Localization of the strychnine binding site on the 48-kilodalton subunit of the glycine receptor

Amino acid residues that participate in antagonist binding to the strychnine-sensitive glycine receptor (GlyR) have been identified by selectively modifying functional groups with chemical reagents. Moreover, a region directly involved with strychnine binding has been localized in the 48-kDa subunit of this receptor by covalent labeling and proteolytic mapping. Modification of tyrosyl or arginyl residues promotes a marked decrease of specific [3H]strychnine binding either to rat spinal cord plasma membranes or to the purified GlyR incorporated into phospholipid vesicles. Occupancy of the receptor by strychnine, but not by glycine, completely protects from the inhibition caused by chemical reagents. Furthermore, these tyrosine- or arginine-specific reagents decrease the number of binding sites (Bmax) for [3H]strychnine binding without affecting the affinity for the ligand (Kd). These observations strongly suggest that such residues are present at, or very close to, the antagonist binding site. In order to localize the strychnine binding domain within the GlyR, purified and reconstituted receptor preparations were photoaffinity labeled with [3H]strychnine. The radiolabeled 48-kDa subunit was then digested with specific chemical proteolytic reagents, and the peptides containing the covalently bound radioligand were identified by fluorography after gel electrophoresis. N-Chlorosuccinimide treatment of [3H]strychnine-labeled 48K polypeptide yielded a single labeled peptide of Mr approximately 7300, and cyanogen bromide gave a labeled peptide of Mr 6200.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and functional analysis of phosphorylated tyrosine residues within EphA2 receptor tyrosine kinase

EphA2 is a member of the Eph family of receptor tyrosine kinases. EphA2 mediates cell-cell communication and plays critical roles in a number of physiological and pathologic responses. We have previously shown that EphA2 is a key regulator of tumor angiogenesis and that tyrosine phosphorylation regulates EphA2 signaling. To understand the role of EphA2 phosphorylation, we have mapped phosphorylated tyrosines within the intracellular region of EphA2 by a combination of mass spectrometry analysis and phosphopeptide mapping using two-dimensional chromatography in conjunction with site-directed mutagenesis. The function of these phosphorylated tyrosine residues was assessed by mutational analysis using EphA2-null endothelial cells reconstituted with EphA2 tyrosine-to-phenylalanine or tyrosine-to-glutamic acid substitution mutants. Phosphorylated Tyr(587) and Tyr(593) bind to Vav2 and Vav3 guanine nucleotide exchange factors, whereas Tyr(P)(734) binds to the p85 regulatory subunit of phosphatidylinositol 3-kinase. Mutations that uncouple EphA2 with Vav guanine nucleotide exchange factors or p85 are defective in Rac1 activation and cell migration. Finally, EphA2 mutations in the juxtamembrane region (Y587F, Y593F, Y587E/Y593E), kinase domain (Y734F), or SAM domain (Y929F) inhibited ephrin-A1-induced vascular assembly. In addition, EphA2-null endothelial cells reconstituted with these mutants were unable to incorporate into tumor vasculature, suggesting a critical role of these phosphorylation tyrosine residues in transducing EphA2 signaling in vascular endothelial cells during tumor angiogenesis.     

Mutational analysis of the cytoplasmic tail of the human transferrin receptor. Identification of a sub-domain that is required for rapid endocytosis

It has been reported that the sequence Tyr20-X-Arg-Phe23 present within the cytoplasmic tail of the transferrin receptor may represent a tyrosine internalization signal (Collawn, J.F., Stangel, M., Kuhn, L.A., Esekogwu, V., Jing, S., Trowbridge, I.S., and Tainer, J. A. (1990) Cell 63, 1061-1072). However, as Tyr20 is not conserved between species (Alvarez, E., Gironès, N., and Davis, R. J. (1990) Biochem. J. 267, 31-35), the functional role of the putative tyrosine internalization signal is not clear. To address this question, we constructed a series of 32 deletions and point mutations within the cytoplasmic tail of the human transferrin receptor. The effect of these mutations on the apparent first order rate constant for receptor endocytosis was examined. It was found that the region of the cytoplasmic tail that is proximal to the transmembrane domain (residues 28-58) is dispensable for rapid endocytosis. In contrast, the distal region of the cytoplasmic tail (residues 1-27) was found to be both necessary and sufficient for the rapid internalization of the transferrin receptor. The region identified includes Tyr20-X-Arg-Phe23, but is significantly larger than this tetrapeptide. It is therefore likely that structural information in addition to the proposed tyrosine internalization signal is required for endocytosis. To test this hypothesis, we investigated whether a heterologous tyrosine internalization signal (from the low density lipoprotein receptor) could function to cause the rapid endocytosis of the transferrin receptor. It was observed that this heterologous tyrosine internalization signal did not allow rapid endocytosis. We conclude that the putative tyrosine internalization signal (Tyr20-Thr-Arg-Phe23) is not sufficient to determine rapid endocytosis of the transferrin receptor. The data reported here indicate that the transferrin receptor internalization signal is formed by a larger cytoplasmic tail structure located at the amino terminus of the receptor.     

The unique extracellular disulfide loop of the glycine receptor is a principal ligand binding element.

A loop structure, formed by the putative disulfide bridging of Cys198 and Cys209, is a principal element of the ligand binding site in the glycine receptor (GlyR). Disruption of the loop's tertiary structure by Ser mutations of these Cys residues either prevented receptor assembly on the cell surface, or created receptors unable to be activated by agonists or to bind the competitive antagonist, strychnine. Mutation of residues Lys200, Tyr202 and Thr204 within this loop reduced agonist binding and channel activation sensitivities by up to 55-, 520- and 190-fold, respectively, without altering maximal current sizes, and mutations of Lys200 and Tyr202 abolished strychnine binding to the receptor. Removal of the hydroxyl moiety from Tyr202 by mutation to Phe profoundly reduced agonist sensitivity, whilst removal of the benzene ring abolished strychnine binding, thus demonstrating that Tyr202 is crucial for both agonist and antagonist binding to the GlyR. Tyr202 also influences receptor assembly on the cell surface, with only large chain substitutions (Phe, Leu and Arg, but not Thr, Ser and Ala) forming functional receptors. Our data demonstrate the presence of a second ligand binding site in the GlyR, consistent with the three-loop model of ligand binding to the ligand-gated ion channel superfamily.     

Role of conserved threonine and tyrosine residues in acetylcholine binding and muscarinic receptor activation. A study with m3 muscarinic receptor point mutants.

Structure-function relationship studies of the m3 muscarinic acetylcholine receptor have recently identified a series of threonine and tyrosine residues (all located within the hydrophobic receptor core) that are critically involved in acetylcholine binding (Wess, J., Gdula, D., and Brann, M.R. (1991) EMBO J. 10, 3729-3734). To gain further insight into the functional roles of these amino acids, the agonist binding properties of six rat m3 muscarinic receptor point mutants, in which the critical threonine and tyrosine residues had been individually replaced by alanine and phenylalanine, respectively, were studied in greater detail following their transient expression in COS-7 cells. The binding profiles of a series of acetylcholine derivatives suggest that the altered threonine and tyrosine residues are primarily involved in the interaction of the acetylcholine ester moiety with the receptor protein. The two m3 receptor point mutants, Thr234----Ala and Tyr506----Phe, which showed the most pronounced decreases in acetylcholine binding affinities (approximately 40-60-fold as compared with the wild-type receptor), were stably expressed in CHO cells for further functional analysis. Both mutant receptors were found to be severely impaired in their ability to stimulate agonist-dependent phosphatidylinositol hydrolysis. Consistent with this observation, acetylcholine binding to the two mutant receptors was not significantly affected by addition of the GTP analog Gpp(NH)p (5'-guanylyl imidodiphosphate). Our data suggest that Thr234 and Tyr506 (located within transmembrane domains V and VI, respectively), which are conserved among all muscarinic receptors (m1-m5), may play an important role in agonist-induced muscarinic receptor activation.     

The cytoplasmic tyrosine motifs in full-length glycoprotein 130 have different roles in IL-6 signal transduction

The function of the signal-transducing receptor subunit glycoprotein 130 (gp130) in the IL-6-receptor complex has previously been studied using carboxyl-terminal deletion mutants or a truncated molecule of approximately 60 membrane-proximal amino acids (containing box 1 and box 2) linked to the individual gp130 tyrosine motifs. However, the redundancy of the tyrosine motifs within the cytoplasmic part of gp130 has been neglected. Here we describe the analysis of the function of the individual cytoplasmic tyrosine residues of gp130 in the context of the full-length receptor protein in IL-6 signaling as measured by STAT activation, acute phase protein induction, and stimulation of proliferation. Add-back receptor mutants containing only one cytoplasmic tyrosine have been generated and tested for their efficiency in IL-6 signal transduction. Our studies revealed that tyrosine motifs which have been described to recruit STAT proteins are not equivalent with respect to their potential to activate STAT factors and acute phase protein gene promoters: the two distal tyrosines, Tyr905 and Tyr915, of gp130 were more potent than Tyr767 and Tyr814. Surprisingly, Tyr905 and Tyr915 mediate acute phase protein gene promoter activation stronger than the wild-type receptor containing all six cytoplasmic tyrosine residues. In contrast, Ba/F3 cells stably transfected with add-back receptors containing Tyr767 or Tyr905 were more sensitive to IL-6-induced proliferation than cells expressing the other add-back receptor mutants. Thus, the tyrosine residues in the cytoplasmic part of gp130 were found to contribute differentially to IL-6 signal transduction in the full- length gp130 protein.     

Helicobacter pylori CagA containing ITAM-like sequences localized to lipid rafts negatively regulates VacA-induced signaling in vivo

Background. Helicobacter pylori CagA is injected into the host cell and tyrosine‐phosphorylated. We examined tyrosine‐phosphorylation sites of CagA, as well as the function of CagA proteins in vivo and in vitro. Methods. After proteolytic digestion of CagA with lysyl endopeptidase, CagA tyrosine‐phosphorylation sites were determined using quadropolar time‐of‐flight (Q‐TOF) mass spectrometry analysis. Specific anti‐pY CagA polyclonal and anti‐CagA monoclonal antibodies were used to examine gastric mucosal biopsy specimens from H. pylori infected patients. Results. Mass spectrometry identified five crucial tyrosine‐phosphorylation sites of CagA at Tyr893, Tyr912, Tyr965, Tyr999, and Tyr1033 within the five repeated EPIYA sequences of H. pylori (NCTC11637)‐infected AGS cells. CagA protein also had an immuno‐receptor tyrosine‐based activation motif (ITAM)‐like amino acid sequences in the 3′ region of the cagA, E PIY ATI x₂₇EIY ATI , which closely resembled the ITAM. CagA proteins: (i) were localized to the 1% TritonX‐100 resistant membrane fraction (lipid rafts); (ii) formed a cluster of phosphorylated CagA protein complexes; (iii) associated with tyrosine‐phosphorylated GIT1/Cat1 (G protein‐coupled receptor kinase‐interactor 1/Cool‐associated tyrosine‐phosphorylated 1), substrate molecules of receptor type protein‐tyrosine phosphatase (RPTPζ/β), which is the receptor of VacA; and (iv) were involved in a delay and negative regulation of VacA‐induced signal. Furthermore, immunohistochemical staining of gastric mucosal biopsy specimens provided strong evidence that tyrosine‐phosphorylated CagA is found together with CagA at the luminal surface of gastric foveola in vivo. Conclusion. These findings suggest an important role for CagA containing ITAM‐like sequences in the pathogenesis of H. pylori‐related disease.     

Peroxynitrite-induced nitration of tyrosine hydroxylase: identification of tyrosines 423, 428, and 432 as sites of modification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and tyrosine-scanning mutagenesis

Tyrosine hydroxylase (TH), the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter dopamine, is inactivated by peroxynitrite. The sites of peroxynitrite-induced tyrosine nitration in TH have been identified by matrix-assisted laser desorption time-of-flight mass spectrometry and tyrosine-scanning mutagenesis. V8 proteolytic fragments of nitrated TH were analyzed by matrix-assisted laser desorption time-of-flight mass spectrometry. A peptide of 3135.4 daltons, corresponding to residues V410-E436 of TH, showed peroxynitrite-induced mass shifts of +45, +90, and +135 daltons, reflecting nitration of one, two, or three tyrosines, respectively. These modifications were not evident in untreated TH. The tyrosine residues (positions 423, 428, and 432) within this peptide were mutated to phenylalanine to confirm the site(s) of nitration and assess the effects of mutation on TH activity. Single mutants expressed wild-type levels of TH catalytic activity and were inactivated by peroxynitrite while showing reduced (30-60%) levels of nitration. The double mutants Y423F,Y428F, Y423F,Y432F, and Y428F,Y432F showed trace amounts of tyrosine nitration (7-30% of control) after exposure to peroxynitrite, and the triple mutant Y423F,Y428F,Y432F was not a substrate for nitration, yet peroxynitrite significantly reduced the activity of each. When all tyrosine mutants were probed with PEO-maleimide activated biotin, a thiol-reactive reagent that specifically labels reduced cysteine residues in proteins, it was evident that peroxynitrite resulted in cysteine oxidation. These studies identify residues Tyr(423), Tyr(428), and Tyr(432) as the sites of peroxynitrite-induced nitration in TH. No single tyrosine residue appears to be critical for TH catalytic function, and tyrosine nitration is neither necessary nor sufficient for peroxynitrite-induced inactivation. The loss of TH catalytic activity caused by peroxynitrite is associated instead with oxidation of cysteine residues.     

Hepatocyte growth factor receptor tyrosine kinase met is a substrate of the receptor protein-tyrosine phosphatase DEP-1

The receptor protein-tyrosine phosphatase (PTP) DEP-1 (CD148/PTP-eta) has been implicated in the regulation of cell growth, differentiation, and transformation, and most recently has been identified as a potential tumor suppressor gene mutated in colon, lung, and breast cancers. We have generated constructs comprising the cytoplasmic segment of DEP-1 fused to the maltose-binding protein to identify potential substrates and thereby suggest a physiological function for DEP-1. We have shown that the substrate-trapping mutant form of DEP-1 interacted with a small subset of tyrosine-phosphorylated proteins from lysates of the human breast tumor cell lines MDA-MB-231, T-47D, and T-47D/Met and have identified the hepatocyte growth factor/scatter factor receptor Met, the adapter protein Gab1, and the junctional component p120 catenin as potential substrates. Following ligand stimulation, phosphorylation of specific tyrosyl residues in Met induces mitogenic, motogenic, and morphogenic responses. When co-expressed in 293 cells, the full-length substrate-trapping mutant form of DEP-1 formed a stable complex with the chimeric receptor colony stimulating factor 1 (CSF)-Met and wild type DEP-1 dephosphorylated CSF-Met. Furthermore, we observed that DEP-1 preferentially dephosphorylated a Gab1 binding site (Tyr(1349)) and a COOH-terminal tyrosine implicated in morphogenesis (Tyr(1365)), whereas tyrosine residues in the activation loop of Met (Tyr(1230), Tyr(1234), and Tyr(1235)) were not preferred targets of the PTP. The ability of DEP-1 preferentially to dephosphorylate particular tyrosine residues that are required for Met-induced signaling suggests that DEP-1 may function in controlling the specificity of signals induced by this PTK, rather than as a simple "off-switch" to counteract PTK activity.     

Distinct signal transduction through the tyrosine-containing domains of the granulocyte colony-stimulating factor receptor.

The receptor for granulocyte colony-stimulating factor (G-CSFR) is a hemopoietic growth factor receptor, which mediates proliferation and differentiation signals. The cytoplasmic region of G-CSFR carries four tyrosine residues in its C-terminal half. We constructed mutant receptors in which each tyrosine residue of G-CSFR was mutated to phenylalanine. Two mutant receptors (Tyr703 and Tyr728) neither transduced the growth-inhibitory signal nor induced the neutrophil-specific myeloperoxidase (MPO) gene. The Tyr703 mutant did not induce morphological changes in cells, whereas transformants expressing the Tyr728 mutant adhered to plates with a macrophage-like morphology upon G-CSF stimulation. Mutation of the most distal tyrosine residue (Tyr763) abolished the ability of G-CSFR to stimulate the tyrosine phosphorylation of a cellular protein with an M(r) of 54 kDa. These results indicated that the regions around the three tyrosine residues of G-CSFR play essential and distinct roles in signal transduction.     

The role of tyrosine residues in fibroblast growth factor receptor 1 signaling in PC12 cells. Systematic site-directed mutagenesis in the endodomain.

To assess the contribution of the intracellular domain tyrosine residues to the signaling capacity of fibroblast growth factor receptor 1 (FGFR1), stably transfected chimeras bearing the ectodomain of the platelet-derived growth factor receptor (PDGFR) and the endodomain of FGFR1 were systematically altered by a tyrosine to phenylalanine bloc and individual conversions. The 15 tyrosine residues of the endodomain of this construct (PFR1) were divided into four linear segments (labeled A, B, C, and D) that contained 4, 4, 2, and 5 tyrosine residues, respectively. When stimulated by platelet-derived growth factor, derivatives in which the A, B, or A + B blocs of tyrosines were mutated were about two-thirds as active as the unmodified chimera at 48 h but achieved full activity by 96 h in a neurite outgrowth assay in transfected PC12 cells. Elimination of only the two activation loop tyrosines (C bloc) also inactivated the receptor. All derivatives in which 4 (or 5) of the D bloc tyrosines were mutated were inactive in producing differentiation but showed low levels of kinase activity in in vitro assays. Derivatives in which 1, 2, or 3 tyrosines of the D bloc in different combinations were systematically changed demonstrated that 2 residues (Tyr(677) and Tyr(701), using hFGFR1 numbering) were essential for bioactivity, but the remaining 3 residues, including Tyr(766), the previously identified site for phospholipase C gamma (PLC gamma) activation, were not. Differentiation activity was paralleled by the activation (phosphorylation) of FRS2, SOS, and ERK1/2. PLC gamma activity was dependent on the presence of Tyr(766) but also required Tyr(677) and/or Tyr(701). Although fully active chimeras did not require PLC gamma, the responses of chimeras showing reduced activation of FRS2 were significantly enhanced by this activity. These results establish that PFR1 does not utilize any tyrosine residues, phosphorylated or not, to activate FRS2. However, it does require Tyr(677) and/or Tyr(701), which may function to stabilize the active conformation directly or indirectly.     

Use of tyrosine-containing polymers to characterize the substrate specificity of insulin and other hormone-stimulated tyrosine kinases

Synthetic copolymers containing tyrosine residues were used to characterize the substrate specificity of the insulin receptor kinase and compare it to tyrosine kinases stimulated by epidermal growth factor, insulin-like growth factor-1 and phorbol ester. In partially purified receptor preparations from eight different tissues insulin best stimulated (highest V) phosphorylation of a random copolymer composed of glutamic and tyrosine residues at a 4:1 ratio (Glu/Tyr, 4:1). The insulin-stimulated phosphorylation of this polymer was highly significant also in receptor preparations from fresh human monocytes, where insulin binding and autophosphorylation were difficult to detect. Other tyrosine-containing polymers Ala/Glu/Lys/Tyr (6:2:5:1) and Glu/Ala/Tyr (6:3:1) were also phosphorylated by the insulin-stimulated kinase but to a lower extent. A tyrosine kinase stimulated by insulin-like growth factor-1, and one stimulated by phorbol ester also best phosphorylated the polymer Glu/Tyr (4:1). The three kinases differed only in their capability to phosphorylate Glu/Ala/Tyr (6:3:1) or Ala/Glu/Lys/Tyr (6:2:5:1). Glu/Tyr (4:1) was a poor substrate for the epidermal growth factor receptor kinase which best phosphorylated the polymer Glu/Ala/Tyr (6:3:1). Three additional polymers: Glu/Tyr (1:1), Glu/Ala/Tyr (1:1:1), and Lys/Tyr (1:1) failed to serve as substrates for all four tyrosine kinases tested. Taken together these findings suggest that. Hormone-sensitive tyrosine kinases have similar yet distinct substrate specificity and are likely to phosphorylate their native substrates on tyrosines adjacent to acidic (glutamic) residues. Tyrosine-containing polymer substrates are highly sensitive and convenient tools to study (hormone-sensitive) tyrosine kinases whose native substrates are unknown or present at low concentrations.     

Mapping of antigenic epitopes on the alpha 1 subunit of the inhibitory glycine receptor.

The inhibitory glycine receptor (GlyR) is a ligand-gated chloride channel protein that occurs in developmentally regulated isoforms in the vertebrate central nervous system. Monoclonal antibodies (mAbs) against the GlyR distinguish neonatal and adult GlyR proteins by identifying distinct alpha subunit variants within these receptor isoforms. Here, bacterially expressed fusion proteins of the rat GlyR alpha 1 subunit were used to localize the major antigenic epitopes of this protein within its N-terminal 105 amino acids. Synthetic peptides allowed further fine mapping of two mAb binding domains. MAb 2b, specific for the adult alpha 1 subunit, bound to a peptide corresponding to amino acids 1-10, whereas mAb 4a, which recognizes both neonatal and adult GlyR isoforms, reacted with a peptide representing residues 96-105 of the alpha 1 polypeptide. These data define unique and common antigenic epitopes on GlyR alpha subunit variants.     

Reaction of hen egg-white lysozyme with tetranitromethane: a new side reaction, oxidative bond cleavage at glycine 104, and sequential nitration of three tyrosine residues

We found that the reaction of hen egg-white lysozyme with an equimolar amount of tetranitromethane (TNM) at pH 8.0 and room temperature yielded derivatives in which the N-C bond of Gly104 is oxidatively cleaved, and a mono-nitrotyrosine lysozyme in which Tyr23 is nitrated. This bond cleavage occurred more predominantly with a decrease in the nitration of Tyr23, when the reaction was carried out under more dilute conditions. A possible mechanism in which a phenoxyl radical of Tyr 23 (an intermediate of nitration) is involved was proposed for this oxidative bond cleavage. When lysozyme was reacted with a 10 times molar excess of TNM, in addition to a mono-nitrotyrosine lysozyme in which only Try23 is nitrated, a di-nitrotyrosine lysozyme in which Tyr20 and Tyr23 are both nitrated and a tri-nitrotyrosine lysozyme in which Tyr20, Tyr23, and Tyr53 are all nitrated were obtained. However, no other possible mono- or di-nitrotyrosine lysozymes could be isolated. Thus, it is concluded that the three tyrosine residues in lysozyme are essentially nitrated sequentially with TNM in the order of Tyr23, Tyr20, and Tyr53. Since the derivatives obtained here were all active, none of the three tyrosine residues or the residues around Gly104 are considered to be very important for the lysozyme activity.     

Identification of the minimal tyrosine residues required for linker for activation of T cell function

The linker for activation of T cells (LAT) is essential for signaling through the T cell receptor (TCR). Following TCR stimulation, LAT becomes tyrosine-phosphorylated, creating docking sites for other signaling proteins such as phospholipase C-gamma(1) (PLC-gamma(1)), Grb2, and Gads. In this study, we have attempted to identify the critical tyrosine residues in LAT that mediate TCR activation-induced mobilization of intracellular Ca(2+) and activation of the MAP kinase Erk2. By using the LAT-deficient Jurkat derivative, J.CaM2, stable cell lines were established expressing various tyrosine mutants of LAT. We show that three specific tyrosine residues (Tyr(132), Tyr(171), and Tyr(191)) are necessary and sufficient to achieve a Ca(2+) flux following TCR stimulation. These tyrosine residues function by reconstituting PLC-gamma(1) phosphorylation and recruitment to LAT. However, these same tyrosines can only partially reconstitute Erk activation. Full reconstitution of Erk requires two additional tyrosine residues (Tyr(110) and Tyr(226)), both of which have the Grb2-binding motif YXN. This reconstitution of Erk activation requires that the critical tyrosine residues be on the same molecule of LAT, suggesting that a single LAT molecule nucleates multiple protein-protein interactions required for optimal signal transduction.     

Molecular determinants of ivermectin sensitivity at the glycine receptor chloride channel

Ivermectin is an anthelmintic drug that works by activating glutamate-gated chloride channel receptors (GluClRs) in nematode parasites. GluClRs belong to the Cys-loop receptor family that also includes glycine receptor (GlyR) chloride channels. GluClRs and A288G mutant GlyRs are both activated by low nanomolar ivermectin concentrations. The crystal structure of the Caenorhabditis elegans α GluClR complexed with ivermectin has recently been published. Here, we probed ivermectin sensitivity determinants on the α1 GlyR using site-directed mutagenesis and electrophysiology. Based on a mutagenesis screen of transmembrane residues, we identified Ala288 and Pro230 as crucial sensitivity determinants. A comparison of the actions of selamectin and ivermectin suggested the benzofuran C05-OH was required for high efficacy. When taken together with docking simulations, these results supported a GlyR ivermectin binding orientation similar to that seen in the GluClR crystal structure. However, whereas the crystal structure shows that ivermectin interacts with the α GluClR via H-bonds with Leu218, Ser260, and Thr285 (α GluClR numbering), our data indicate that H-bonds with residues homologous to Ser260 and Thr285 are not important for high ivermectin sensitivity or direct agonist efficacy in A288G α1 GlyRs or three other GluClRs. Our data also suggest that van der Waals interactions between the ivermectin disaccharide and GlyR M2-M3 loop residues are unimportant for high ivermectin sensitivity. Thus, although our results corroborate the ivermectin binding orientation as revealed by the crystal structure, they demonstrate that some of the binding interactions revealed by this structure do not pertain to other highly ivermectin-sensitive Cys-loop receptors.     

Identification of the insulin receptor tyrosine residues undergoing insulin-stimulated phosphorylation in intact rat hepatoma cells

Tyr(P)-containing proteins were purified from extracts of insulin-treated rat hepatoma cells (H4-II-E-C3) by antiphosphotyrosine immunoaffinity chromatography. Two major insulin-stimulated, Tyr(P) proteins were recovered: an Mr 95,000 protein (identified as the insulin receptor beta subunit by its immunoprecipitation by a patient-derived anti-insulin receptor serum and several anti-insulin receptor (peptide) antisera) and an Mr 180,000 protein (which was unreactive with all anti-insulin receptor antibodies). After purification and tryptic digestion of the Mr 95,000 protein, tryptic peptides containing Tyr(P) were purified by sequential antiphosphotyrosine immunoaffinity, reversed-phase, anion-exchange chromatography. The partial amino acid sequence obtained by gas- and solid-phase Edman degradation was compared to the amino acid sequence of the intracellular extension of the rat insulin receptor deduced from the genomic sequence. Approximately 80% of all beta subunit [32P]Tyr(P) resides on two tryptic peptides: 50-60% of [32P]Tyr(P) is found on the tryptic peptide Asp-Ile-Tyr-Glu-Thr-Asp-Tyr-Tyr-Arg from the tyrosine kinase domain, which is recovered mainly as the double phosphorylated species (predominantly in the form with Tyr(P) at residues 3 and 7 from the amino terminus; the remainder with Tyr(P) at residues 3 and 8), with 10-15% as the triple phosphorylated species. A second tryptic peptide is located near the carboxyl terminus, contains 2 tyrosines, and has the sequence, Thr-Tyr-Asp-Glu-His-Ile-Pro-Tyr-Thr-; this contains 20-30% of beta subunit [32P]Tyr(P) and is identified primarily in a double phosphorylated form. Approximately 10% of beta subunit [32P]Tyr(P) resides on an unidentified tryptic peptide of Mr 4,000-5,000. The insulin-stimulated tyrosine phosphorylation of the insulin receptor in intact rat hepatoma cells thus involves at least 6 of the 13 tyrosine residues located on the beta subunit intracellular extension. These tyrosines are clustered in several domains in a distribution virtually identical to that previously found for partially purified human insulin receptor autophosphorylated in vitro in the presence of insulin. This multisite regulatory tyrosine phosphorylation is the initial intracellular event in insulin action.     

Protein phosphatase 2A activity associated with Golgi membranes during the G2/M phase may regulate phosphorylation of ERK2

The extracellular signal-regulated kinase (ERK) 1 and 2 proteins are mitogen-activated protein kinase (MAPK) members that regulate cell proliferation and differentiation. ERK proteins are activated exclusively by MAPK kinase 1 and 2 phosphorylation of threonine and tyrosine residues located within the conserved TXY MAPK activation motif. Although dual phosphorylation of Thr and Tyr residues confers full activation of ERK, in vitro studies suggest that a single phosphorylation on either Thr or Tyr may yield partial ERK activity. Previously, we have demonstrated that phosphorylation of the tyrosine residue (Tyr(P) ERK) may be involved in regulating the Golgi complex structure during the G2 and M phases of the cell cycle (Cha, H., and Shapiro, P. (2001) J. Cell Biol. 153, 1355-1368). In the present study, we examined mechanisms for generating Tyr(P) ERK by determining cell cycle-dependent changes in localized phosphatase activity. Using fractionated nuclei-free cell lysates, we find increased serine/threonine phosphatase activity associated with Golgi-enriched membranes in cells synchronized in the late G2/early M phase as compared with G1 phase cells. The addition of phosphatase inhibitors in combination with immunodepletion assays identified this activity to be related to protein phosphatase 2A (PP2A). The increased activity was accounted for by elevated PP2A association with mitotic Golgi membranes as well as increased catalytic activity after normalization of PP2A protein levels in the phosphatase assays. These data indicate that localized changes in PP2A activity may be involved in regulating proteins involved in Golgi disassembly as cells enter mitosis.     

Trapped tyrosyl radical populations in modified reaction centers from Rhodobacter sphaeroides

The photosynthetic reaction center from the purple bacterium Rhodobacter sphaeroides has been modified such that the bacteriochlorophyll dimer, when it becomes oxidized after light excitation, is capable of oxidizing tyrosine residues. One factor in this ability is a high oxidation-reduction midpoint potential for the dimer, although the location and protein environment of the tyrosine residue appear to be critical as well. These factors were tested in a series of mutants, each of which contains changes, at residues L131, M160, M197, and M210, that give rise to a bacteriochlorophyll dimer with a midpoint potential of at least 800 mV. The protein environment was altered near tyrosine residues that are either present in the wild type or introduced by mutagenesis, focusing on residues that could act as acceptors for the phenolic proton of the tyrosine upon oxidation. These mutations include Ser M190 to His, which is near Tyr L162, the combination of His M193 to Tyr and Arg M164 to His, which adds a Tyr-His pair, and the combinations of Arg L135 to Tyr with Tyr L164 to His, Arg L135 to Tyr with Tyr L144 to Glu, and Arg L135 to Tyr with Tyr L164 to Phe. Radicals were produced in the mutants by using light to initiate electron transfer. The radicals were trapped by freezing the samples, and the relative populations of the oxidized dimer and tyrosyl radicals were determined by analysis of low-temperature electron paramagnetic resonance spectra. The mutants all showed evidence of tyrosyl radical formation at high pH, and the extent of radical formation at Tyr L135 with pH differed depending on the identity of L144 and L164. The results show that tyrosine residues within approximately 10 A of the dimer can become oxidized when provided with a suitable protein environment.