Modulation of arsenic-induced epidermal growth factor receptor pathway signalling by resveratrol

Arsenic (As) is both a human carcinogen and an effective anticancer drug. These aspects of arsenic toxicity develop as a consequence of arsenic-induced oxidative stress and modifications to signal pathway activity which alter gene expression. Resveratrol (RVL) a food antioxidant found in grapes and other fruits, exhibits anti-carcinogenic properties by reducing oxidative stress and restoring signal pathway control. This study investigated the impact of RVL on arsenite [As(III)]-induced cell signalling in HaCaT keratinocytes by assaying phosphorylation status of epidermal growth factor receptor (EGFR) signalling intermediates and measuring changes in expression of Phase II and DNA repair biomarkers. As(III) exposure produced dose-dependent toxicity which was associated with increased activation of EGFR pathway intermediates, cSrc, Rac1 and extracellular signal-regulated kinases 1 and 2 (ERK1/2). Arsenic-mediated ERK1/2 activation negatively regulated DNA polymerase beta expression and up regulated heme-oxygenase-1 at toxic concentrations. RVL treatment modulated As(III)-mediated ERK1/2 activation by shifting the balance of cSrc regulatory domain phosphorylation. These effects significantly altered the response of the EGFR pathway to growth factor-induced stimulation. Our research provides evidence that treatment with pharmacologically relevant doses of RVL influences cellular responses to As(III), largely due to RVL-mediated changes to Src and ERK1/2 activation.     

Fibroblast growth factor receptor-1 interacts with the T-cell receptor signalling pathway

Fibroblast growth factor receptors are expressed by some T cells, and provide costimulation for these cells. Such receptors allow T cells to respond to fibroblast growth factors expressed in response to injury and inflammation and may provide a mechanism for 'context-dependent' responses to antigens within the local microenvironment. The mechanisms by which fibroblast growth factor receptors might interact with the TCR signalling pathway are not defined. Here we show that the TCR and fibroblast growth factor receptors co-localize during combined stimulation. Signalling via fibroblast growth factor receptors alone results in phosphorylation of Lck and induces nuclear translocation of nuclear factors of activated T cells. Combined stimulation via fibroblast growth factor receptors and the TCR synergistically enhances the activation of nuclear factors of activated T cells. The results suggest that peptide growth factors produced at sites of injury and inflammation can contribute to the outcome of T-cell encounters with antigen.     

Identification of an agonist-induced conformational change occurring adjacent to the ligand-binding pocket of the M(3) muscarinic acetylcholine receptor

To study the conformational changes that convert G protein-coupled receptors (GPCRs) from their resting to their active state, we used the M(3) muscarinic acetylcholine receptor, a prototypical class A GPCR, as a model system. Specifically, we employed a recently developed in situ disulfide cross-linking strategy that allows the formation of disulfide bonds in Cys-substituted mutant M(3) muscarinic receptors present in their native membrane environment. At present, little is known about the conformational changes that GPCR ligands induce in the immediate vicinity of the ligand-binding pocket. To address this issue, we generated 11 Cys-substituted mutant M(3) muscarinic receptors and characterized these receptors in transfected COS-7 cells. All analyzed mutant receptors contained an endogenous Cys residue (Cys-532(7.42)) located within the exofacial segment of transmembrane domain (TM) VII, close to the agonist-binding site. In addition, all mutant receptors harbored a second Cys residue that was introduced into the exofacial segment of TM III, within the sequence Leu-142(3.27)-Asn-152(3.37). Disulfide cross-linking studies showed that muscarinic agonists, but not antagonists, promoted the formation of a disulfide bond between S151(3.36)C and Cys-532. A three-dimensional model of the inactive state of the M(3) muscarinic receptor indicated that Cys-532 and Ser-151 face each other in the center of the TM receptor core. Our cross-linking data therefore support the concept that agonist activation pulls the exofacial segments of TMs VII and III closer to each other. This structural change may represent one of the early conformational events triggering the more pronounced structural reorganization of the intracellular receptor surface. To the best of our knowledge, this is the first direct demonstration of a conformational change occurring in the immediate vicinity of the binding site of a GPCR activated by a diffusible ligand.     

Expression of a glycosylphosphatidylinositol-anchored ligand,growth hormone,blocks receptor signalling

We have investigated the interaction between GH (growth hormone) and GHR (GH receptor). We previously demonstrated that a truncated GHR that possesses a transmembrane domain but no cytoplasmic domain blocks receptor signalling. Based on this observation we investigated the impact of tethering the receptor''s extracellular domain to the cell surface using a native lipid GPI (glycosylphosphatidylinositol) anchor. We also investigated the effect of tethering GH, the ligand itself, to the cell surface and demonstrated that tethering either the ecGHR (extracellular domain of GHR) or the ligand itself to the cell membrane via a GPI anchor greatly attenuates signalling. To elucidate the mechanism for this antagonist activity, we used confocal microscopy to examine the fluorescently modified ligand and receptor. GH–GPI was expressed on the cell surface and formed inactive receptor complexes that failed to internalize and blocked receptor activation. In conclusion, contrary to expectation, tethering an agonist to the cell surface can generate an inactive hormone receptor complex that fails to internalize.     

Ligand-induced conformational change in the minimized insulin receptor

Within the class of insulin and insulin-like growth factor receptors, detailed information about the molecular recognition event at the hormone-receptor interface is limited by the absence of suitable co-crystals. We describe the use of a biologically active insulin derivative labeled with the NBD fluorophore (B29NBD-insulin) to characterize the mechanism of reversible 1:1 complex formation with a fragment of the insulin receptor ectodomain. The accompanying 40 % increase in the fluorescence quantum yield of the label provides the basis for a dynamic study of the hormone-receptor binding event. Stopped-flow fluorescence experiments show that the kinetics of complex formation are biphasic comprising a bimolecular binding event followed by a conformational change. Displacement with excess unlabeled insulin gave monophasic kinetics of dissociation. The rate data are rationalized in terms of available experiments on mutant receptors and the X-ray structure of a non-binding fragment of the receptor of the homologous insulin-like growth factor (IGF-1).     

Insulin inhibits growth hormone signaling via the growth hormone receptor/JAK2/STAT5B pathway

Insulin is important for maintaining the responsiveness of the liver to growth hormone (GH). Insulin deficiency results in a decrease in liver GH receptor (GHR) expression, which can be reversed by insulin administration. In osteoblasts, continuous insulin treatment decreases the fraction of cellular GHR localized to the plasma membrane. Thus, it is not clear whether hyperinsulinemia results in an enhancement or inhibition of GH action. We asked whether continuous insulin stimulation, similar to what occurs in hyperinsulinemic states, results in GH resistance. Our present studies suggest that insulin treatment of hepatoma cells results in a time-dependent inhibition of acute GH-induced phosphorylation of STAT5B. Whereas total protein levels of JAK2 were not reduced after insulin pretreatment for 16 h, GH-induced JAK2 phosphorylation was inhibited. There was a concomitant decrease in GH binding and a reduction in immunoreactive GHR levels following pretreatment with insulin for 8-24 h. In summary, continuous insulin treatment in rat H4 hepatoma cells reduces GH binding, immunoreactive GHR, GH-induced phosphorylation of JAK2, and GH-induced tyrosine phosphorylation of STAT5B. These findings suggest that hepatic GH resistance may develop when a patient exhibits chronic hyperinsulinemia, a condition often observed in patients with obesity and in the early stage of Type 2 diabetes.     

An overview of the Notch signalling pathway

The Notch receptor plays a key role in modulating cell fate decisions throughout the development of invertebrate and vertebrate species. Four Notch homologues have been identified in the human genome and aberrant Notch function has been associated with a number of human diseases. An intriguing pathway of Notch signalling has been elucidated involving multiple proteolytic steps and this pathway offers several targets for potential therapeutic intervention. While a consensus model has emerged, in the details there is much that is contentious. This review will primarily focus on our current understanding of the proteolytic events involved in generating and regulating Notch signalling.     

An actin-dependent conformational change in myosin

Conformational changes within myosin lead to its movement relative to an actin filament. Several crystal structures exist for myosin bound to various nucleotides, but none with bound actin. Therefore, the effect of actin on the structure of myosin is poorly understood. Here we show that the swing of smooth muscle myosin lever arm requires both ADP and actin. This is the first direct observation that a conformation of myosin is dependent on actin. Conformational changes within myosin were monitored using fluorescence resonance energy transfer techniques. A cysteine-reactive probe is site-specifically labeled on a 'cysteine-light' myosin variant, in which the native reactive cysteines were removed and a cysteine engineered at a desired position. Using this construct, we show that the actin-dependent ADP swing causes an 18 A change in distance between a probe on the 25/50 kDa loop on the catalytic domain and a probe on the regulatory light chain, corresponding to a 23 degrees swing of the light-chain domain.     

Epidermal growth factor binding induces a conformational change in the external domain of its receptor.

To study the properties of the extracellular epidermal growth factor (EGF) binding domain of the human EGF receptor, we have infected insect cells with a suitably engineered baculovirus vector containing the cDNA encoding the entire ectodomain of the parent molecule. This resulted in a correctly folded, stable, 110 kd protein which possessed an EGF binding affinity of 200 nM. The protein was routinely purified in milligram amounts from 1 litre insect cell cultures using a series of three standard chromatographic steps. The properties of the ectodomain were studied before and after the addition of different EGF ligands, using both circular dichroism and fluorescence spectroscopic techniques. A secondary structural analysis of the far UV CD spectrum of the ectodomain indicated significant proportions of alpha-helix and beta-sheet in agreement with a published model of the EGF receptor. The ligand additions to the receptor showed differences in both the near- and far-UV CD spectra, and were similar for each ligand used, suggesting similar conformational differences between uncomplexed and complexed receptor. Steady-state fluorescence measurements indicated that the tryptophan residues present in the ectodomain are buried and that the solvent-accessible tryptophans in the ligands become buried on binding the receptor. The rotational correlation times measured by fluorescence anisotropy decay for the receptor-ligand complexes were decreased from 6 to 2.5 ns in each case. This may indicate a perturbation of the tryptophan environment of the receptor on ligand binding. Ultracentrifugation studies showed that no aggregation occurred on ligand addition, so this could not explain the observed differences from CD or fluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)     

pH-induced conformational states of bovine growth hormone

The folding behavior of bovine growth hormone (bGH) is examined by chemical and pH denaturation using several spectroscopic probes of protein secondary and tertiary structure. Partially denaturing concentrations of urea eliminate the native-state quenching of intrinsic tryptophan fluorescence, from the single protein tryptophan, but the fluorescence emission spectrum is not red-shifted like the unfolded state, and the protein retains substantial secondary structure. A neutral-to-acid pH shift also eliminates tryptophan quenching; however, the loss of quenching is not accompanied by an emission red-shift. In addition, the protein undergoes a pH-dependent UV absorbance transition; the changes in absorptivity have the same midpoint as the transition associated with the change in intrinsic tryptophan fluorescence. The magnitude of the absorption transition is similar to that observed previously for urea denaturation of the protein. In a similar fashion, a pH-dependent CD transition is also observed; however, the transition occurs at a higher pH. The behavior of the various optical probes indicates that the pH-induced conformational transition produces a highly populated species in which the microenvironment surrounding the single protein tryptophan residue resembles that observed during the urea-induced unfolding/refolding transition. The pH-induced changes in tertiary structure occur at a lower pH than the changes associated with a portion of the secondary structure. Proton NMR of the low-pH intermediate indicates that the three His and six Tyr resonances are indistinguishable from the unfolded state. The intermediate(s) observed by either chemical or pH-induced denaturation resemble(s) a molten globule state which contains significant secondary structure. The residual secondary structure present in the intermediate could be nonnative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Compartmentalization of growth factor receptor signalling

Spatial and temporal separation of signal transduction pathways often determines the specificity in cellular responses. Recent advances have improved our understanding of how growth factor signalling is influenced by the formation of molecular complexes (signalosomes) in distinct cellular compartments. There has also been new insight into the mechanisms that determine the signalling competence of these complexes and their role in receptor endocytosis, retrograde trafficking in neurons and restricted protein biosynthesis, and many examples have been found where signalosome deregulation leads to disease.     

Hormone-induced conformational change of the purified soluble hormone binding domain of follitropin receptor complexed with single chain follitropin

Human follicle-stimulating hormone receptor (hFSHR) belongs to family I of G protein-coupled receptors. FSHR extracellular domain (ECD) is predicted to have 8-9 alphabeta or leucine-rich repeat motif elements. The objective of this study was to identify elements of the FSHR ECD involved in ligand binding. Preincubation of recombinant hFSHR ECD with rabbit antisera raised against synthetic peptides of hFSHR ECD primary sequence abolished follitropin binding primarily in the region of amino acids 150-254. Accessibility of hFSHR ECD after hormone binding, captured by monoclonal antibodies against either ECD or FSH, was decreased for the region of amino acids 150-220 but additionally for amino acids 15-100. Thus, when hFSH bound first, accessibility of antibody binding was decreased to a much larger extent than if antibody was bound first. This suggestion of a conformational change upon binding was examined further. Circular dichroism spectra were recorded for purified single chain hFSH, hFSHR ECD, and hFSHR ECD-single chain hFSH complex. A spectral change indicated a small but consistent conformational change in the ECD.FSH complex after hormone binding. Taken together, these data demonstrate that FSH binding requires elements within the leucine-rich repeat motifs that form a central region of hFSHR ECD, and a conformational change occurs upon hormone binding.     

Ghrelin and the growth hormone secretagogue receptor constitute a novel autocrine pathway in astrocytoma motility

Originally thought of as a stomach-derived endocrine peptide acting via its receptors in the central nervous system to stimulate food intake and growth hormone expression, ghrelin and its receptor (growth hormone secretagogue receptor (GHS-R)) are widely expressed in a number of organ systems, including cancer cells. However, the direct functional role of ghrelin and its receptor in tumors of central nervous system origin remains to be defined. Here, we demonstrate that the human astrocytoma cell lines U-118, U-87, CCF-STTG1, and SW1088 express 6-, 11-, 15-, and 29-fold higher levels of GHS-R compared with primary normal human astrocytes. The ligation of GHS-R by ghrelin on these cells resulted in an increase in intracellular calcium mobilization, protein kinase C activation, actin polymerization, matrix metalloproteinase-2 activity, and astrocytoma motility. In addition, ghrelin led to actin polymerization and membrane ruffling on cells, with the specific co-localization of the small GTPase Rac1 with GHS-R on the leading edge of the astrocytoma cells and imparting the tumor cells with a motile phenotype. Disruption of the endogenous ghrelin/GHS-R pathway by RNA interference resulted in diminished motility, matrix metalloproteinase activity, and Rac expression, whereas tumor cells stably overexpressing GHS-R exhibited increased cell motility. The relevance of ghrelin and GHS-R expression was verified in clinically relevant tissues from 20 patients with oligodendrogliomas and grade II-IV astrocytomas. Analysis of a central nervous system tumor tissue microarray revealed that strong GHS-R and ghrelin expression was significantly more common in high grade tumors compared with low grade ones. Together, these findings suggest a novel role for the ghrelin/GHS-R axis in astrocytoma cell migration and invasiveness of cancers of central nervous system origin.     

Luteinizing hormone signaling in preovulatory follicles involves early activation of the epidermal growth factor receptor pathway

LH activates a cascade of signaling events that are propagated throughout the ovarian preovulatory follicle to promote ovulation of a mature egg. Critical to LH-induced ovulation is the induction of epidermal growth factor (EGF)-like growth factors and transactivation of EGF receptor (EGFR) signaling. Because the timing of this transactivation has not been well characterized, we investigated the dynamics of LH regulation of the EGF network in cultured follicles. Preovulatory follicles were cultured with or without recombinant LH and/or specific inhibitors. EGFR and MAPK phosphorylation were examined by immunoprecipitation and Western blot analyses. By semiquantitative RT-PCR, increases in amphiregulin and epiregulin mRNAs were detected 30 min after recombinant LH stimulation of follicles and were maximal after 2 h. LH-induced EGFR phosphorylation also increased after 30 min and reached a maximum at 2 h. EGFR activation precedes oocyte maturation and is cAMP dependent, because forskolin similarly activated EGFR. LH-induced EGFR phosphorylation was sensitive to AG1478, an EGFR kinase inhibitor, and to inhibitors of matrix metalloproteases GM6001 and TNFalpha protease inhibitor-1 (TAPI-1), suggesting the involvement of EGF-like growth factor shedding. LH- but not amphiregulin-induced oocyte maturation and EGFR phosphorylation were sensitive to protein synthesis inhibition. When granulosa cells were cultured with a combination of neutralizing antibodies against amphiregulin, epiregulin, and betacellulin, EGFR phosphorylation and MAPK activation were inhibited. In cultured follicles, LH-induced MAPK activation was partially inhibited by AG1478 and GM6001, indicating that this pathway is regulated in part by the EGF network but also involves additional pathways. Thus, complex mechanisms are involved in the rapid amplification and propagation of the LH signal within preovulatory follicles and include the early activation of the EGF network.     

Muscarinic receptor activity change after prolonged treatment with growth hormone and somatostatin

1. The effect of 10-day treatment with growth hormone (GH) (1 mg/kg body weight day) and somatostatin (SRIF) (0.25 mg/kg body weight day) subcutaneously on the activity of muscarinic (M) receptors in rat hypothalamic, pituitary and cerebral cortical membrane fractions was studied using (3H)quinuclidinyl benzylate [(3H)QNB] as radioligand. 2. The administration of GH and SRIF significantly decreased the M-receptor binding affinity in the hypothalamus. 3. In the pituitary the M-receptor affinity was increased after both GH and SRIF treatment. 4. In the hypothalamus and the pituitary the binding capacity of muscarinic receptors was unchanged. 5. In the cerebral cortex the chronical GH injection induced an increase in the number of antagonist binding sites and a decrease of their affinity, while the similar SRIF treatment led to an increase of the binding affinity without any change of M-receptor capacity. 6. These results indicate that GH and SRIF selectively and region-specifically modulate muscarinic receptor binding affinity and capacity and provide new insight into the feedback regulatory mechanisms of GH secretion.     

Linkage of the ubiquitin-conjugating system and the endocytic pathway in ligand-induced internalization of the growth hormone receptor.

The major function of the ubiquitin-conjugating system is the targeting of cytosolic and nuclear proteins for degradation by the proteasome. Recently, ubiquitin conjugation has been implicated in the downregulation of signalling receptors such as the mammalian growth hormone receptor (GHR) and the alpha-factor receptor in yeast. By examining truncated receptors, the internalization-deficient receptor mutant F327A and conditions under which clathrin-mediated GHR endocytosis is inhibited, we show here that GHR ubiquitination and ligand-induced GHR internalization are coupled events. Previously, we had shown that GHR endocytosis is dependent on an intact ubiquitination system. Here we present evidence that GHR ubiquitination depends on an intact endocytic pathway. Our data indicate that the ubiquitin-conjugating system and the endocytic pathway interact at the cytoplasmic tail of the GHR at the plasma membrane, where they cooperate to regulate internalization of the GHR.     

Molecular recognition events involved in the activation of the growth hormone receptor by growth hormone

Binding of growth hormone (GH) to its receptor (GHR) is a well-studied example of molecular recognition between a cytokine and its receptor. Extensive mutagenesis studies and several crystal structures have defined the key interactive amino acid residues that are involved in binding and subsequent receptor dimerization. This review encompasses each of the three molecular recognition events involved in GHR activation, namely binding of GH to its two receptors and the interactions that occur between these receptors. Particular attention is given to species and ligand specificity of hormone binding and to the molecular recognition events involved in receptor activation, including the possibility that a conformational change in the receptor is required.