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

LDL receptor-related protein-1 regulates NFκB and microRNA-155 in macrophages to control the inflammatory response

LDL receptor-related protein-1 (LRP1) is an endocytic and cell-signaling receptor. In mice in which LRP1 is deleted in myeloid cells, the response to lipopolysaccharide (LPS) was greatly exacerbated. LRP1 deletion in macrophages in vitro, under the control of tamoxifen-activated Cre-ER(T) fusion protein, robustly increased expression of proinflammatory cytokines and chemokines. In LRP1-expressing macrophages, proinflammatory mediator expression was regulated by LRP1 ligands in a ligand-specific manner. The LRP1 agonists, α2-macroglobulin and tissue-type plasminogen activator, attenuated expression of inflammatory mediators, even in the presence of LPS. The antagonists, receptor-associated protein (RAP) and lactoferrin (LF), and LRP1-specific antibody had the entirely opposite effect, promoting inflammatory mediator expression and mimicking LRP1 deletion. NFκB was rapidly activated in response to RAP and LF and responsible for the initial increase in expression of proinflammatory mediators. RAP and LF also significantly increased expression of microRNA-155 (miR-155) after a lag phase of about 4 h. miR-155 expression reflected, at least in part, activation of secondary cell-signaling pathways downstream of TNFα. Although miR-155 was not involved in the initial induction of cytokine expression in response to LRP1 antagonists, miR-155 was essential for sustaining the proinflammatory response. We conclude that LRP1, NFκB, and miR-155 function as members of a previously unidentified system that has the potential to inhibit or sustain inflammation, depending on the continuum of LRP1 ligands present in the macrophage microenvironment

Fee Arrangements and Fee Shifting: Lessons From the Experience in Ontario

About one-third of oestrogen receptor alpha-positive breast cancer patients treated with tamoxifen relapse. Here we identify the nuclear receptor retinoic acid receptor alpha as a marker of tamoxifen resistance. Using quantitative mass spectrometry-based proteomics, we show that retinoic acid receptor alpha protein networks and levels differ in a tamoxifen-sensitive (MCF7) and a tamoxifen-resistant (LCC2) cell line. High intratumoural retinoic acid receptor alpha protein levels also correlate with reduced relapse-free survival in oestrogen receptor alpha-positive breast cancer patients treated with adjuvant tamoxifen solely. A similar retinoic acid receptor alpha expression pattern is seen in a comparable independent patient cohort. An oestrogen receptor alpha and retinoic acid receptor alpha ligand screening reveals that tamoxifen-resistant LCC2 cells have increased sensitivity to retinoic acid receptor alpha ligands and are less sensitive to oestrogen receptor alpha ligands compared with MCF7 cells. Our data indicate that retinoic acid receptor alpha may be a novel therapeutic target and a predictive factor for oestrogen receptor alpha-positive breast cancer patients treated with adjuvant tamoxifen

Spatial intensity distribution analysis quantifies the extent and regulation of homodimerization of the secretin receptor

Previous studies have indicated that the G protein-coupled secretin receptor is present as a homo-dimer, organized through symmetrical contacts in transmembrane domain IV, and that receptor dimerization is critical for high potency signalling by secretin. However, whether all of the receptor exists in the dimeric form or if this is regulated, is unclear. We used measures of quantal brightness of the secretin receptor tagged with monomeric enhanced green fluorescent protein (mEGFP) and Spatial Intensity Distribution Analysis to assess this. Calibration using cells expressing plasma membrane-anchored forms of mEGFP initially allowed demonstration that the Epidermal Growth Factor receptor is predominantly monomeric in the absence of ligand and whilst wild type receptor was rapidly converted to a dimeric form by ligand, a mutated form of this receptor remained monomeric. Equivalent studies showed that at moderate expression levels the secretin receptor exists as a mixture of monomeric and dimeric forms, with little evidence of higher-order complexity. However, sodium butyrate induced up-regulation of the receptor resulted in a shift from monomeric towards oligomeric organization. By contrast, a form of the secretin receptor containing a pair of mutations on the lipid-facing side of transmembrane domain IV was almost entirely monomeric. Down-regulation of the secretin receptor-interacting G protein Gαs did not alter receptor organization, indicating that dimerization is defined specifically by direct protein-protein interactions between copies of the receptor polypeptide, whilst short term treatment with secretin had no effect on organization of the wild type receptor but increased the dimeric proportion of the mutated receptor variant

Androgen receptor as a mediator and biomarker of radioresistance in triple-negative breast cancer.

Increased rates of locoregional recurrence have been observed in triple-negative breast cancer despite chemotherapy and radiation therapy. Thus, approaches that combine therapies for radiosensitization in triple-negative breast cancer are critically needed. We characterized the radiation therapy response of 21 breast cancer cell lines and paired this radiation response data with high-throughput drug screen data to identify androgen receptor as a top target for radiosensitization. Our radiosensitizer screen nominated bicalutamide as the drug most effective in treating radiation therapy-resistant breast cancer cell lines. We subsequently evaluated the expression of androgen receptor in >2100 human breast tumor samples and 51 breast cancer cell lines and found significant heterogeneity in androgen receptor expression with enrichment at the protein and RNA level in triple-negative breast cancer. There was a strong correlation between androgen receptor RNA and protein expression across all breast cancer subtypes (R2 = 0.72, p < 0.01). In patients with triple-negative breast cancer, expression of androgen receptor above the median was associated with increased risk of locoregional recurrence after radiation therapy (hazard ratio for locoregional recurrence 2.9-3.2)) in two independent data sets, but there was no difference in locoregional recurrence in triple-negative breast cancer patients not treated with radiation therapy when stratified by androgen receptor expression. In multivariable analysis, androgen receptor expression was most significantly associated with worse local recurrence-free survival after radiation therapy (hazard ratio of 3.58) suggesting that androgen receptor expression may be a biomarker of radiation response in triple-negative breast cancer. Inhibition of androgen receptor with MDV3100 (enzalutamide) induced radiation sensitivity (enhancement ratios of 1.22-1.60) in androgen receptor-positive triple-negative breast cancer lines, but did not affect androgen receptor-negative triple-negative breast cancer or estrogen-receptor-positive, androgen receptor-negative breast cancer cell lines. androgen receptor inhibition with MDV3100 significantly radiosensitized triple-negative breast cancer xenografts in mouse models and markedly delayed tumor doubling/tripling time and tumor weight. Radiosensitization was at least partially dependent on impaired dsDNA break repair mediated by DNA protein kinase catalytic subunit. Our results implicate androgen receptor as a mediator of radioresistance in breast cancer and identify androgen receptor inhibition as a potentially effective strategy for the treatment of androgen receptor-positive radioresistant tumors

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

Orexin-1 receptor-cannabinoid CB1 receptor heterodimerization results in both ligand-dependent and -independent coordinated alterations of receptor localization and function

Following inducible expression in HEK293 cells, the human orexin-1 receptor was targeted to the cell surface but became internalized following exposure to the peptide agonist orexin A. By contrast, constitutive expression of the human cannabinoid CB1 receptor resulted in a predominantly punctate, intracellular distribution pattern consistent with spontaneous, agonistindependent internalization. Expression of the orexin-1 receptor in the presence of the CB1 receptor resulted in both receptors displaying the spontaneous internalization phenotype. Single cell fluorescence resonance energy transfer imaging indicated the two receptors were present as heterodimers/oligomers in intracellular vesicles. Addition of the CB1 receptor antagonist SR-141716A to cells expressing only the CB1 receptor resulted in re-localization of the receptor to the cell surface. Although SR-141716A has no significant affinity for the orexin-1 receptor, in cells co-expressing the CB1 receptor, the orexin-1 receptor was also re-localized to the cell surface by treatment with SR-141716A. Treatment of cells co-expressing the orexin-1 and CB1 receptors with the orexin-1 receptor antagonist SB-674042 also resulted in re-localization of both receptors to the cell surface. Treatment with SR-141716A resulted in decreased potency of orexin A to activate the mitogen-activated protein kinases ERK1/2 only in cells co-expressing the two receptors. Treatment with SB-674042 also reduced the potency of a CB1 receptor agonist to phosphorylate ERK1/2 only when the two receptors were co-expressed. These studies introduce an entirely novel pharmacological paradigm, whereby ligands modulate the function of receptors for which they have no significant inherent affinity by acting as regulators of receptor heterodimers

Dynamic regulation of quaternary organization of the M1 muscarinic receptor by subtype-selective antagonist drugs

Although rhodopsin-like G protein-coupled receptors can exist as both monomers and non-covalently associated dimers/oligomers, the steady-state proportion of each form and whether this is regulated by receptor ligands is unknown. Herein we address these topics for the M1 muscarinic acetylcholine receptor, a key molecular target for novel cognition enhancers, by employing Spatial Intensity Distribution Analysis. This method can measure fluorescent particle concentration and assess oligomerization states of proteins within defined regions of living cells. Imaging and analysis of the basolateral surface of cells expressing some 50 molecules.microm-2 of the human muscarinic M1 receptor identified an ~75/25 mixture of receptor monomers and dimers/oligomers. Both sustained and shorter-term treatment with the selective M1 antagonist pirenzepine resulted in a large shift in the distribution of receptor species to favor the dimeric/oligomeric state. Although sustained treatment with pirenzepine also resulted in marked upregulation of the receptor, simple mass-action effects were not the basis for ligand-induced stabilization of receptor dimers/oligomers. The related antagonist telenzepine also produced stabilization and enrichment of the M1 receptor dimer population but the receptor subtype non-selective antagonists atropine and N-methylscopolamine did not. In contrast, neither pirenzepine nor telenzepine altered the quaternary organization of the related M3 muscarinic receptor. These data provide unique insights into the selective capacity of receptor ligands to promote and/or stabilize receptor dimers/oligomers and demonstrate that the dynamics of ligand regulation of the quaternary organization of G protein-coupled receptors is markedly more complex than previously appreciated. This may have major implications for receptor function and behavior

High throughput mutagenesis for identification of residues regulating human prostacyclin (hIP) receptor

The human prostacyclin receptor (hIP receptor) is a seven-transmembrane G protein-coupled receptor (GPCR) that plays a critical role in vascular smooth muscle relaxation and platelet aggregation. hIP receptor dysfunction has been implicated in numerous cardiovascular abnormalities, including myocardial infarction, hypertension, thrombosis and atherosclerosis. Genomic sequencing has discovered several genetic variations in the PTGIR gene coding for hIP receptor, however, its structure-function relationship has not been sufficiently explored. Here we set out to investigate the applicability of high throughput random mutagenesis to study the structure-function relationship of hIP receptor. While chemical mutagenesis was not suitable to generate a mutagenesis library with sufficient coverage, our data demonstrate error-prone PCR (epPCR) mediated mutagenesis as a valuable method for the unbiased screening of residues regulating hIP receptor function and expression. Here we describe the generation and functional characterization of an epPCR derived mutagenesis library compromising >4000 mutants of the hIP receptor. We introduce next generation sequencing as a useful tool to validate the quality of mutagenesis libraries by providing information about the coverage, mutation rate and mutational bias. We identified 18 mutants of the hIP receptor that were expressed at the cell surface, but demonstrated impaired receptor function. A total of 38 non-synonymous mutations were identified within the coding region of the hIP receptor, mapping to 36 distinct residues, including several mutations previously reported to affect the signaling of the hIP receptor. Thus, our data demonstrates epPCR mediated random mutagenesis as a valuable and practical method to study the structurefunction relationship of GPCRs. © 2014 Bill et al