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 D-4 Receptor Polymorphic Variants : Additionally, we show that the dopamine D-4.7 receptor variant, which is associated with a predisposition to develop attention deficient hyperactivity disorder, is differentially ubiquitinated compared to the other common receptor variants D-4.2 and D-4.4. Together, our study suggests that GPCR ubiquitination is a complex and variable process

Heteroreceptor complexes and their allosteric receptor-receptor interactions in the central nervous system. Focus on examples from Dopamine D2 and Serotonin 5-HT1a receptors

GPCR interacting proteins (specially β- arrestin) and their receptor-protein interactions are also covered but their interactions with the allosteric receptor-receptor interactions in heteroreceptor complexes remain to be elucidated. The physiological and pathological relevance of the allosteric receptor-receptor interactions in heteroreceptor complexes is emphasized and novel strategies for treatment of mental and neurological disease are introduced based on this new biological principle of integration. This work gives further experimental evidences which strongly support the current view that allosteric receptor–receptor interactions in heteroreceptor complexes appear to represent a new principle in biology making possible integration of signals already at the level of the plasma membrane. These heteroreceptor complexes and their dynamics may be part of the molecular basis of learning and memory. The receptor protomers and their allosteric receptor-receptor interactions can be disturbed in neurological and mental disorders, and in diseases of peripheral tissues like the endocrine, cardiovascular and immune systems. The dopamine (DA) neuron system most relevant for schizophrenia and Parkinson s diseases is the meso-limbic-cortical DA system inter alia densely innervating subcortical limbic regions as well as the dorsal striatum. The field of dopamine D2Rs changed significantly with the discovery of many types of D2R heteroreceptor complexes in the ventral and dorsal striatum. The results indicate that the D2R is a hub receptor (www.gpcr-hetnet.com) which interacts not only with many other GPCRs including DA isoreceptors but also with ion-channel receptors, receptor tyrosine kinases, scaffolding proteins and DA transporters. Disturbances in several of these D2R heteroreceptor complexes may contribute to the development of schizophrenia and Parkinson s diseases through changes in the balance of diverse D2R homo- and heteroreceptor complexes mediating the DA signal, especially to the ventral striato-pallidal GABA pathway. In schizophrenia, this will have consequences for the control of this pathway of the glutamate drive to the prefrontal cortex via the mediodorsal thalamic nucleus which can contribute to psychotic processes. Allosteric receptor-receptor interactions in GPCR heteromers appeared to introduce an intermolecular allosteric mechanism contributing to the diversity and bias in the GPCR protomers. In A2A-D2R heteroreceptor complexes allosteric A2A-D2R receptor-receptor interaction brings about a biased modulation of the D2R protomer signalling (Chapter 1). A conformational state of the D2R is induced which moves away from Gi/o signaling and instead favours b-arrestin2 mediated signalling which may be the main mechanism for its atypical antipsychotic properties especially linked to the limbic A2AR-D2R heteroreceptor complexes. Furthermore, D2R-NTS1R heterocomplexes also exist in the ventral and dorsal striatum (Chapter 2) and likely also in midbrain DA nerve cells as D2R-NTS1R autoreceptor complexes where neurotensin produces antipsychotic and propsychotic actions, respectively. D2R protomer appeared to bias the specificity of the NT orthosteric binding site towards neuromedin N vs neurotensin in the heteroreceptor complex. There is a new awareness that Receptor tyrosine kinases (RTK) and transmitter activated GPCR possess the capacity for transactivation not only via GPCR induced release of neurotrophic factors, but also during signal initiation and propagation, using shared signaling pathways or using themselves as signaling platforms via direct allosteric receptor–receptor interactions. RTK are a family of transmembrane- spanning receptors that mediate the signaling from ligands such as growth factors, like the platelet-derived growth factor (PDGF), epidermal growth factor (EGF), the brain derived neurotrophic factor (BDNF), and the fibroblast growth factor (FGF). This hypothesis on direct GPCR-RTK receptor-receptor interactions in heteroreceptor complexes was introduced by Fuxe et al 1983. They also proposed the existence of 5- HT1A-FGFR1 heteroreceptor complexes having a role in depression. The hypothesis was introduced that the neurotrophic system FGF-2/FGFR1 may be a good candidate to mediate antidepressant induced improvement in 5-HT neuronal communication and neurotrophism with regeneration of connections lost during depression. RTK transactivation in response to antidepressant drug treatment was postulated to take place via a new allosteric receptor–receptor between distinct serotonin receptor subtypes and FGFR1 in heteroreceptor complexes. The discovery of brain FGFR1-5-HT1A heteroreceptor complexes and their enhancement of neuroplasticity offers an integration of the serotonin and the neurotrophic factor hypotheses of depression at the molecular level. These heteroreceptor complexes were found in the hippocampus and midbrain raphe 5-HT nerve cells, enriched in 5-HT1A autoreceptors. Based on the triplet puzzle theory several sets of triplet homologies were identified that may be part of the receptor interface. Combined FGF-2 and 5-HT1A agonist treatment increased the formation of these heterocomplexes and the facilitatory allosteric receptor-receptor interactions within them leading to an enhancement of FGFR1 signaling (Chapter 3). This integrative phenomenon is reciprocal and RTK signaling can be placed downstream of GPCRs. Formation of such heterocomplexes involving two major classes of membrane receptors can be involved in regulating all aspects of receptor protomer function including recognition, signaling, trafficking, desensitization, and downregulation (Chapter 3). These events were associated with development of rapid antidepressant effects. These heteroreceptor complexes are a novel target for antidepressant drugs. These examples, based on solid experimental evidences, serve to illustrate that allosteric receptor-receptor interactions in GPCR heteroreceptor complexes play a significant role in receptor diversity and bias of the participating GPCR protomers.G-protein coupled receptors (GPCR)-mediated signalling is a more complicated process than described previously since every GPCR and GPCR heteromer requires a set of G protein interacting proteins (GIP) which interacts with the receptor in an orchestrated spatio-temporal fashion. Therefore, there is a high interest in understanding the dynamics of the receptor-receptor and receptor-protein interactions in space and time, and specially, their integration in GPCR heterocomplexes of the Central Nervous System (CNS). Also, pathological protein-protein interactions in homocomplexes and heterocomplexes of Aβ, Tau, and α-Syn are at the heart of the development of conformational protein disorders. Along this work, experimental evidences are given to illustrate that GPCR interactions have relevance for neurological and mental diseases and are targets for drug development. GPCR containing heteromers and higher order heteromers through allosteric receptor- receptor interactions have become major integrative centers at the molecular level and their receptor protomers act as moonlighting proteins. They have become exciting new targets for neurotherapeutics in e.g. Parkinson’s disease, schizophrenia, drug addiction, anxiety and depression opening up a new field in neuropsychopharmacology. Along this work, the allosteric receptor-receptor interactions over the interfaces in A2AR-D2R, D2R-NTS1R, D2R-Sigma1R and 5-HT1A-FGFR1 heteroreceptor complexes will be explored and their biochemical, pharmacological and functional integrative implications in the CNS described. Methodologies for studies on receptor- receptor interactions are discussed including the use of FRET and BRET-based techniques in the analysis of G protein coupled receptor (GPCR) dimerization in living cells. In situ proximity ligation assay is performed to establish the existence of native heteroreceptor complexes in the CNS

The role of ECL2 in CGRP receptor activation: a combined modelling and experimental approach

The calcitonin gene-related peptide (CGRP) receptor is a complex of a calcitonin receptor-like receptor (CLR), which is a family B G-protein-coupled receptor (GPCR) and receptor activity modifying protein 1. The role of the second extracellular loop (ECL2) of CLR in binding CGRP and coupling to Gs was investigated using a combination of mutagenesis and modelling. An alanine scan of residues 271–294 of CLR showed that the ability of CGRP to produce cAMP was impaired by point mutations at 13 residues; most of these also impaired the response to adrenomedullin (AM). These data were used to select probable ECL2-modelled conformations that are involved in agonist binding, allowing the identification of the likely contacts between the peptide and receptor. The implications of the most likely structures for receptor activation are discussed.</jats:p

Evaluation of a novel virtual screening strategy using receptor decoy binding sites

Virtual screening is used in biomedical research to predict the binding affinity of a large set of small organic molecules to protein receptor targets. This report shows the development and evaluation of a novel yet straightforward attempt to improve this ranking in receptor-based molecular docking using a receptor-decoy strategy. This strategy includes defining a decoy binding site on the receptor and adjusting the ranking of the true binding-site virtual screen based on the decoy-site screen. The results show that by docking against a receptor-decoy site with Autodock Vina, improved Receiver Operator Characteristic Enrichment (ROCE) was achieved for 5 out of fifteen receptor targets investigated, when up to 15 % of a decoy site rank list was considered. No improved enrichment was seen for 7 targets, while for 3 targets the ROCE was reduced. The extent to which this strategy can effectively improve ligand prediction is dependent on the target receptor investigated

Vitamin D3 receptor is highly expressed in Hodgkin's lymphoma

Background: Hodkin s lymphoma is one of the most frequent lymphoma in western world. Despite an overall good prognosis some patients suffer relapsing tumors which are difficult to cure. Over a long period Vitamin D has been shown to be a potential treatment for cancer. Vitamin D acts via the vitamin D receptor, a nuclear receptor, acting as an inducible transcription factor. We aimed to investigate the expression of vitamin D receptor as potential therapeutic target structure in Hodgkin s lymphoma as well as in non Hodgkin s lymphoma. Methods: We used a panel of 193 formalin fixed tissues of lymphoma cases consisting of 55 cases of Hodgkin s lymphoma and 138 cases on several non Hodgkin s lymphoma entities. Results: Vitamin D receptor is strongly expressed in Hodgkin s lymphoma, regardless of the subentity with an overall positivity of 80% of all Hodgkin lymphoma cases. In contrast, only about 17% of the analyzed non Hodgkin s lymphoma of B-cell origin showed positivity for vitamin D receptor. Predominant nuclear localization of vitamin D receptor in Hodgkin s lymphoma suggests activated status of the vitamin D receptor. Conclusions: From this study, we conclude that vitamin D receptor plays a potentially important role in pathogenesis of Hodgkin s lymphoma but not in non Hodgkin s lymphoma. Further investigations of mutational status and functional studies may shed some light in functional relevance of vitamin D receptor signaling in Hodgkin s lymphoma

Expression of the insulin-like growth factor-II/mannose-6-phosphate receptor in multiple human tissues during fetal life and early infancy

The insulin like growth factor-II/mannose-6-phosphate (IGF-II/M6P) receptor has been detected in many cells and tissues. In the rat, there is a dramatic developmental regulation of IGF-II/M6P receptor expression, the receptor being high in fetal and neonatal tissues and declining thereafter. We have systematically studied the expression of the human IGF-II/M6P receptor protein in tissues from 10 human fetuses and infants (age 23 weeks gestation to 24 months postnatal). We have asked 1) whether there is differential expression among different organs, and 2) whether or not the human IGF-II/M6P receptor is developmentally regulated from 23 weeks gestation to 24 months postnatal. Protein was extracted from human tissues using a buffer containing 2% sodium dodecyl sulfate and 2% Triton X-100. Aliquots of the protein extracts were analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis and immunoblotting using an anti-IGF- II/M6P receptor antiserum (no. 66416) and 125I-protein A or an immunoperoxidase stain. IGF-II/M6P receptor immunoreactivity was detected in all tissues studied with the highest amount of receptor being expressed in heart, thymus, and kidney and the lowest receptor content being measured in brain and muscle. The receptor content in ovary, testis, lung, and spleen was intermediate. The apparent molecular weight of the IGF-II/M6P receptor (220,000 kilos without reduction of disulfide bonds) varied among the different tissues: in brain the receptor was of lower molecular weight than in other organs. Immunoquantitation experiments employing 125I-protein A and protein extracts from human kidney at different ages revealed a small, albeit not significant, difference of the receptor content between fetal and postnatal tissues: as in other species, larger amounts of receptor seemed to be present in fetal than in postnatal organs. In addition, no significant difference of the receptor content between human fetal liver and early postnatal liver was measured employing 125I-protein A- immunoquantitation in three fetal and five postnatal liver tissue samples. The distribution of IGF-binding protein (IGEBP) species, another abundant and major class of IGF binding principles, was also measured in human fetal and early postnatal lung, liver, kidney, muscle, and brain using Western ligand blotting with 125I-IGF-II: as with IGF-II/M6P receptor immunoreactivity there was differential expression of the different classes of IGFBPs in the various organs

Regulation of Neuromodulator Receptor Efficacy - Implications for Whole-Neuron and Synaptic Plasticity

Membrane receptors for neuromodulators (NM) are highly regulated in their distribution and efficacy - a phenomenon which influences the individual cell's response to central signals of NM release. Even though NM receptor regulation is implicated in the pharmacological action of many drugs, and is also known to be influenced by various environmental factors, its functional consequences and modes of action are not well understood. In this paper we summarize relevant experimental evidence on NM receptor regulation (specifically dopamine D1 and D2 receptors) in order to explore its significance for neural and synaptic plasticity. We identify the relevant components of NM receptor regulation (receptor phosphorylation, receptor trafficking and sensitization of second-messenger pathways) gained from studies on cultured cells. Key principles in the regulation and control of short-term plasticity (sensitization) are identified, and a model is presented which employs direct and indirect feedback regulation of receptor efficacy. We also discuss long-term plasticity which involves shifts in receptor sensitivity and loss of responsivity to NM signals. Finally, we discuss the implications of NM receptor regulation for models of brain plasticity and memorization. We emphasize that a realistic model of brain plasticity will have to go beyond Hebbian models of long-term potentiation and depression. Plasticity in the distribution and efficacy of NM receptors may provide another important source of functional plasticity with implications for learning and memory.Comment: 35 page

Nothing Besides Remains: Preserving the Scientific and Cultural Value of Paleontological Resources in the United States

Receptor occupancy assessed by Positron Emission Tomography (PET) can provide important translational information to help bridge information from one drug to another or from animal to man. The aim of this thesis was to develop nonlinear mixed effects methods for estimation of the relationship between drug exposure and receptor occupancy for the two mGluR5 antagonists AZD9272 and AZD2066 and for the 5HT1B receptor antagonist AZD3783. Also the optimal design for improved estimation of the relationship between drug exposure and receptor occupancy as well as for improved dose finding in neuropathic pain treatment, was investigated. Different modeling approaches were applied. For AZD9272, the radioligand kinetics and receptor occupancy was simultaneously estimated using arterial concentrations as input function and including two brain regions of interest. For AZD2066, a model was developed where brain/plasma partition coefficients from ten different brain regions were included simultaneously as observations. For AZD3783, the simplified reference tissue model was extended to allow different non-specific binding in the reference region and brain regions of interest and the possibility of using white matter as reference was also evaluated. The optimal dose-selection for improved precision of receptor occupancy as well as for improved precision of the minimum effective dose of a neuropathic pain treatment was assessed, using the D-optimal as well as the Ds-optimal criteria. Simultaneous modelling of radioligand and occupancy provided a means to avoid simplifications or approximations and provided the possibility to tests or to relax assumptions. Inclusion of several brain regions of different receptor density simultaneously in the analysis, markedly improved the precision of the affinity parameter. Higher precision was achieved in relevant parameters with designs based on the Ds compared to the D-optimal criterion. The optimal design for improved precision of the relationship between dose and receptor occupancy depended on the number of brain regions and the receptor density of these regions. In conclusion, this thesis presents novel non-linear mixed effects models estimating the relationship between drug exposure and receptor occupancy, providing useful translational information, allowing for a better informed drug-development

The relationship between insulin binding, insulin activation of insulin-receptor tyrosine kinase, and insulin stimulation of glucose uptake in isolated rat adipocytes

We have studied the relationship between insulin activation of insulin-receptor kinase and insulin stimulation of glucose uptake in isolated rat adipocytes. Glucose uptake was half-maximally or maximally stimulated, respectively, when only 4% or 14% of the maximal kinase activity had been reached. To investigate this relationship also under conditions where the insulin effect on activation of receptor kinase was decreased, the adipocytes were exposed to 10 microM-isoprenaline alone or with 5 micrograms of adenosine deaminase/ml. An approx. 30% (isoprenaline) or approx. 50% (isoprenaline + adenosine deaminase) decrease in the insulin effect on receptor kinase activity was found at insulin concentrations between 0.4 and 20 ng/ml, and this could not be explained by decreased insulin binding. The decreased insulin-effect on kinase activity was closely correlated with a loss of insulin-sensitivity of glucose uptake. Moreover, our data indicate that the relation between receptor kinase activity and glucose uptake (expressed as percentage of maximal uptake) remained unchanged. The following conclusions were drawn. (1) If activation of receptor kinase stimulates glucose uptake, only 14% of the maximal kinase activity is sufficient for maximal stimulation. (2) Isoprenaline decreases the coupling efficiency between insulin binding and receptor-kinase activation, this being accompanied by a corresponding decrease in sensitivity of glucose uptake. (3) Our data indicate that the signalling for glucose uptake is closely related to receptor-kinase activity, even when the coupling efficiency between insulin binding and kinase activation is altered. They thus support the hypothesis that receptor-kinase activity reflects the signal which originates from the receptor and which is transduced to the glucose-transport system