A single mutation in arrestin-2 prevents ERK1/2 activation by reducing c-Raf1 binding

Arrestins regulate the signaling and trafficking of G protein-coupled receptors (GPCRs). GPCR complexes with both nonvisual arrestins channel signaling to G protein-independent pathways, one of which is the activation of extracellular signal regulated kinase 1/2 (ERK1/2). Here we used alanine-scanning mutagenesis of residues on the nonreceptor-binding surface conserved between arrestin-2 and arrestin-3. We show that an Arg307Ala mutation significantly reduced arrestin-2 binding to c-Raf1, whereas the binding of the mutant to active phosphorylated receptor and downstream kinases MEK1 and ERK2 was not affected. In contrast to wild-type arrestin-2, the Arg307Ala mutant failed to rescue arrestin-dependent ERK1/2 activation via β2-adrenergic receptor in arrestin-2/3 double knockout mouse embryonic fibroblasts. Thus, Arg307 plays a specific role in arrestin-2 binding to c-Raf1 and is indispensable in the productive scaffolding of c-Raf1-MEK1-ERK1/2 signaling cascade. Arg307Ala mutation specifically eliminates arrestin-2 signaling through ERK, which makes arrestin-2-Arg307Ala the first signaling-biased arrestin mutant constructed. In the crystal structure the side chain of homologous arrestin-3 residue Lys308 points in a different direction. Alanine substitution of Lys308 does not significantly affect c-Raf1 binding to arrestin-3 and its ability to promote ERK1/2 activation, suggesting that the two nonvisual arrestins perform the same function via distinct molecular mechanisms.     

Both Th1 and Th17 are immunopathogenic but differ in other key biological activities

The role of Th17 lymphocytes in immunopathogenic processes has been well established, but little is known about their basic cell features. In this study, we compared polarized Th1 and Th17 for key biological activities related to pathogenicity and trafficking. Th1 and Th17 lineages were derived from TCR-transgenic CD4 murine cells specific against hen egg lysozyme. When adoptively transferred into mice expressing hen egg lysozyme in their eyes, both Th1 and Th17 induced ocular inflammation but with slight differences in histological pathology. PCR analysis revealed selective expression of IFN-gamma or IL-17 in eyes of Th1 or Th17 recipients, respectively. Additionally, Th1 and Th17 were found to differ in three other key activities: 1) Th17 cells were inferior to Th1 cells in their capacity to trigger massive lymphoid expansion and splenomegaly; 2) the proportion of Th1 cells among infiltrating cells in inflamed recipient eyes declined rapidly, becoming a minority by day 7, whereas Th17 cells remained in the majority throughout this period; and 3) remarkable differences were noted between Th1 and Th17 cells in their expression of certain surface markers. In particular, reactivated Th1 expressed higher levels of CD49d and alpha(4)beta(7) (mucosal homing) in vitro and higher levels of CXCR3 (Th1 trafficking) in vivo. Reactivated Th17, however, expressed higher levels of alpha(E)beta(7) (epithelial tissue homing) and CD38 (activation, maturation and trafficking) in vitro, but in vivo Th17 expressed higher levels of alpha(4)beta(7) and CCR6 (lymphocyte trafficking). These data reveal that Th1 and Th17 cells differ in several key biological activities influencing migration and pathogenic behavior during inflammatory disease.     

Constitutively active mutants of the alpha 1B-adrenergic receptor: role of highly conserved polar amino acids in receptor activation.

Site-directed mutagenesis and molecular dynamics simulations of the alpha 1B-adrenergic receptor (AR) were combined to explore the potential molecular changes correlated with the transition from R (inactive state) to R (active state). Using molecular dynamics analysis we compared the structural/dynamic features of constitutively active mutants with those of the wild type and of an inactive alpha 1B-AR to build a theoretical model which defines the essential features of R and R. The results of site-directed mutagenesis were in striking agreement with the predictions of the model supporting the following hypothesis. (i) The equilibrium between R and R depends on the equilibrium between the deprotonated and protonated forms, respectively, of D142 of the DRY motif. In fact, replacement of D142 with alanine confers high constitutive activity to the alpha 1B-AR. (ii) The shift of R143 of the DRY sequence out of a conserved 'polar pocket' formed by N63, D91, N344 and Y348 is a feature common to all the active structures, suggesting that the role of R143 is fundamental for mediating receptor activation. Disruption of these intramolecular interactions by replacing N63 with alanine constitutively activates the alpha 1B-AR. Our findings might provide interesting generalities about the activation process of G protein-coupled receptors.     

Role of phosphorylated aminoacyl residues in generating atypical consensus sequences which are recognized by casein kinase-2 but not by casein kinase-1.

Casein kinase-2 (CK-2) is a ubiquitous Ser/Thr specific protein kinase that recognizes phosphorylatable residues located upstream of acidic determinants, its consensus sequence being Ser(Thr)-Xaa-Xaa-Acidic. Here we show that the phosphotetrapeptide AcSer(P)-Ser(P)-Ser-Ser(P), which is devoid of the canonical consensus sequence, is nevertheless phosphorylated by CK-2 with rates comparable to that of typical peptide substrates Ser-Glu-Glu-Glu-Glu-Glu and Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu routinely employed for assaying CK-2 activity. The phosphopeptide AcSer(P)-Ser-Ser(P) [but not Ac-Ser-Ser(P)-Ser(P) or AcSer(P)-Ser(P)-Ser] is also phosphorylated albeit less efficiently than AcSer(P)-Ser(P)-Ser-Ser(P). Further N-terminal elongation with additional phosphoseryl residues to give the peptides AcSer(P)-Ser(P)-Ser(P)-Ser-Ser(P) and AcSer(P)-Ser(P)-Ser(P)-Ser(P)-Ser-Ser(P) does not improve but rather slightly decreases the phosphorylation efficiency by CK-2. These two peptides are conversely excellent substrates for CK-1, which does not appreciably phosphorylate either AcSer(P)-Ser-Ser(P) or AcSer-(P)-Ser(P)-Ser-Ser(P). Either individual or multiple replacement of the phosphorylated residues with glutamic acid in the peptide AcSer(P)-Ser(P)-Ser-Ser(P) drastically reduces the phosphorylation efficiency by CK-2, the phosphoseryl residue at position -2 playing an especially crucial role which cannot be surrogated by glutamyl residues.(ABSTRACT TRUNCATED AT 250 WORDS)     

GUCY2D mutations in retinal guanylyl cyclase 1 provide biochemical reasons for dominant cone–rod dystrophy but not for stationary night blindness

Mutations in the GUCY2D gene coding for the dimeric human retinal membrane guanylyl cyclase (RetGC) isozyme RetGC1 cause various forms of blindness, ranging from rod dysfunction to rod and cone degeneration. We tested how the mutations causing recessive congenital stationary night blindness (CSNB), recessive Leber''s congenital amaurosis (LCA1), and dominant cone–rod dystrophy-6 (CORD6) affected RetGC1 activity and regulation by RetGC-activating proteins (GCAPs) and retinal degeneration-3 protein (RD3). CSNB mutations R666W, R761W, and L911F, as well as LCA1 mutations R768W and G982VfsX39, disabled RetGC1 activation by human GCAP1, -2, and -3. The R666W and R761W substitutions compromised binding of GCAP1 with RetGC1 in HEK293 cells. In contrast, G982VfsX39 and L911F RetGC1 retained the ability to bind GCAP1 in cyto but failed to effectively bind RD3. R768W RetGC1 did not bind either GCAP1 or RD3. The co-expression of GUCY2D allelic combinations linked to CSNB did not restore RetGC1 activity in vitro. The CORD6 mutation R838S in the RetGC1 dimerization domain strongly dominated the Ca²⁺ sensitivity of cyclase regulation by GCAP1 in RetGC1 heterodimer produced by co-expression of WT and the R838S subunits. It required higher Ca²⁺ concentrations to decelerate GCAP-activated RetGC1 heterodimer—6-fold higher than WT and 2-fold higher than the Ser⁸³⁸-harboring homodimer. The heterodimer was also more resistant than homodimers to inhibition by RD3. The observed biochemical changes can explain the dominant CORD6 blindness and recessive LCA1 blindness, both of which affect rods and cones, but they cannot explain the selective loss of rod function in recessive CSNB.     

Repeated phosphopeptide motifs in human Claspin are phosphorylated by Chk1 and mediate Claspin function

Claspin is a checkpoint protein involved in ATR (ataxia telangiectasia mutated- and Rad3-related)-dependent Chk1 activation in Xenopus and human cells. In Xenopus, Claspin interacts with Chk1 after DNA damage through a region containing two highly conserved repeats, which becomes phosphorylated during the checkpoint response. Because this region is also conserved in human Claspin, we investigated the regulation and function of these potential phosphorylation sites in human Claspin. We found that Claspin is phosphorylated in vivo at Thr-916 in response to replication stress and UV damage. Mutation of these phosphorylation sites on Claspin inhibited Claspin-Chk1 interaction in vivo, impaired Chk1 activation, and induced premature chromatin condensation in cells, indicating a defect in replication checkpoint. In addition, we found that Thr-916 on Claspin is phosphorylated by Chk1, suggesting that Chk1 regulates Claspin during checkpoint response. These results together indicate that phosphorylation of Claspin repeats in human Claspin is important for Claspin function and the regulation of Claspin-Chk1 interaction in human cells.     

Constitutively active mutants of the alpha(1a)- and the alpha(1b)-adrenergic receptor subtypes reveal coupling to different signaling pathways and physiological responses in rat cardiac myocytes

Activation of alpha(1)-adrenergic receptors influences both the contractile activity and the growth potential of cardiac myocytes. However, the signaling pathways linking activation of specific alpha(1)-adrenergic receptor (AR) subtypes to these physiological responses remain controversial. In the present study, a molecular approach was used to identify conclusively the signaling pathways activated in response to the individual alpha(1A)- and alpha(1B)-AR subtypes in cardiac myocytes. For this purpose, a mutant alpha(1a)-AR subtype (alpha(1a)-S(290/293)-AR) was constructed based on analogy to the previously described constitutively active mutant alpha(1b)-AR subtype (alpha(1b)-S(288-294)-AR). The mutant alpha(1a)-S(290/293)-AR subtype displayed constitutive activity based on four criteria. To introduce the constitutively active alpha(1)-AR subtypes into cardiac myocytes, recombinant Sindbis viruses encoding either the alpha(1a)-S(290/293)-AR or alpha(1b)-S(288-294)-AR subtype were used to infect the whole cell population with >90% efficiency, thereby allowing the biochemical activities of the various signaling pathways to be measured. When expressed at comparable levels, the alpha(1a)-S(290/293)-AR subtype exhibited a significantly elevated basal level as well as agonist-stimulated level of inositol phosphate accumulation, coincident with activation of atrial natriuretic factor-luciferase gene expression. By contrast, the alpha(1b)-S(288-294)-AR subtype displayed a markedly increased serum response element-luciferase gene expression but no activation of atrial natriuretic factor-luciferase gene expression. Taken together, this study provides the first molecular evidence for coupling of the alpha(1a)-AR and the alpha(1b)-AR subtypes to different signaling pathways in cardiac myocytes.     

Formation of tight junction strands by expression of claudin-1 mutants in their ZO-1 binding site in MDCK cells

To investigate the formation mechanism of tight junctions (TJs), we constructed three claudin-1 mutants which varied in their COOH-termini and expressed them in MDCK cells under the control of doxycycline. The differences between these constructs are that a putative ZO-1 binding sequence (KDYV) at the COOH-terminus of claudin-1 was deleted (DeltaCmyc) or present (1CLmyc and DeltaCmycYV), or that a myc-epitope was added at the COOH-terminus (1CLmyc and DeltaCmyc) or inserted just before the KDYV sequence (DeltaCmycYV). All three constructs caused the formation of aberrant TJ strands along the lateral plasma membranes. However, when their expression levels were reduced by adding 0.2 ng/ml doxycycline, they were located at apical TJs and colocalized with ZO-1, even in the KDYV-deleted construct. These results suggest that, although the addition of the myc-epitope at or near the COOH-terminus of claudin-1 interfered with the binding to ZO-1 and induced aberrant TJ strand formation, endogenous claudins which could bind to ZO-1 might recruit these deformed claudin-1s expressed at a low level to apical TJs. A calcium switch assay revealed that claudin-1 was transported to cadherin-based cell-cell contacts where ZO-1 had already accumulated, and was then concentrated at apical TJs together with ZO-1. Crosslinking between claudin-1 and the perijunctional actin ring through ZO-1 may be necessary for TJ strands to be localized or retained at apical TJs.     

HDLs in apoA-I transgenic Abca1 knockout mice are remodeled normally in plasma but are hypercatabolized by the kidney

Patients homozygous for Tangier disease have a near absence of plasma HDL as a result of mutations in ABCA1 and hypercatabolize normal HDL particles. To determine the relationship between ABCA1 expression and HDL catabolism, we investigated intravascular remodeling, plasma clearance, and organ-specific uptake of HDL in mice expressing the human apolipoprotein A-I (apoA-I) transgene in the Abca1 knockout background. Small HDL particles (7.5 nm), radiolabeled with (125)I-tyramine cellobiose, were injected into recipient mice to quantify plasma turnover and the organ uptake of tracer. Small HDL tracer was remodeled to 8.2 nm diameter particles within 5 min in human apolipoprotein A-I transgenic (hA-I(Tg)) mice (control) and knockout mice. Decay of tracer from plasma was 1.6-fold more rapid in knockout mice (P < 0.05) and kidney uptake was twice that of controls, with no difference in liver uptake. We also observed 2-fold greater hepatic expression of ABCA1 protein in hA-I(Tg) mice compared with nontransgenic mice, suggesting that overexpression of human apoA-I stabilized hepatic ABCA1 protein in vivo. We conclude that ABCA1 is not required for in vivo remodeling of small HDLs to larger HDL subfractions and that the hypercatabolism of normal HDL particles in knockout mice is attributable to a selective catabolism of HDL apoA-I by the kidney.     

Selection of beta-lactamases and penicillin binding mutants from a library of phage displayed TEM-1 beta-lactamase randomly mutated in the active site omega-loop

A combinatorial library of mutants of the phage displayed TEM-1 lactamase was generated in the region encompassing residues 163 to 171 of the active site Omega-loop. Two in vitro selection protocols were designed to extract from the library phage-enzymes characterised by a fast acylation by benzyl-penicillin (PenG) to yield either stable or very unstable acyl-enzymes. The critical step of the selections was the kinetically controlled labelling of the phages by reaction with either a biotinylated penicillin derivative or a biotinylated penicillin sulfone, i.e. a beta-lactamase suicide substrate; the biotinylated phages were recovered by panning on immobilised streptavidin. As labelling with biotinylated suicide substrates tends to select enzymes that do not turnover, a counter-selection against penicillin binding mutants was introduced to extract the beta-lactamases. The selected phage-enzymes were characterised by sequencing to identify conserved residues and by kinetic analysis of the reaction with benzyl-penicillin. Several penicillin binding mutants, in which the essential Glu166 is replaced by Asn, were shown to be acylated very fast by PenG, the acylation being characterised by biphasic kinetics. These data are interpreted by a kinetic scheme in which the enzymes exist in two interconvertible conformations. The rate constant of the conformational change suggests that it involves an isomerisation of the peptide bond between residues 166 and 167 and controls a conformation of the Omega-loop compatible with fast acylation of the active site serine residue.     

Cell division defects of Schizosaccharomyces pombe liz1- mutants are caused by defects in pantothenate uptake

The liz1⁺ gene of the fission yeast Schizosaccharomyces pombe was previously identified by complementation of a mutation that causes abnormal mitosis when ribonucleotide reductase is inhibited. Liz1 has similarity to transport proteins from Saccharomyces cerevisiae, but the potential substrate and its connection to the cell division cycle remain elusive. We report here that liz1⁺ encodes a plasma membrane-localized active transport protein for the vitamin pantothenate, the precursor of coenzyme A (CoA). Liz1 is required for pantothenate uptake at low extracellular concentrations. A lack of pantothenate uptake results in three phenotypes: (i) slow growth, (ii) delayed septation, and (iii) aberrant mitosis in the presence of hydroxyurea (HU). All three phenotypes are suppressed by high extracellular concentrations of pantothenate, where pantothenate uptake occurs by passive diffusion. liz1Δ mutants are viable because they can synthesize pantothenate from uracil as an endogenous source. The use of uracil for both pantothenate biosynthesis and deoxyribonucleotide generation provides an explanation for the aberrant mitosis in the presence of HU. HU blocks ribonucleotide reductase, and we propose that the accumulation of ribonucleotides reduces uracil biosynthesis by feedback inhibition of aspartate transcarbamoylase. Thus, the addition of HU to liz1Δ mutants results in a shortage of pantothenate. Because liz1Δ mutants show striking similarities to mutants with defects in fatty acid biosynthesis, we propose that the shortage of pantothenate compromises fatty acid synthesis, resulting in slow growth and mitotic defects.     

YXXL motifs in SH2-Bbeta are phosphorylated by JAK2, JAK1, and platelet-derived growth factor receptor and are required for membrane ruffling

SH2-Bbeta binds to the activated form of JAK2 and various receptor tyrosine kinases. It is a potent stimulator of JAK2, is required for growth hormone (GH)-induced membrane ruffling, and increases mitogenesis stimulated by platelet-derived growth factor (PDGF) and insulin-like growth factor I. Its domain structure suggests that SH2-Bbeta may act as an adapter protein to recruit downstream signaling proteins to kinase.SH2-Bbeta complexes. SH2-Bbeta is tyrosyl-phosphorylated in response to GH and interferon-gamma, stimulators of JAK2, as well as in response to PDGF and nerve growth factor. To begin to elucidate the role of tyrosyl phosphorylation in the function of SH2-Bbeta, we used phosphopeptide mapping, mutagenesis, and a phosphotyrosine-specific antibody to identify Tyr-439 and Tyr-494 in SH2-Bbeta as targets of JAK2 both in vitro and in intact cells. SH2-Bbeta lacking Tyr-439 and Tyr-494 inhibits GH-induced membrane ruffling but still activates JAK2. We provide evidence that JAK1, like JAK2, phosphorylates Tyr-439 and Tyr-494 in SH2-Bbeta and that PDGF receptor phosphorylates SH2-Bbeta on Tyr-439. Therefore, phosphorylated Tyr-439 and/or Tyr-494 in SH2-Bbeta may provide a binding site for one or more proteins linking cytokine receptor.JAK2 complexes and/or receptor tyrosine kinases to the actin cytoskeleton.     

HIV-1-specific CD4+ T cells are detectable in most individuals with active HIV-1 infection, but decline with prolonged viral suppression

The role of HIV-1-specific CD4+ T-cell responses in controlling HIV-1 infection remains unclear. Previous work has suggested that such cells are eliminated in the early stages of infection in most subjects, and thus cannot substantially contribute to host defense against HIV-1. Here, using flow cytometric detection of antigen-induced intracellular cytokines, we show that significant frequencies of gag specific, T-helper-1 CD4+ memory T cells are detectable in most subjects with active/progressive HIV-1 infection (median frequency, 0.12% of memory subset; range, 0-0.66%). Median frequencies of these cells were considerably higher in nonprogressive HIV-1 disease (0.40%), but there was substantial overlap between the two groups (range of nonprogressors, 0.10-1.7%). Continuous HIV-1 suppression with anti-retroviral therapy was associated with a time-dependent reduction in median frequencies of gag-specific CD4+ memory T cells: 0.08% in subjects treated for 4-24 weeks, and 0.03% in subjects treated for 47-112 weeks. Thus, functional HIV-1-specific CD4+ T cells are commonly available for support of anti-HIV-1 effector responses in active disease, but their decline with anti-retroviral therapy indicates that immunologic participation in long-term HIV-1 control will probably require effective vaccination strategies.     

Persistent and acute central nervous system infections are caused by Theiler''s murine encephalomyelitis viruses which differ in RNA composition but code for only slightly different proteins

The RNA and proteins for four representatives of the two subgroups of Theiler's murine encephalomyelitis viruses were studied. The large RNase T1-resistant oligonucleotides, when mapped along the RNA molecules, were found to be differently distributed in the two subgroups. Replicative form RNAs of two representatives were partially denatured, and the denaturation maps obtained were found to be similar but not identical. In addition, the analysis of the tryptic maps of the capsid proteins of all four isolates revealed that only small differences in the peptide map patterns exist among these viruses. The correlation of these findings with the pathogenicity of Theiler's viruses is discussed.     

The active site of thrombin is altered upon binding to thrombomodulin. Two distinct structural changes are detected by fluorescence, but only one correlates with protein C activation.

The association of thrombin with thrombomodulin, a non-enzymatic endothelial cell surface receptor, alters the substrate specificity of thrombin. Complex formation converts thrombin from a procoagulant to an anticoagulant enzyme. Structure-function analysis of this change in specificity is facilitated by the availability of two soluble proteolytic derivatives of thrombomodulin, one consisting of the six repeated growth factor-like domains of thrombomodulin (GF1-6) and the other containing only the fifth and sixth such domains (GF5-6). Both derivatives can bind to thrombin and block fibrinogen clotting activity, though only the larger GF1-6 can stimulate the activation of protein C. To ascertain whether the substrate specificity change from fibrinogen to protein C is accompanied by structural changes in the active site of the enzyme, fluorescent dyes were positioned at different locations within the active site. A 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) dye was covalently attached to the active site serine to form dansyl-thrombin, while either a fluorescein or an anilinonaphthalene-6-sulfonic acid (ANS) dye was attached covalently to the active site histidine of thrombin via a D-Phe-Pro-Arg linkage. The environment of the dansyl dye was altered in a similar fashion when either GF1-6 or GF5-6 bound to thrombin, since a similar reduction in dansyl emission intensity was elicited by these two thrombomodulin derivatives (25 and 32%, respectively). These spectral changes, and all others in this study, were saturable and reached a maximum when the ratio of thrombomodulin derivative to thrombin was close to 1. The environments of the fluorescein and ANS dyes were also altered when GF1-6 bound to thrombin because binding resulted in emission intensity changes of -13% and +18%, respectively. In contrast, no fluorescence changes were observed when the fluorescein and ANS thrombin derivatives were titrated with GF5-6. Thus, the structure of the active site was altered by thrombomodulin both immediately adjacent to the active site serine and also more than 15 A away from it. However, the structural change far from Ser-195 was only elicited by thrombomodulin species that stimulate thrombin-dependent activation of protein C.     

HLA-B51 and HLA-Bw52 differ by only two amino acids which are in the helical region of the alpha 1 domain

Genes encoding the serologically cross-reactive HLA-B51 and HLA-Bw52 molecules were isolated and the exons sequenced. HLA-B51 genes obtained from Caucasian and Oriental individuals were identical. HLA-Bw52 differs from HLA-B51 by four nucleotide substitutions in exon 2 encoding the alpha 1 domain. These comprise one isolated silent substitution in codon 23 and a cluster of three coding substitutions in codons 63 and 67. Amino acid substitutions of N----E at position 63 and F----S at position 67 are the only differences between HLA-B51 and HLA-Bw52 and these residues are postulated to form HLA-B51 specific epitopes. HLA-B51 could have been formed from HLA-Bw52 by the combination of a genetic exchange with HLA-B8 and a point mutation. Similarity of HLA-B51 and HLA-Bw52 with HLA-Bw58 suggest they also share a common ancestor.