Glucocorticoid receptors in lung. Comparison between nonactivated and activated forms of the cytoplasmic glucocorticoid binding protein and their relationship to the nuclear binding protein of fetal rabbit lung.

In the absence of salt the cytoplasmic glucocorticoid receptor of fetal rabbit lung sediments at 7 S while the nuclear receptor sediments at 4 S. However, if nuclear extracts are mixed with receptor-depleted cytosol preparations in dilute buffer solutions without added salt, the nuclear 4 S receptor sediments as a 7 S species similar to that observed for the cytoplasmic form under the same conditions suggesting an interaction of the nuclear receptor with other cytosol proteins rather than with itself. In addition, both cytoplasmic and nuclear receptors sediment at 4 S in 0.4 M KCl and a major fraction of the nuclear receptor has an agarose elution profile identical to that of the cytoplasmic receptor. Thus a major fraction of the nuclear receptors is indistinguishable from the cytoplasmic receptors by the methods used. Since the cytoplasmic receptor sediments at 4 S in 0.15 M KCl, it is suggested that in vivo the glucocorticoid receptor may exist as a 4 S species and that the 7 S form described previously may result from an interaction of the 4 S component with other cytosol proteins in hypotonic media. About 25% of the receptor present in nuclear extracts has an agarose elution profile different from that of the cytoplasmic receptor in 0.4 M KCl. This suggests that either the nuclear receptor associates with itself or other nuclear proteins or that more than one form of nuclear receptor exists. Earlier observations suggested that in the absence of hormone the glucocorticoid receptor is localized exclusively in the cytoplasm of lung cells and that the nuclear receptor is formed by a transfer of the cytoplasmic steroid-receptor complex into the nucleus. A prerequisite for this transfer seems to be a modification of the receptor to an active form which can bind to nuclei. This receptor transfomration, referred to in this paper as activation of the receptor, can occur in the absence of nuclei and is highly dependent on temperature and ionic strength. Cytoplasmic receptors activated either by heating or by exposure to high ionic strength are indistinguishable from nonactivated receptors by sucrose density gradient analysis or by agarose gel filtration in solutions containing 0.4 M KCl. Simiarly, no significant difference in the absence of salt is observed after activation by heating. These results suggest that activation of the cytoplasmic glucocorticoid receptor involves conformational changes which favor its transfer and/or binding to nuclear sites rather than conversion of a 4 S species to a faster-sedimenting form by dimerization or by addition of another protein unit as has been proposed for the activation of the estrogen receptor of the rat uterus.     

Homologous and heterologous phosphorylation of the vasopressin V1a receptor

The vasopressin V1a receptor undergoes homologous and heterologous desensitizations which can be mimicked by activation of protein kinase C. This suggests that phosphorylation of the V1a receptor may be involved in the desensitization mechanisms. Such a phosphorylation was presently investigated in HEK 293 cells stably transfected with rat vasopressin V1a receptor. Metabolic labelling and immunoprecipitation of epitope-tagged V1a receptor evidenced a 52-kDa band and a 92-kDa band. Glycosidase treatments and immunoblotting experiments suggest that the 52-kDa band corresponds to an immature unprocessed receptor protein, whereas the 92-kDa band would correspond to a highly glycosylated form of the mature V1a receptor. Exposure of the cells to vasopressin induced a selective 32P phosphate incorporation in the 92-kDa form of the receptor. This homologous ligand-induced phosphorylation was dose dependent with maximal phosphate incorporation corresponding to four times the basal level. Stimulation of the endogenous phospholipase C-coupled m3 muscarinic receptor by carbachol-induced heterologous phosphorylation of the V1a receptor whose amplitude was half that of the homologous phosphorylation. This heterologous phosphorylation was associated with a reduced vasopressin-dependent increase in intracellular calcium.     

Transmembrane domains 4 and 7 of the M(1) muscarinic acetylcholine receptor are critical for ligand binding and the receptor activation switch.

Activation of the muscarinic acetylcholine receptors requires agonist binding followed by a conformational change, but the ligand binding and conformation-switching residues have not been completely identified. Systematic alanine-scanning mutagenesis has been used to assess residues 142-164 in transmembrane helix 4 and 402-421 in transmembrane helix 7 of the M(1) muscarinic acetylcholine receptor. Several inward-facing amino acid side chains in the exofacial parts of transmembrane helices 4 and 7 contribute to acetylcholine binding. Alanine substitution of the aromatic residues in this group reduced signaling efficacy, suggesting that they may form part of a charge-stabilized aromatic cage, which triggers rotation and movement of the transmembrane helices. The mutation of adjacent residues modulated receptor activation, either reducing signaling or causing constitutive activation. In the buried endofacial section of transmembrane helix 7, alanine substitution mutants of the conserved NSXXNPXXY motif displayed strongly reduced signaling efficacy, despite having increased or unchanged acetylcholine affinity. These residues may have dual functions, forming intramolecular contacts that stabilize the receptor in the inactive ground state, but that are broken, allowing them to form new intramolecular bonds in the activated state. This conformational rearrangement is critical to produce a G protein binding site and may represent a key mechanism of receptor activation.     

Leptin activates AMP-activated protein kinase in hepatic cells via a JAK2-dependent pathway

AMP-activated protein kinase (AMPK) plays a key role in the regulation of energy homeostasis within the individual cell. Recent reports have suggested that leptin, an adipocyte-secreted hormone, phosphorylates AMPK in skeletal muscle directly. However, little is known about the interaction between leptin signaling and AMPK activation. Here, we report that the leptin-induced phosphorylation of AMPK was detected in Huh7 cells expressing long form leptin receptor (OBRb) as well as short form leptin receptor (OBRa). In addition, we demonstrate that AMPK activation does not require the phosphorylation of either Tyr-985 or Tyr-1138 within the OBRb and may occur via a STAT3-independent signaling pathway. We also show that Huh7 cells expressing OBRb and SOCS3 (inhibitor of JAK2) resulted in a marked reduction of AMPK activation in response to leptin. These findings suggest that the activation of JAK2, but not STAT3, may play a critical role in leptin-induced AMPK activation in Huh7 cells.     

Epidermal growth factor directs sex-specific steroid signaling through Src activation

Estrogens and androgens exert many biological effects that do not require interactions of their receptors with chromosomal DNA. However, it has been a long-standing question how the sex steroid receptors provoke signal transduction outside the nucleus. Here we have shown that epidermal growth factor (EGF) directs sex-specific steroid signaling through Src activation. We have revealed that estrogen (E2)-induced Src activation takes place in, not only plasma, but also endomembranes. This was found ascribed to the existence of EGF and the occurrence of EGF receptor (EGFR)-involved endocytosis of estrogen receptor together with Src. EGFR, estrogen receptor, and Src were found to form a complex upon E2 stimulation. The cell growth of breast cancer-derived MCF-7 cells was found to remarkably increase through the above EGF-involved estrogen-signaling process. In contrast, the androgen 5alpha-dihydrotestosterone-induced Src activation occurs only in the plasma membrane free from the interaction of EGFR with androgen receptor, irrespective of EGF. The cell growth occurred only moderately as a result. The spatial difference in Src activation between E2 and 5alpha-dihydrotestosterone may be responsible for the different extent of observed cell growth.     

Constitutive TrkA activity in receptor-overexpressing PC12 clones

We have studied ligand-independent signaling by the nerve growth factor receptor TrkA in PC12 clones, under conditions of receptor overexpression. Our results indicate that TrkA-overexpressing PC12 clones display constitutive receptor activation, involving both the mature, 140-kDa form and the immature, intracellular 110-kDa form of the receptor. Phosphorylation of Tyr 674/675, located in the activation loop domain and reflecting TrkA kinase activity, appears particularly prominent in the immature form of the receptor. Constitutive receptor activation is able to chronically stimulate the PI-3 kinase/Akt as well as the mitogen-activated protein kinase pathways, leading to ligand-independent neurite extension. Under conditions of overexpression, a significant fraction of the receptor is retained intracellularly by thiol-mediated mechanisms. Exposure of the cells to reducing agents promotes translocation of the intracellular pool of the receptor to the plasma membrane and suppresses ligand-independent neurite outgrowth. Our results suggest that the levels of expression of TrkA, both intracellularly and at the cell surface, may act to modulate its kinase activity and generate ligand-independent downstream signaling.     

Epidermal growth factor induces rapid, reversible aggregation of the purified epidermal growth factor receptor

Epidermal growth factor (EGF) receptor from A-431 cells was purified by affinity chromatography with monoclonal anti-receptor antibodies. The purified radiolabeled receptor was incubated with EGF and then analyzed by gel electrophoresis under nondenaturing conditions. In these gels, the EGF receptor migrates in two forms: a fast-migrating (low) form and an EGF-induced slow-migrating (high) form. On the basis of the various control and calibration experiments described, it is concluded that the low form represents the monomeric 170-kilodalton EGF receptor and the high form represents an EGF receptor dimer. The binding of EGF causes a rapid, temperature-sensitive dimerization of the EGF receptor. Receptor dimerization is fully reversible and involves saturable, noncovalent interactions that are stable at neutral pH and in nonionic detergents. Both the monomeric and dimeric forms of the receptor bind EGF and undergo self-phosphorylation. The dimeric form of the receptor may possess higher ligand binding affinity, and it seems to be phosphorylated earlier than the monomeric form following the addition of EGF and [gamma-32P]ATP. On the basis of these results, it is concluded that receptor oligomerization is an intrinsic property of the occupied EGF receptor and that it may play a role in the activation of the kinase function and the subsequent transmembrane signaling process.     

Activation of the Met receptor by cell attachment induces and sustains hepatocellular carcinomas in transgenic mice

Overexpression is the most common abnormality of receptor tyrosine kinases (RTKs) in human tumors. It is presumed that overexpression leads to constitutive activation of RTKs, but the mechanism of that activation has been uncertain. Here we show that overexpression of the Met RTK allows activation of the receptor by cell attachment and that this form of activation can be tumorigenic. Transgenic mice that overexpressed Met in hepatocytes developed hepatocellular carcinoma (HCC), one of the human tumors in which Met has been implicated previously. The tumorigenic Met was activated by cell attachment rather than by ligand. Inactivation of the transgene led to regression of even highly advanced tumors, apparently mediated by apoptosis and cessation of cellular proliferation. These results reveal a previously unappreciated mechanism by which the tumorigenic action of RTKs can be mediated, provide evidence that Met may play a role in both the genesis and maintenance of HCC, and suggest that Met may be a beneficial therapeutic target in tumors that overexpress the receptor.     

Desensitization of NMDA receptor channels is modulated by glutamate agonists

Two distinct forms of desensitization have been characterized for N-methyl-D-aspartate (NMDA) receptors. One form results from a weakening of agonist affinity when channels are activated whereas the other form of desensitization results when channels enter a long-lived nonconducting state. A weakening of glycine affinity upon NMDA receptor activation has been reported. Cyclic reaction schemes for NMDA receptor activation require that a concomitant affinity shift should be observed for glutamate agonists. In this study, measurements of peak and steady-state NMDA receptor currents yielded EC50 values for glutamate that differed by 1.9-fold, but no differences were found for another agonist, L-cysteine-S-sulfate (LCSS). Simulations show that shifts in EC50 values may be masked by significant degrees of desensitization resulting from channels entering a long-lived nonconducting state. Simulations also show that a decrease in the degree of desensitization with increasing agonist concentration is a good indicator for the existence of desensitization resulting from a weakening of agonist affinity. Both glutamate and LCSS exhibited this trend. An affinity difference of three- to eightfold between high-and low-affinity agonist-binding states was estimated from fitting of dose-response data with models containing both types of desensitization. This indicates that activation of NMDA receptors causes a reduction in both glutamate and glycine affinities.     

The conserved C-terminal domain of the bovine papillomavirus E5 oncoprotein can associate with an alpha-adaptin-like molecule: a possible link between growth factor receptors and viral transformation.

The bovine papillomavirus E5 gene encodes an oncoprotein that can independently transform rodent fibroblasts. This small 44-amino-acid protein is thought to function through the activation of growth factor receptors. E5 activation of the epidermal growth factor receptor results in an increase in the number of activated receptors at the cell surface. This finding suggests that E5 may act by inhibiting the normal down regulation of activated epidermal growth factor receptor via coated pit-mediated endocytosis. We have constructed a fusion protein consisting of glutathione S-transferase and the conserved C-terminal domain of E5 (GST-E5) in order to identify E5-associated cellular proteins that may be involved in its transforming activity. We have identified a 125-kDa cellular protein with a strong associated serine kinase activity that specifically associated with GST-E5 in the reduced form but not with GST-E5 fusions that contained changes in several conserved amino acids. Microsequence and biochemical analyses suggest that p125 is a novel member of the alpha-adaptin family. Since alpha-adaptins have previously been shown to be involved in coated pit-mediated cell surface receptor endocytosis and down regulation, these results suggest that p125 may be an alpha-adaptin-like molecule involved in growth factor receptor down regulation and that E5 may act by inhibiting its activity.     

A common model for cytokine receptor activation: combined scissor-like rotation and self-rotation of receptor dimer induced by class I cytokine

The precise mechanism by which the binding of a class I cytokine to the extracellular domain of its corresponding receptor transmits a signal through the cell membrane remains unclear. Receptor activation involves a cytokine-receptor complex with a 1∶2 stoichiometry. Previously we used our transient-complex theory to calculate the rate constant of the initial cytokine-receptor binding to form a 1∶1 complex. Here we computed the binding pathway leading to the 1∶2 activation complex. Three cytokine systems (growth hormone, erythropoietin, and prolactin) were studied, and the focus was on the binding of the extracellular domain of the second receptor molecule after forming the 1∶1 complex. According to the transient-complex theory, translational and rotation diffusion of the binding entities bring them together to form a transient complex, which has near-native relative separation and orientation but not the short-range specific native interactions. Subsequently conformational rearrangement leads to the formation of the native complex. We found that the changes in relative orientations between the two receptor molecules from the transient complex to the 1∶2 native complex are similar for the three cytokine-receptor systems. We thus propose a common model for receptor activation by class I cytokines, involving combined scissor-like rotation and self-rotation of the two receptor molecules. Both types of rotations seem essential: the scissor-like rotation separates the intracellular domains of the two receptor molecules to make room for the associated Janus kinase molecules, while the self-rotation allows them to orient properly for transphosphorylation. This activation model explains a host of experimental observations. The transient-complex based approach presented here may provide a strategy for designing antagonists and prove useful for elucidating activation mechanisms of other receptors.     

Isolation and characterization of two novel forms of the human prolactin receptor generated by alternative splicing of a newly identified exon 11

We have identified a novel exon 11 of the human prolactin receptor (hPRLR) gene that is distinct from its rodent counterparts and have demonstrated the presence of two novel short forms of the hPRLR (S1(a) and S1(b)), which are derived from alternative splicing of exons 10 and 11. S1(a) encodes 376 amino acids (aa) that contain partial exon 10 and a unique 39-aa C-terminal region encoded by exon 11. S1(b) encodes 288 aa that lack the entire exon 10 and contains 3 amino acids at the C terminus derived from exon 11 using a shifted reading frame. These short forms, which were found in several normal tissues and in breast cancer cell lines, were expressed as cell surface receptors and possessed binding affinities comparable with the long form. Unlike the long form, neither short form was able to mediate the activation of the beta-casein gene promoter induced by prolactin. Instead they acted as dominant negative forms when co-expressed with the long form in transfected cells. Due to a marked difference in the cellular levels between the two short forms in transfected cells, S1(b) was more effective in inhibiting the prolactin-induced activation of the beta-casein gene promoter mediated by the long form of the receptor. The low cellular level of S1(a) was due to its more rapid turnover than the S1(b) protein. This is attributable to specific residues within the C-terminal unique 39 amino acids of the S1(a) form and may represent a new mechanism by which the hPRLR is modulated at the post-translational level. Since both short forms contain abbreviated cytoplasmic domains with unique C termini, they may also exhibit distinct signaling pathways in addition to modulating the signaling from the long form of the receptor. These receptors may therefore play important roles in the diversified actions of prolactin in human tissues.     

ATP-dependent activation of glucocorticoid receptor from rat liver cytosol.

The glucocorticoid--receptor complex from freshly prepared rat liver cytosol is in a non-activated form, with very little affinity to bind to isolated nuclei. When such preparations were incubated with 5--10 mM-ATP at 4 degrees C, the receptor complex acquired the properties of an 'activated' transformed form, which readily bound to nuclei, ATP--Sepharose, phosphocellulose and DNA--cellulose. This transformation was comparable with the activation achieved by warming the steroid--receptor complex at 23 degrees C. The effect of ATP was specific, as it was more effective than ADP, whereas AMP had no such effect on activation. The process of receptor activation was sensitive to the presence of 10 mM-sodium molybdate; the latter blocked activation by both ATP and heat. Bivalent cations had no observable effect on the receptor activation at low temperature, but they decreased the extent of activation by ATP. The steroid-binding properties of glucocorticoid receptor remained intact under the above conditions. However, a significant increase in steroid binding occurred when ATP was preincubated with cytosol receptor before the addition of [3H]triamcinolone acetonide. ATP also stabilized the glucocorticoid--receptor complexes at 23 degrees C. These results suggest a role for ATP in receptor function and offer a convenient method of studying the activation process of glucocorticoid receptor under mild assay conditions.     

A basic molecular model for the H2 histamine receptor. Part 1

A 3D model of the canine H2 receptor was built and analysed. This model was constructed using primary sequence comparisons and three-dimensional homology building with bacteriorhodopsin serving as a template. The energy analysis of the interaction between the N3H⁺ form and the N1H⁺ form of histamine with the receptor shows that both have the same binding affinity for the H2 receptor, but only the N3H⁺ form provokes structural changes. The calculated potential energies are consistent with the published binding data and suggest that Asp 98 is the principal residue for ligand recognition. On the basis of sequence alignment studies we postulate that Glu 270 in helix 7 may be important for activation of the H2 receptor. Docking studies of the N3H⁺ folded conformation in our model show that an intramolecular hydrogen bond between N3 and the amino group of the histamine molecule is broken, and the histamine then adopts a conformation similar to the N3H⁺ extended form to interact optimally with the H2 receptor. Mutations were made in the H2 receptor model to mimic published experimental point mutations. The interactions of the mutated receptor models with histamine are consistent with the experimental data.     

N-formyl peptide receptors cluster in an active raft-associated state prior to phosphorylation

In response to ligand binding, G protein-coupled receptors undergo phosphorylation and activate cellular internalization machinery. An important component of this process is the concentration of receptors into clusters on the plasma membrane. Aside from organizing the receptor in anticipation of internalization, little is known of the function of ligand-mediated G protein-coupled receptor clustering, which has traditionally been thought of as being a phosphorylation-dependent event prior to receptor internalization. We now report that following receptor activation, the N-formyl peptide receptor (FPR) forms distinct membrane clusters prior to its association with arrestin. To determine whether this clustering is dependent upon receptor phosphorylation, we used a mutant form of the FPR, DeltaST-FPR, which lacks all phosphorylation sites in the carboxyl-terminal domain. We found that activation of the signaling-competent DeltaST-FPR resulted in rapid receptor clustering on the plasma membrane independent of Gi protein activation. This clustering required receptor activation since the D71A mutant receptor, which binds ligand but is incapable of transitioning to an active state, failed to induce receptor clustering. Furthermore we demonstrated that FPR-mediated clustering and signaling were cholesterol-dependent processes, suggesting that translocation of the active receptor to lipid rafts may be required for maximal signaling activity. Finally we showed that FPR stimulation in the absence of receptor phosphorylation resulted in translocation of FPR to GM1-rich clusters. Our results demonstrate for the first time that formation of a clustered activated receptor state precedes receptor phosphorylation, arrestin binding, and internalization.     

Extracellular oxidation by taurine chloramine activates ERK via the epidermal growth factor receptor

Taurine is present in high concentrations in neutrophils, and when the cells are stimulated taurine can react with hypochlorous acid (HOCl) to form taurine-chloramine (Tau-Cl). This compound retains oxidant activity and can affect the neutrophil itself or surrounding tissue cells. We have investigated the effects of Tau-Cl on MAPK signaling in human umbilical vein endothelial cells (HUVEC). Tau-Cl caused no loss in intracellular glutathione or inactivation of the thiol-sensitive enzyme glyceraldehyde-3-phosphate dehydrogenase, indicating that it had not entered the cells. However, stimulation of HUVEC with Tau-Cl (20-100 microM) induced the rapid activation of ERK within 10 min. This activation was abolished by inhibition of MEK by U0126, indicating that it was not because of direct oxidation of ERK. No activation of p38 was detected. These results suggest that Tau-Cl reacts with a cell membrane target that results in intracellular ERK activation. Tau-Cl over the same concentration range and time scale stimulated epidermal growth factor (EGF) receptor tyrosine phosphorylation in A431 cells and HUVEC. The EGF receptor inhibitor PD158780 significantly attenuated Tau-Cl-induced phosphorylation of both the EGF receptor and ERK. This implicates the EGF receptor in the upstream activation of ERK. The Src tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine had no effect on Tau-Cl-induced EGF receptor or ERK activation. We propose that Tau-Cl acts on an oxidant-sensitive target on the cell surface, this being either the EGF receptor itself or another target that can interact with the EGF receptor, with consequential activation of ERK.     

Transformation (4S to 5S) of the nuclear estrogen receptor is reversible but not accompanied by a change in the affinity for DNA

The nuclear estrogen receptor from calf uterus was used to investigate the possible relationship between receptor transformation (4S to 5S) and receptor activation (DNA binding). Receptors extracted from nuclei after exposure of uterine tissue tc [3H]estradiol sedimented at 5.2S, the characteristic value of the transformed receptor. After storage at -20 degrees C the receptor sedimented at 4.0S, indicating conversion of the 5S form into the non-transformed 4S form. Upon reincubation at 28 degrees C the 4S form transformed into the 5S form following second-order kinetics. The rate constant obtained was 4.3 x 10(7) M-1 min-1, a value identical to that reported for the cytosol receptor. These data show that receptor transformation is reversible. Molybdate (10-50 mM) was not able to prevent receptor transformation in the nuclear extract, but was inhibitory in cytosol. This suggests that molybdate does not prevent receptor transformation, but rather inhibits disaggregation of the 8S oligomer into the 4S monomer. In DNA-binding assays (DNA-cellulose or nuclei) the non-transformed (4S) and transformed (5S) states of the nuclear estrogen receptors displayed identical affinities for DNA. The present data show that 4S to 5S transformation of nuclear receptors follows a readily reversible process, but this process is not an essential step for the exposure of the receptors' DNA-binding site. Although the physiological function of the 5S form remains unclear it may be important for the recognition of specific gene regulatory sites.     

Syntaxin 1A and Receptor for Activated C Kinase Interact with the N-Terminal Region of Human Dopamine Transporter

The dopamine transporter (DAT) regulates the extent and duration of dopamine receptor activation through sodium-dependant reuptake of dopamine into presynaptic neurons, resulting in termination of dopaminergic neurotransmission. Using the yeast two-hybrid system, we have identified novel interactions between DAT, the SNARE protein syntaxin 1A, and the receptor for activated C kinases (RACK1). This association involves the intracellular N-terminal domain of human DAT (hDAT). Our data suggest that hDAT may exist as dimers or oligomers and that its protein-protein interactions with syntaxin 1A and RACK1 form functional regulatory complexes that may mediate DAT trafficking through modulation of hDAT phosphorylation by PKC.