Role of endocytosis in the activation of the extracellular signal-regulated kinase cascade by sequestering and nonsequestering G protein-coupled receptors

Acting through a number of distinct pathways, many G protein-coupled receptors (GPCRs) activate the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) cascade. Recently, it has been shown that in some cases, clathrin-mediated endocytosis is required for GPCR activation of the ERK/MAPK cascade, whereas in others it is not. Accordingly, we compared ERK activation mediated by a GPCR that does not undergo agonist-stimulated endocytosis, the α2A adrenergic receptor (α2A AR), with ERK activation mediated by the β2 adrenergic receptor (β2 AR), which is endocytosed. Surprisingly, we found that in COS-7 cells, ERK activation by the α2A AR, like that mediated by both the β2 AR and the epidermal growth factor receptor (EGFR), is sensitive to mechanistically distinct inhibitors of clathrin-mediated endocytosis, including monodansylcadaverine, a mutant dynamin I, and a mutant β-arrestin 1. Moreover, we determined that, as has been shown for many other GPCRs, both α2A and β2 AR-mediated ERK activation involves transactivation of the EGFR. Using confocal immunofluorescence microscopy, we found that stimulation of the β2 AR, the α2A AR, or the EGFR each results in internalization of a green fluorescent protein-tagged EGFR. Although β2 AR stimulation leads to redistribution of both the β2 AR and EGFR, activation of the α2A AR leads to redistribution of the EGFR but the α2A AR remains on the plasma membrane. These findings separate GPCR endocytosis from the requirement for clathrin-mediated endocytosis in EGFR transactivation-mediated ERK activation and suggest that it is the receptor tyrosine kinase or another downstream effector that must engage the endocytic machinery.

Interaction of the poliovirus receptor with poliovirus

The structure of the extracellular, three-domain poliovirus receptor (CD155) complexed with poliovirus (serotype 1) has been determined to 22-Å resolution by means of cryo-electron microscopy and three-dimensional image-reconstruction techniques. Density corresponding to the receptor was isolated in a difference electron density map and fitted with known structures, homologous to those of the three individual CD155 Ig-like domains. The fit was confirmed by the location of carbohydrate moieties in the CD155 glycoprotein, the conserved properties of elbow angles in the structures of cell surface molecules with Ig-like folds, and the concordance with prior results of CD155 and poliovirus mutagenesis. CD155 binds in the poliovirus “canyon” and has a footprint similar to that of the intercellular adhesion molecule-1 receptor on human rhinoviruses. However, the orientation of the long, slender CD155 molecule relative to the poliovirus surface is quite different from the orientation of intercellular adhesion molecule-1 on rhinoviruses. In addition, the residues that provide specificity of recognition differ for the two receptors. The principal feature of receptor binding common to these two picornaviruses is the site in the canyon at which binding occurs. This site may be a trigger for initiation of the subsequent uncoating step required for viral infection.

Analysis of receptor signaling pathways by mass spectrometry: Identification of Vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors

Oligomerization of receptor protein tyrosine kinases such as the epidermal growth factor receptor (EGFR) by their cognate ligands leads to activation of the receptor. Transphosphorylation of the receptor subunits is followed by the recruitment of signaling molecules containing src homology 2 (SH2) or phosphotyrosine interaction domains (PID). Additionally, several cytoplasmic proteins that may or may not associate with the receptor undergo tyrosine phosphorylation. To identify several components of the EGFR signaling pathway in a single step, we have immunoprecipitated molecules that are tyrosine phosphorylated in response to EGF and analyzed them by one-dimensional gel electrophoresis followed by mass spectrometry. Combining matrix-assisted laser desorption/ionization (MALDI) and nanoelectrospray tandem mass spectrometry (MS/MS) led to the identification of nine signaling molecules, seven of which had previously been implicated in EGFR signaling. Several of these molecules were identified from low femtomole levels of protein loaded onto the gel. We identified Vav-2, a recently discovered guanosine nucleotide exchange factor that is expressed ubiquitously, as a substrate of the EGFR. We demonstrate that Vav-2 is phosphorylated on tyrosine residues in response to EGF and associates with the EGFR in vivo. Binding of Vav-2 to the EGFR is mediated by the SH2 domain of Vav-2. In keeping with its ubiquitous expression, Vav-2 seems to be a general signaling molecule, since it also associates with the platelet-derived growth factor (PDGF) receptor and undergoes tyrosine phosphorylation in fibroblasts upon PDGF stimulation. The strategy suggested here can be used for routine identification of downstream components of cell surface receptors in mammalian cells.

NMR structure of the bovine prion protein

The NMR structures of the recombinant 217-residue polypeptide chain of the mature bovine prion protein, bPrP(23–230), and a C-terminal fragment, bPrP(121–230), include a globular domain extending from residue 125 to residue 227, a short flexible chain end of residues 228–230, and an N-terminal flexibly disordered “tail” comprising 108 residues for the intact protein and 4 residues for bPrP(121–230), respectively. The globular domain contains three α-helices comprising the residues 144–154, 173–194, and 200–226, and a short antiparallel β-sheet comprising the residues 128–131 and 161–164. The best-defined parts of the globular domain are the central portions of the helices 2 and 3, which are linked by the only disulfide bond in bPrP. Significantly increased disorder and mobility is observed for helix 1, the loop 166–172 leading from the β-strand 2 to helix 2, the end of helix 2 and the following loop, and the last turn of helix 3. Although there are characteristic local differences relative to the conformations of the murine and Syrian hamster prion proteins, the bPrP structure is essentially identical to that of the human prion protein. On the other hand, there are differences between bovine and human PrP in the surface distribution of electrostatic charges, which then appears to be the principal structural feature of the “healthy” PrP form that might affect the stringency of the species barrier for transmission of prion diseases between humans and cattle.

Unraveling the mechanism of the lactose permease of Escherichia coli

We studied the effect of pH on ligand binding in wild-type lactose permease or mutants in the four residues—Glu-269, Arg-302, His-322, and Glu-325—that are the key participants in H+ translocation and coupling between sugar and H+ translocation. Although wild-type permease or mutants in Glu-325 and Arg-302 exhibit marked decreases in affinity at alkaline pH, mutants in either His-322 or Glu-269 do not titrate. The results offer a mechanistic model for lactose/H+ symport. In the ground state, the permease is protonated, the H+ is shared between His-322 and Glu-269, Glu-325 is charge-paired with Arg-302, and substrate is bound with high affinity at the outside surface. Substrate binding induces a conformational change that leads to transfer of the H+ from His-322/Glu-269 to Glu-325 and reorientation of the binding site to the inner surface with a decrease in affinity. Glu-325 then is deprotonated on the inside because of rejuxtaposition with Arg-302. The His-322/Glu-269 complex then is reprotonated from the outside surface to reinitiate the cycle.

Measuring the thickness of the human cerebral cortex from magnetic resonance images

Accurate and automated methods for measuring the thickness of human cerebral cortex could provide powerful tools for diagnosing and studying a variety of neurodegenerative and psychiatric disorders. Manual methods for estimating cortical thickness from neuroimaging data are labor intensive, requiring several days of effort by a trained anatomist. Furthermore, the highly folded nature of the cortex is problematic for manual techniques, frequently resulting in measurement errors in regions in which the cortical surface is not perpendicular to any of the cardinal axes. As a consequence, it has been impractical to obtain accurate thickness estimates for the entire cortex in individual subjects, or group statistics for patient or control populations. Here, we present an automated method for accurately measuring the thickness of the cerebral cortex across the entire brain and for generating cross-subject statistics in a coordinate system based on cortical anatomy. The intersubject standard deviation of the thickness measures is shown to be less than 0.5 mm, implying the ability to detect focal atrophy in small populations or even individual subjects. The reliability and accuracy of this new method are assessed by within-subject test–retest studies, as well as by comparison of cross-subject regional thickness measures with published values.

Protein fluctuations are sensed by stimulated infrared echoes of the vibrations of carbon monoxide and azide probes

The correlation functions of the fluctuations of vibrational frequencies of azide ions and carbon monoxide in proteins are determined directly from stimulated photon echoes generated with femtosecond infrared pulses. The asymmetric stretching vibration of azide bound to carbonic anhydrase II exhibits a pronounced evolution of its vibrational frequency distribution on the time scale of a few picoseconds, which is attributed to modifications of the ligand structure through interactions with the nearby Thr-199. When azide is bound in hemoglobin, a more complex evolution of the protein structure is required to interchange the different ligand configurations, as evidenced by the much slower relaxation of the frequency distribution in this case. The time evolution of the distribution of frequencies of carbon monoxide bound in hemoglobin occurs on the ≈10-ps time scale and is very nonexponential. The correlation functions of the frequency fluctuations determine the evolution of the protein structure local to the probe and the extent to which the probe can navigate those parts of the energy landscape where the structural configurations are able to modify the local potential energy function of the probe.

Mechanism for fetal globin gene expression: Role of the soluble guanylate cyclase–cGMP-dependent protein kinase pathway

Despite considerable concerns with pharmacological stimulation of fetal hemoglobin (Hb F) as a therapeutic option for the β-globin disorders, the molecular basis of action of Hb F-inducing agents remains unclear. Here we show that an intracellular pathway including soluble guanylate cyclase (sGC) and cGMP-dependent protein kinase (PKG) plays a role in induced expression of the γ-globin gene. sGC, an obligate heterodimer of α- and β-subunits, participates in a variety of physiological processes by converting GTP to cGMP. Northern blot analyses with erythroid cell lines expressing different β-like globin genes showed that, whereas the β-subunit is expressed at similar levels, high-level expression of the α-subunit is preferentially observed in erythroid cells expressing γ-globin but not those expressing β-globin. Also, the levels of expression of the γ-globin gene correlate to those of the α-subunit. sGC activators or cGMP analogs increased expression of the γ-globin gene in erythroleukemic cells as well as in primary erythroblasts from normal subjects and patients with β-thalassemia. Nuclear run-off assays showed that the sGC activator protoporphyrin IX stimulates transcription of the γ-globin gene. Furthermore, increased expression of the γ-globin gene by well known Hb F-inducers such as hemin and butyrate was abolished by inhibiting sGC or PKG activity. Taken together, these results strongly suggest that the sGC–PKG pathway constitutes a mechanism that regulates expression of the γ-globin gene. Further characterization of this pathway should permit us to develop new therapeutics for the β-globin disorders.

Highly expressed and alien genes of the Synechocystis genome

Comparisons of codon frequencies of genes to several gene classes are used to characterize highly expressed and alien genes on the Synechocystis PCC6803 genome. The primary gene classes include the ensemble of all genes (average gene), ribosomal protein (RP) genes, translation processing factors (TF) and genes encoding chaperone/degradation proteins (CH). A gene is predicted highly expressed (PHX) if its codon usage is close to that of the RP/TF/CH standards but strongly deviant from the average gene. Putative alien (PA) genes are those for which codon usage is significantly different from all four classes of gene standards. In Synechocystis, 380 genes were identified as PHX. The genes with the highest predicted expression levels include many that encode proteins vital for photosynthesis. Nearly all of the genes of the RP/TF/CH gene classes are PHX. The principal glycolysis enzymes, which may also function in CO2 fixation, are PHX, while none of the genes encoding TCA cycle enzymes are PHX. The PA genes are mostly of unknown function or encode transposases. Several PA genes encode polypeptides that function in lipopolysaccharide biosynthesis. Both PHX and PA genes often form significant clusters (operons). The proteins encoded by PHX and PA genes are described with respect to functional classifications, their organization in the genome and their stoichiometry in multi-subunit complexes.

Arg-302 facilitates deprotonation of Glu-325 in the transport mechanism of the lactose permease from Escherichia coli

A mechanistic model for lactose/H+ symport via the lactose permease of Escherichia coli proposed recently indicates that the permease must be protonated to bind ligand with high affinity. Moreover, in the ground state, the symported H+ is shared between His-322 (helix X) and Glu-269 (helix VIII), whereas Glu-325 (helix X) is charge-paired with Arg-302 (helix IX). Substrate binding at the outer surface induces a conformational change that leads to transfer of the H+ to Glu-325 and reorientation of the binding site to the inner surface. After release of the substrate, Glu-325 is deprotonated on the inside because of rejuxtapositioning with Arg-302. To test the role of Arg-302 in the mechanism, the catalytic properties of mutants Arg-302→Ala and Arg-302→Ser were studied. Both mutants are severely defective in active lactose transport, as well as in efflux or influx down a concentration gradient, translocation modes that involve net H+ movement. In marked contrast, the mutants catalyze equilibrium exchange of lactose and bind ligand with high affinity. These characteristics are remarkably analogous to those of permease mutants with neutral replacements for Glu-325, a residue that plays a direct role in H+ translocation. These observations lend strong support for the argument that Arg-302 interacts with Glu-325 to facilitate deprotonation of the carboxylic acid during turnover.

Three-dimensional reconstruction of the recombinant type 3 ryanodine receptor and localization of its amino terminus

Recombinant type 3 ryanodine receptor (RyR3) has been purified in quantities sufficient for structural characterization by cryoelectron microscopy and three-dimensional (3D) reconstruction. Two cDNAs were prepared and expressed in HEK293 cells, one encoding the wild-type RyR3 and the other encoding RyR3 containing glutathione S-transferase (GST) fused to its amino terminus (GST-RyR3). RyR3 was purified from detergent-solubilized transfected cells by affinity chromatography using 12.6-kDa FK506-binding protein in the form of a GST fusion as the affinity ligand. Purification of GST-RyR3 was achieved by affinity chromatography by using glutathione-Sepharose. Purified recombinant RyR3 and GST-RyR3 proteins exhibited high-affinity [3H]ryanodine binding that was sensitive to activation by Ca2+ and caffeine and to inhibition by Mg2+. 3D reconstructions of both recombinant RyR3 and GST-RyR3 appeared very similar to that of the native RyR3 purified from bovine diaphragm. Comparison of the 3D reconstructions of RyR3 and GST-RyR3 revealed that the GST domains and, hence, the amino termini of the RyR3 subunits are located in the “clamp” structures that form the corners of the square-shaped cytoplasmic region of homotetrameric RyR3. This study describes the 3D reconstruction of a recombinant ryanodine receptor and it demonstrates the potential of this technology for characterizing functional and structural perturbations introduced by site-directed mutagenesis.

Regulation of the stem cell leukemia (SCL) gene: A tale of two fishes

The stem cell leukemia (SCL) gene encodes a tissue-specific basic helix–loop–helix (bHLH) protein with a pivotal role in hemopoiesis and vasculogenesis. Several enhancers have been identified within the murine SCL locus that direct reporter gene expression to subdomains of the normal SCL expression pattern, and long-range sequence comparisons of the human and murine SCL loci have identified additional candidate enhancers. To facilitate the characterization of regulatory elements, we have sequenced and analyzed 33 kb of the SCL genomic locus from the pufferfish Fugu rubripes, a species with a highly compact genome. Although the pattern of SCL expression is highly conserved from mammals to teleost fish, the genes flanking pufferfish SCL were unrelated to those known to flank both avian and mammalian SCL genes. These data suggest that SCL regulatory elements are confined to the region between the upstream and downstream flanking genes, a region of 65 kb in human and 8.5 kb in pufferfish. Consistent with this hypothesis, the entire 33-kb pufferfish SCL locus directed appropriate expression to hemopoietic and neural tissue in transgenic zebrafish embryos, as did a 10.4-kb fragment containing the SCL gene and extending to the 5′ and 3′ flanking genes. These results demonstrate the power of combining the compact genome of the pufferfish with the advantages that zebrafish provide for studies of gene regulation during development. Furthermore, the pufferfish SCL locus provides a powerful tool for the manipulation of hemopoiesis and vasculogenesis in vivo.

Role of the Ascorbate-Glutathione Cycle of Mitochondria and Peroxisomes in the Senescence of Pea Leaves1

We investigated the relationship between H2O2 metabolism and the senescence process using soluble fractions, mitochondria, and peroxisomes from senescent pea (Pisum sativum L.) leaves. After 11 d of senescence the activities of Mn-superoxide dismutase, dehydroascorbate reductase (DHAR), and glutathione reductase (GR) present in the matrix, and ascorbate peroxidase (APX) and monodehydroascorbate reductase (MDHAR) activities localized in the mitochondrial membrane, were all substantially decreased in mitochondria. The mitochondrial ascorbate and dehydroascorbate pools were reduced, whereas the oxidized glutathione levels were maintained. In senescent leaves the H2O2 content in isolated mitochondria and the NADH- and succinate-dependent production of superoxide (O2·−) radicals by submitochondrial particles increased significantly. However, in peroxisomes from senescent leaves both membrane-bound APX and MDHAR activities were reduced. In the matrix the DHAR activity was enhanced and the GR activity remained unchanged. As a result of senescence, the reduced and the oxidized glutathione pools were considerably increased in peroxisomes. A large increase in the glutathione pool and DHAR activity were also found in soluble fractions of senescent pea leaves, together with a decrease in GR, APX, and MDHAR activities. The differential response to senescence of the mitochondrial and peroxisomal ascorbate-glutathione cycle suggests that mitochondria could be affected by oxidative damage earlier than peroxisomes, which may participate in the cellular oxidative mechanism of leaf senescence longer than mitochondria.

Identification of a Novel Isoform of the Chloroplast-Coupling Factor α-Subunit 1

Studies were conducted to identify a 64-kD thylakoid membrane protein of unknown function. The protein was extracted from chloroplast thylakoids under low ionic strength conditions and purified to homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Four peptides generated from the proteolytic cleavage of the wheat 64-kD protein were sequenced and found to be identical to internal sequences of the chloroplast-coupling factor (CF1) α-subunit. Antibodies for the 64-kD protein also recognized the α-subunit of CF1. Both the 64-kD protein and the 61-kD CF1 α-subunit were present in the monocots barley (Hordeum vulgare), maize (Zea mays), oat (Avena sativa), and wheat (Triticum aestivum); but the dicots pea (Pisum sativum), soybean (Glycine max Merr.), and spinach (Spinacia oleracea) contained only a single polypeptide corresponding to the CF1 α-subunit. The 64-kD protein accumulated in response to high irradiance (1000 μmol photons m−2 s−1) and declined in response to low irradiance (80 μmol photons m−2 s−1) treatments. Thus, the 64-kD protein was identified as an irradiance-dependent isoform of the CF1 α-subunit found only in monocots. Analysis of purified CF1 complexes showed that the 64-kD protein represented up to 15% of the total CF1 α-subunit.

Reovirus reverse genetics: Incorporation of the CAT gene into the reovirus genome

We have modified the infectious reovirus RNA system so as to generate a reovirus reverse genetics system. The system consists of (i) the plus strands of nine wild-type reovirus genome segments; (ii) transcripts of the genetically modified cDNA form of the tenth genome segment; and (iii) a cell line transformed so as to express the protein normally encoded by the tenth genome segment. In the work described here, we have generated a serotype 3 reovirus into the S2 double-stranded RNA genome segment of which the CAT gene has been cloned. The virus is stable, replicates in cells that have been transformed (so as to express the S2 gene product, protein σ2), and expresses high levels of CAT activity. This technology can be extended to members of the orbivirus and rotavirus genera. This technology provides a powerful system for basic studies of double-stranded RNA virus replication; a nonpathogenic viral vector that replicates to high titers and could be used for clinical applications; and a system for providing nonselectable viral variants (the result of mutations, insertions, and deletions) that could be valuable for the construction of viral vaccine strains against human and animal pathogens.