Orexin/Hypocretin Activates mTOR Complex 1 (mTORC1) via an Erk/Akt-independent and Calcium-stimulated Lysosome v-ATPase Pathway

The lack of the neuropeptide orexin, also known as hypocretin, results in narcolepsy, a chronic sleep disorder characterized by frequent sleep/cataplexy attacks and rapid eye movement sleep abnormalities. However, the downstream pathways of orexin signaling are not clearly understood. Here, we show that orexin activates the mTOR pathway, a central regulator of cell growth and metabolism, in the mouse brain and multiple recombinant cell lines that express the G protein-coupled receptors (GPCRs), orexin 1 receptor (OX1R) or orexin 2 receptor (OX2R). This orexin/GPCR-stimulated mTOR activation is sensitive to rapamycin, an inhibitor of mTOR complex 1 (mTORC1) but is independent of two well known mTORC1 activators, Erk and Akt. Rather, our studies indicate that orexin activates mTORC1 via extracellular calcium influx and the lysosome pathway involving v-ATPase and Rag GTPases. Moreover, a cytoplasmic calcium transient is sufficient to mimic orexin/GPCR signaling to mTORC1 activation in a v-ATPase-dependent manner. Together, our studies suggest that the mTORC1 pathway functions downstream of orexin/GPCR signaling, which plays a crucial role in many physiological and metabolic processes.     

Mapping of the localization of type 1 angiotensin receptor in membrane microdomains using bioluminescence resonance energy transfer-based sensors

Initiation and termination of signaling of the type I angiotensin receptor (AT(1)-R) can lead to dynamic changes in its localization in plasma membrane microdomains. Several markers were recently developed to investigate membrane microdomains. Here, we used several YFP-labeled fusion constructs (i.e. raft or non-raft plasma membrane markers) to analyze the agonist-induced changes in compartmentalization of AT(1)-R, including internalization or lateral movement between plasma membrane compartments in response to stimulation using bioluminescence resonance energy transfer measurements. Our data demonstrate that angiotensin II (AngII) stimulus changes the microdomain localization of wild type or mutated (DRY → AAY or TSTS → AAAA) AT(1)-Rs co-expressed with the fluorescent probes in HEK293 cells. The comparison of the trafficking of AT(1)-R upon AngII stimulus with those of [Sar(1),Ile(8)]AngII or [Sar(1),Ile(4),Ile(8)]AngII stimulus revealed different types of changes, depending on the nature of the ligand. The observed changes in receptor compartmentalization of the AT(1)-R are strikingly different from those of 5HT-2C and EGF receptors, which demonstrate the usefulness of the bioluminescence resonance energy transfer-based measurements in the investigation of receptor trafficking in the plasma membrane in living cell experiments.     

Discovery of 2,5-diarylnicotinamides as selective orexin-2 receptor antagonists (2-SORAs)

The orexin (or hypocretin) system has been identified as a novel target for the treatment of insomnia due to the wealth of biological and genetic data discovered over the past decade. Recently, clinical proof-of-concept was achieved for the treatment of primary insomnia using dual (OX₁R/OX₂R) orexin receptor antagonists. However, elucidation of the pharmacology associated with selective orexin-2 receptor antagonists (2-SORAs) has been hampered by the lack of orally bioavailable, highly selective small molecule probes. Herein, the discovery and optimization of a novel series of 2,5-diarylnicotinamides as potent and orally bioavailable orexin-2 receptor selective antagonists is described. A compound from this series demonstrated potent sleep promotion when dosed orally to EEG telemetrized rats.     

Novel role for proteinase-activated receptor 2 (PAR2) in membrane trafficking of proteinase-activated receptor 4 (PAR4)

Proteinase-activated receptors 4 (PAR(4)) is a class A G protein-coupled receptor (GPCR) recognized through the ability of serine proteases such as thrombin and trypsin to mediate receptor activation. Due to the irreversible nature of activation, a fresh supply of receptor is required to be mobilized to the cell surface for responsiveness to agonist to be sustained. Unlike other PAR subtypes, the mechanisms regulating receptor trafficking of PAR(4) remain unknown. Here, we report novel features of the intracellular trafficking of PAR(4) to the plasma membrane. PAR(4) was poorly expressed at the plasma membrane and largely retained in the endoplasmic reticulum (ER) in a complex with the COPI protein subunit β-COP1. Analysis of the PAR(4) protein sequence identified an arginine-based (RXR) ER retention sequence located within intracellular loop-2 (R(183)AR → A(183)AA), mutation of which allowed efficient membrane delivery of PAR(4). Interestingly, co-expression with PAR(2) facilitated plasma membrane delivery of PAR(4), an effect produced through disruption of β-COP1 binding and facilitation of interaction with the chaperone protein 14-3-3ζ. Intermolecular FRET studies confirmed heterodimerization between PAR(2) and PAR(4). PAR(2) also enhanced glycosylation of PAR(4) and activation of PAR(4) signaling. Our results identify a novel regulatory role for PAR(2) in the anterograde traffic of PAR(4). PAR(2) was shown to both facilitate and abrogate protein interactions with PAR(4), impacting upon receptor localization and cell signal transduction. This work is likely to impact markedly upon the understanding of the receptor pharmacology of PAR(4) in normal physiology and disease.     

From molecular details of the interplay between transmembrane helices of the thyrotropin receptor to general aspects of signal transduction in family a G-protein-coupled receptors (GPCRs)

Transmembrane helices (TMHs) 5 and 6 are known to be important for signal transduction by G-protein-coupled receptors (GPCRs). Our aim was to characterize the interface between TMH5 and TMH6 of the thyrotropin receptor (TSHR) to gain molecular insights into aspects of signal transduction and regulation. A proline at TMH5 position 5.50 is highly conserved in family A GPCRs and causes a twist in the helix structure. Mutation of the TSHR-specific alanine (Ala-593^5.50) at this position to proline resulted in a 20-fold reduction of cell surface expression. This indicates that TMH5 in the TSHR might have a conformation different from most other family A GPCRs by forming a regular α-helix. Furthermore, linking our own and previous data from directed mutagenesis with structural information led to suggestions of distinct pairs of interacting residues between TMH5 and TMH6 that are responsible for stabilizing either the basal or the active state. Our insights suggest that the inactive state conformation is constrained by a core set of polar interactions among TMHs 2, 3, 6, and 7 and in contrast that the active state conformation is stabilized mainly by non-polar interactions between TMHs 5 and 6. Our findings might be relevant for all family A GPCRs as supported by a statistical analysis of residue properties between the TMHs of a vast number of GPCR sequences.     

Few residues within an extensive binding interface drive receptor interaction and determine the specificity of arrestin proteins

Arrestins bind active phosphorylated forms of G protein-coupled receptors, terminating G protein activation, orchestrating receptor trafficking, and redirecting signaling to alternative pathways. Visual arrestin-1 preferentially binds rhodopsin, whereas the two non-visual arrestins interact with hundreds of G protein-coupled receptor subtypes. Here we show that an extensive surface on the concave side of both arrestin-2 domains is involved in receptor binding. We also identified a small number of residues on the receptor binding surface of the N- and C-domains that largely determine the receptor specificity of arrestins. We show that alanine substitution of these residues blocks the binding of arrestin-1 to rhodopsin in vitro and of arrestin-2 and -3 to β2-adrenergic, M2 muscarinic cholinergic, and D2 dopamine receptors in intact cells, suggesting that these elements critically contribute to the energy of the interaction. Thus, in contrast to arrestin-1, where direct phosphate binding is crucial, the interaction of non-visual arrestins with their cognate receptors depends to a lesser extent on phosphate binding and more on the binding to non-phosphorylated receptor elements.     

Constitutive homo- and hetero-oligomerization of TbetaRII-B, an alternatively spliced variant of the mouse TGF-beta type II receptor

Transforming growth factor (TGF)-beta ligands signal through transmembrane type I and type II serine/threonine kinase receptors, which form heteromeric signalling complexes upon ligand binding. Type II TGF-beta receptors (TbetaRII) are reported to exist as homodimers at the cell surface, but the oligomerization pattern and dynamics of TbetaRII splice variants in live cells has not been demonstrated thus far. Using co-immunoprecipitation and bioluminescence resonance energy transfer (BRET), we demonstrate that the mouse TbetaRII receptor splice variant TbetaRII-B is capable of forming ligand-independent homodimers and heterodimers with TbetaRII. The homomeric interaction of mouse (m)TbetaRII-B isoforms, however, is less robust than the heteromeric interactions of mTbetaRII-B with wild-type TbetaRII, which indicates that these receptors may be more likely to heterodimerize when both receptors are expressed. Moreover, we demonstrate that mTbetaRII-B is a signalling receptor with ubiquitous tissue expression. Our study thus demonstrates previously unappreciated complex formation of TGF-beta type II receptors, and suggests that mTbetaRII-B can direct TGF-beta-induced signalling in vitro and in vivo.     

Discovery process and pharmacological characterization of a novel dual orexin 1 and orexin 2 receptor antagonist useful for treatment of sleep disorders

The hypothalamic peptides orexin-A and orexin-B are potent agonists of two G-protein coupled receptors, namely the OX(1) and the OX(2) receptor. These receptors are widely distributed, though differentially, in the rat brain. In particular, the OX(1) receptor is highly expressed throughout the hypothalamus, whilst the OX(2) receptor is mainly located in the ventral posterior nucleus. A large body of compelling evidence, both pre-clinical and clinical, suggests that the orexin system is profoundly implicated in sleep disorders. In particular, modulation of the orexin receptors activation by appropriate antagonists was proven to be an efficacious strategy for the treatment of insomnia in man. A novel, drug-like bis-amido piperidine derivative was identified as potent dual OX(1) and OX(2) receptor antagonists, highly effective in a pre-clinical model of sleep.     

Regulation of interleukin-21 receptor expression and its signal transduction by WSB-2

Interleukin-21 (IL-21) is a pleiotropic cytokine that regulates T-cell, B-cell, NK-cell, and myeloid-cell functions. IL-21 binds with its cognate receptor complex, which consists of the IL-21 receptor (IL-21R) and the common gamma chain (γc) receptor subunit. We identified novel IL-21R-binding molecule, WD-40 repeats containing SOCS-box-2, WSB-2. WSB-2 associated with the membrane-proximal intracytoplasmic region of IL-21R, including box1 and box2. Overexpression study of WSB-2 showed the reduction of IL-21R expression and IL-21-induced signal transduction. On the other hand, small interfering RNA for WSB-2 enhanced the expression level of IL-21R and IL-21-induced STAT3 activation, indicating that WSB-2 negatively controls the receptor expression. This report provides the first evidence that WSB-2 is a regulator of IL-21R expression and IL-21-induced signal transduction.     

Oxysterols and calcium signal transduction

Ionised calcium (Ca²⁺) is a key second messenger, regulating almost every cellular process from cell death to muscle contraction. Cytosolic levels of this ion can be increased via gating of channel proteins located in the plasma membrane, endoplasmic reticulum and other membrane-delimited organelles. Ca²⁺ can be removed from cells by extrusion across the plasma membrane, uptake into organelles and buffering by anionic components. Ca²⁺ channels and extrusion mechanisms work in concert to generate diverse spatiotemporal patterns of this second messenger, the distinct profiles of which determine different cellular outcomes. Increases in cytoplasmic Ca²⁺ concentration are one of the most rapid cellular responses upon exposure to certain oxysterol congeners or to oxidised low-density lipoprotein, occurring within seconds of addition and preceding increases in levels of reactive oxygen species, or changes in gene expression. Furthermore, exposure of cells to oxysterols for periods of hours to days modulates Ca²⁺ signal transduction, with these longer-term alterations in cellular Ca²⁺ homeostasis potentially underlying pathological events within atherosclerotic lesions, such as hyporeactivity to vasoconstrictors observed in vascular smooth muscle, or ER stress-induced cell death in macrophages. Despite their candidate roles in physiology and disease, little is known about the molecular mechanisms that couple changes in oxysterol concentrations to alterations in Ca²⁺ signalling. This review examines the ways in which oxysterols could influence Ca²⁺ signal transduction and the potential roles of this in health and disease.     

TNF-alpha and IL-4 regulate expression of IL-13 receptor alpha2 on human fibroblasts

Two interleukin 13 receptors (IL-13Rs) have been identified as IL-13Ralpha1 and IL-13Ralpha2. IL-13Ralpha1 is composed of a heterodimer consisting of IL-13Ralpha1 and IL-4 receptor alpha (IL-4Ralpha) as a signaling subunit. In contrast, IL-13Ralpha2 is known as a decoy receptor for IL-13. In this study, we investigated the expression of IL-13Rs on human fibroblasts. IL-13Ralpha2 was significantly up-regulated after stimulation with tumor necrosis factor-alpha (TNF-alpha) and/or IL-4. In contrast, IL-13Ralpha1 was constitutively detectable and was not up-regulated. After the induction of IL-13alpha2 by IL-4, STAT6 phosphorylation through IL-13Ralpha1 by IL-13 was inhibited. We also detected large intracellular pools of IL-13Ralpha2 in fibroblasts quantitatively. Furthermore, mobilization of the IL-13Ralpha2 protein stores from the cytoplasm to the cell surface was prevented by an inhibitor of protein transport, brefeldin-A. These results indicate that TNF-alpha and IL-4 synergistically up-regulate the expression of IL-13Ralpha2 decoy receptor on human fibroblasts by inducing gene expression and mobilizing intracellular receptors, and thus may down-regulate the IL-13 signaling.     

Differential interactions of fluorescent agonists and antagonists with the yeast G protein coupled receptor Ste2p

We describe a rapid method to probe for mutations in cell surface ligand-binding proteins that affect the environment of bound ligand. The method uses fluorescence-activated cell sorting to screen randomly mutated receptors for substitutions that alter the fluorescence emission spectrum of environmentally sensitive fluorescent ligands. When applied to the yeast α-factor receptor Ste2p, a G protein-coupled receptor, the procedure identified 22 substitutions that red shift the emission of a fluorescent agonist, including substitutions at residues previously implicated in ligand binding and at additional sites. A separate set of substitutions, identified in a screen for mutations that alter the emission of a fluorescent α-factor antagonist, occurs at sites that are unlikely to contact the ligand directly. Instead, these mutations alter receptor conformation to increase ligand-binding affinity and provide signaling in response to antagonists of normal receptors. These results suggest that receptor-agonist interactions involve at least two sites, of which only one is specific for the activated conformation of the receptor.     

The human bitter taste receptor hTAS2R39 is the primary receptor for the bitterness of theaflavins

We purified several hundred mgs of four major theaflavins (theaflavin, theaflavin-3-O-gallate, theaflavin-3′-O-gallate, and theaflavin-3,3′-O-digallate). Among the 25 hTAS2Rs expressed in HEK293T cells, hTAS2R39 and hTAS2R14 were activated by theaflavins. Both hTAS2R39 and hTAS2R14 responded to theaflavin-3′-O-gallate. In addition, hTAS2R39 was activated by theaflavin and theaflavin-3,3′-O-gallate, but not by theaflavin-3-O-gallate. In contrast, hTAS2R14 responded to theaflavin-3-O-gallate.     

Relationship between homo-oligomerization of a mammalian olfactory receptor and its activation state demonstrated by bioluminescence resonance energy transfer

G-protein-coupled receptor homo-oligomerization has been increasingly reported. However, little is known regarding the relationship between activation of the receptor and its association/conformational states. The mammalian olfactory receptors (ORs) belong to the G protein-coupled receptor superfamily. In this study, the homo-oligomerization status of the human OR1740 receptor and its involvement in receptor activation upon odorant ligand binding were addressed by co-immunoprecipitation and bioluminescence resonance energy transfer approaches using crude membranes or membranes from different cellular compartments. For the first time, our data clearly show that mammalian ORs constitutively self-associate into homodimers at the plasma membrane level. This study also demonstrates that ligand binding mediates a conformational change and promotes an inactive state of the OR dimers at high ligand concentrations. These findings support and validate our previously proposed model of OR activation/inactivation based on the tripartite odorant-binding protein-odorant-OR partnership.     

Hydrolytic instability of the important orexin 1 receptor antagonist SB-334867: Possible confounding effects on in vivo and in vitro studies

SB-334867 has been an important ligand for the study of the orexin 1 (OX1) receptor due to its high OX1/OX2 selectivity and bioavailability. This ligand however, contains a 2-methylbenzoxazole ring system which is known to undergo hydrolysis, particularly under acidic or basic conditions. The possibility that SB-334867 would be susceptible to significant hydrolysis was evaluated in various formulations and in the solid state. SB-334867 was found to be unstable under conditions commonly employed to prepare stock solutions for in vitro and in vivo studies. In addition, and most alarmingly, the hydrochloride salt of SB-334867 was found to quantitatively decompose to an OX1-inactive product even in the solid state. These findings combine to suggest that studies using SB-334867 (and any other 2-methylbenzoxazole-containing compound) should be performed with great care to avoid the confounding effects of the rapid hydrolytic decomposition of this susceptible structure.     

Noncanonical Control of Vasopressin Receptor Type 2 Signaling by Retromer and Arrestin

The vasopressin type 2 receptor (V2R) is a critical G protein-coupled receptor (GPCR) for vertebrate physiology, including the balance of water and sodium ions. It is unclear how its two native hormones, vasopressin (VP) and oxytocin (OT), both stimulate the same cAMP/PKA pathway yet produce divergent antinatriuretic and antidiuretic effects that are either strong (VP) or weak (OT). Here, we present a new mechanism that differentiates the action of VP and OT on V2R signaling. We found that vasopressin, as opposed to OT, continued to generate cAMP and promote PKA activation for prolonged periods after ligand washout and receptor internalization in endosomes. Contrary to the classical model of arrestin-mediated GPCR desensitization, arrestins bind the VP-V2R complex yet extend rather than shorten the generation of cAMP. Signaling is instead turned off by the endosomal retromer complex. We propose that this mechanism explains how VP sustains water and Na⁺ transport in renal collecting duct cells. Together with recent work on the parathyroid hormone receptor, these data support the existence of a novel “noncanonical” regulatory pathway for GPCR activation and response termination, via the sequential action of β-arrestin and the retromer complex.     

ACTH receptor: Ectopic expression, activity and signaling

Failure in obtaining expression of functional adrenocorticotropic hormone receptor (ACTHR, or melanocortin 2 receptor, MC2R) in non-adrenal cells has hindered molecular analysis of ACTH signaling pathways. Here, we ectopically expressed the mouse ACTHR in Balb/c mouse 3T3 fibroblasts to analyze ACTH signaling pathways involved in induction of fos and jun genes. Natural constitutive expression of the MC2R accessory protein (MRAP) in Balb3T3 and other mouse 3T3 fibroblasts (NIH, Swiss and 3T3-L1) renders these fibroblastic lines suitable for ectopic expression of ACTHR in its active form properly inserted into the plasma membrane at levels similar to those found in mouse Y1 adrenocortical tumor cells. The Y1 cell line is a cultured cell system well known for stably displaying normal adrenal specific metabolic pathways, ACTHR expression and ACTH functional responses. Thirty-nine sub-lines expressing ACTHR (3T3-AR transfectants) were selected for geneticin-resistance and clonally isolated after transfection of ACTHR-cDNA (in the pSVK3 mammalian plasmidial vector) into Balb3T3 fibroblasts. In addition, sixteen clonal sub-lines of Balb3T3 (3T3-0 transfectants) carrying the pSVK3 empty vector were likewise isolated. Fourteen 3T3-AR and four 3T3-0 clones were screened for response to ACTH₃₉ in comparison with Y1 adrenocortical cells. Eight 3T3-AR clones responded to ACTH₃₉ with activation of adenylate cyclase and induction of c-Fos protein, but the levels of, respectively, activation and induction were not strictly correlated. Other fos and jun genes were also induced by ACTH₃₉ in 3T3-AR transfectants, which express levels of ACTHR protein similar to parental Y1 cells. Signaling pathways relevant to c-Fos induction was extensively investigated in 3 clones: 3T3-AR01 and –07 and 3T3-04. In Y1 cells, specific inhibitors (H89/PKA; PD98059/MEK; Go6983/PKC and SP600125/JNK) show that signals initiated in the ACTH/ACTHR-system activate 4 pathways to induce the c-fos gene, namely: (a) cAMP/PKA/CREB; (b) MEK/ERK1/2; (c) PKC and d) JNK1/2. In 3T3-AR transfectants, both inhibitors PD98059 and Go6983 proved completely ineffective to inhibit c-Fos induction by ACTH₃₉, implying that MEK/ERK and PKC pathways are not involved in this process. On the other hand, SP600125 caused 85% inhibition of c-Fos induction by ACTH₃₉ and, in addition, ACTH₃₉ promotes JNK1/2 phosphorylation, suggesting that JNK is a major signaling pathway mediating c-Fos induction by ACTH₃₉ in these cells. In addiction, PKA inhibitor H89 also inhibits c-Fos induction in 3T3-AR7 cells by ACTH₃₉, implicating activation of the cAMP/PKA/CREB pathway in c-Fos induction by ACTH₃₉. However, the cAMP derivatives db-cAMP and 8Br-cAMP, do not promote CREB phosphorylation and c-Fos induction in parental Balb3T3 and 3T3-AR transfectants, confirming previous report by others. In conclusion, expression of active ACTHR in Balb3T3 fibroblasts renders these cells responsive to ACTH with activation of cAMP/PKA/CREB and JNK pathways and, also, induction of genes from the fos and jun families. These results show that Balb 3T3-AR sublines are useful cellular systems for genetic analysis of ACTH-signaling pathways. However, activation of cAMP/PKA/CREB and JNK pathways and induction of fos and jun genes are not yet sufficient to enable ACTH for interference in morphology, migration and proliferation of Balb3T3 fibroblasts as it does in Y1 adrenocortical cells.     

Regulation of membrane cholecystokinin-2 receptor by agonists enables classification of partial agonists as biased agonists

Given the importance of G-protein-coupled receptors as pharmacological targets in medicine, efforts directed at understanding the molecular mechanism by which pharmacological compounds regulate their presence at the cell surface is of paramount importance. In this context, using confocal microscopy and bioluminescence resonance energy transfer, we have investigated internalization and intracellular trafficking of the cholecystokinin-2 receptor (CCK2R) in response to both natural and synthetic ligands with different pharmacological features. We found that CCK and gastrin, which are full agonists on CCK2R-induced inositol phosphate production, rapidly and abundantly stimulate internalization. Internalized CCK2R did not rapidly recycle to plasma membrane but instead was directed to late endosomes/lysosomes. CCK2R endocytosis involves clathrin-coated pits and dynamin and high affinity and prolonged binding of β-arrestin1 or -2. Partial agonists and antagonists on CCK2R-induced inositol phosphate formation and ERK1/2 phosphorylation did not stimulate CCK2R internalization or β-arrestin recruitment to the CCK2R but blocked full agonist-induced internalization and β-arrestin recruitment. The extreme C-terminal region of the CCK2R (and more precisely phosphorylatable residues Ser(437)-Xaa(438)-Thr(439)-Thr(440)-Xaa(441)-Ser(442)-Thr(443)) were critical for β-arrestin recruitment. However, this region and β-arrestins were dispensable for CCK2R internalization. In conclusion, this study allowed us to classify the human CCK2R as a member of class B G-protein-coupled receptors with regard to its endocytosis features and identified biased agonists of the CCK2R. These new important insights will allow us to investigate the role of internalized CCK2R·β-arrestin complexes in cancers expressing this receptor and to develop new diagnosis and therapeutic strategies targeting this receptor.     

Characterization of an antiserum against the glucocorticoid receptor

An immunoglobulin (IgG) fraction from serum of a rabbit immunized with a highly purified preparation of glucocorticoid receptor from rat liver cytosol contained specific antibodies to glucocorticoid receptor. This was shown following incubation of the [³H]triamcinolone acetonide-glucocorticoid receptor (TA-GR) complex with the IgG fraction by (I) adsorption of the [³H]TA-GR-antibody complex to protein A linked to Sepharose, (II) an increased sedimentation rate of the [³H]TA-GR-antibody complex compared to that of the [³H]TA-GR complex, and (III) an increased molecular size of the [³H]TA-GR-antibody complex when compared to that of the [³H]TA-GR complex as judged from gel filtration. The antibody fraction was characterized with regard to titer, cross-reactivity and specificity. The antibodies cross-reacted with the glucocorticoid receptor from various rat tissues (liver, thymus and hippocampus), as well as with the glucocorticoid receptor from human normal lymphocytes, chronic lymphatic leukemia cells and human hippocampus. In the rat liver, the antibody bound to both the nuclear and the cytosolic glucocorticoid receptor (Stokes radius 6.1 nm). It did not cross-react with the proteolytic fragments of the glucocorticoid receptor, the 3.6 nm complex or the 1.9 nm complex. Binding of the antibodies was not seen to the androgen, estrogen or progestin receptors in rat to rat serum transcortin. With an indirect competitive ELISA (enzyme-linked immunosorbent assay) combined with various separation techniques, based on different physiocochemical principles, it was shown that the glucocorticoid receptor was the only detectable antibody binding protein from rat liver cytosol using this assay system. These findings also indicate an immunochemical similarity between glucocorticoid receptors in different tissues as well as in different species, but not between glucocorticoid receptors and other steroid hormone receptor proteins. The cytosolic and nuclear glucocorticoid receptors in rat liver were shown to be immunochemically similar.     

The tethering arm of the EGF receptor is required for negative cooperativity and signal transduction

The EGF receptor is a classical receptor-tyrosine kinase. In the absence of ligand, the receptor adopts a closed conformation in which the dimerization arm of subdomain II interacts with the tethering arm in subdomain IV. Following the binding of EGF, the receptor opens to form a symmetric, back-to-back dimer. Although it is clear that the dimerization arm of subdomain II is central to the formation of receptor dimers, the role of the tethering arm of subdomain IV (residues 561-585) in this configuration is not known. Here we use (125)I-EGF binding studies to assess the functional role of the tethering arm in the EGF receptor dimer. Mutation of the three major residues that contribute to tethering (D563A,H566A,K585A-EGF receptor) did not significantly alter either the ligand binding properties or the signaling properties of the EGF receptor. By contrast, breaking the Cys(558)-Cys(567) disulfide bond through double alanine replacements or deleting the loop entirely led to a decrease in the negative cooperativity in EGF binding and was associated with small changes in downstream signaling. Deletion of the Cys(571)-Cys(593) disulfide bond abrogated cooperativity, resulting in a high affinity receptor and increased sensitivity of downstream signaling pathways to EGF. Releasing the Cys(571)-Cys(593) disulfide bond resulted in extreme negative cooperativity, ligand-independent kinase activity, and impaired downstream signaling. These data demonstrate that the tethering arm plays an important role in supporting cooperativity in ligand binding. Because cooperativity implies subunit-subunit interactions, these results also suggest that the tethering arm contributes to intersubunit interactions within the EGF receptor dimer.