Src kinase phosphorylates vascular endothelial-cadherin in response to vascular endothelial growth factor: identification of tyrosine 685 as the unique target site.

Src-family tyrosine kinases are regulatory proteins that play a pivotal role in the disorganization of cadherin-dependent cell-cell contacts. We previously showed that Src was associated with vascular endothelial (VE)-cadherin and that tyrosine phosphorylation level of VE-cadherin was dramatically increased in angiogenic tissues as compared to quiescent tissues. Here, we examined whether VE-cadherin was a direct substrate for Src in vascular endothelial growth factor (VEGF)-induced VE-cadherin phosphorylation, and we identified the target tyrosine sites. Co-transfections of Chinese hamster ovary cells (CHO) cells with VE-cadherin and constitutively active Src (Y530F) resulted in a robust tyrosine phosphorylation of VE-cadherin that was not detected with kinase-dead Src (K298M). In an in vitro Src assay, the VE-cadherin cytoplasmic domain is directly phosphorylated by purified Src as well as the tyrosine residue 685 (Tyr)685-containing peptide RPSLY(685)AQVQ. VE-cadherin peptide mapping from human umbilical vein endothelial cells stimulated by VEGF and VE-cadherin-CHO cells transfected with active Src revealed that Y685 was the unique phosphorylated site. The presence of PhosphoY685 was confirmed by its ability to bind to C-terminal Src kinase-SH2 domain in a pull-down assay. Finally, we found that in a VEGF-induced wound-healing assay, cadherin adhesive activity was impaired by Src kinase inhibitors. These data identify that VEGF-induced-VE-cadherin tyrosine phosphorylation is mediated by Src on Y685, a process that appears to be critical for VEGF-induced endothelial cell migration.

Determination of bradykinin B2 receptor in vivo phosphorylation sites and their role in receptor function.

Reversible phosphorylation plays important roles in G protein-coupled receptor signaling, desensitization, and endocytosis, yet the precise location and role of in vivo phosphorylation sites is unknown for most receptors. Using metabolic 32P labeling and phosphopeptide sequencing we provide a complete phosphorylation map of the human bradykinin B2 receptor in its native cellular environment. We identified three serine residues, Ser(339), Ser(346), and Ser(348), at the C-terminal tail as principal phosphorylation sites. Constitutive phosphorylation occurs at Ser(348), while ligand-induced phosphorylation is found at Ser(339) and Ser(346)/Ser(348) that could be executed by several G protein-coupled receptor kinases. In addition, we found a protein kinase C-dependent phosphorylation of Ser(346) that was mutually exclusive with the basal phosphorylation at Ser(348) and therefore may be implicated in differential regulation of B2 receptor activation. Functional analysis of receptor mutants revealed that a low phosphorylation stoichiometry is sufficient to initiate receptor sequestration while a clustered phosphorylation around Ser(346) is necessary for desensitization of the B2 receptor-induced phospholipase C activation. This was further supported by the specifically reduced Ser(346)/Ser(348) phosphorylation observed upon stimulation with a nondesensitizing B2 receptor agonist. The differential usage of clustered phosphoacceptor sites points to distinct roles of multiple kinases in controlling G protein-coupled receptor function.

Chromatin and histones from Giardia lamblia: a new puzzle in primitive eukaryotes.

The three deepest eukaryote lineages in small subunit ribosomal RNA phylogenies are the amitochondriate Microsporidia, Metamonada, and Parabasalia. They are followed by either the Euglenozoa (e.g., Euglena and Trypanosoma) or the Percolozoa as the first mitochondria-containing eukaryotes. Considering the great divergence of histone proteins in protozoa we have extended our studies of histones from Trypanosomes (Trypanosoma cruzi, Crithidia fasciculata and Leishmania mexicana) to the Metamonada Giardia lamblia, since Giardia is thought to be one of the most primitive eukaryotes. In the present work, the structure of G. lamblia chromatin and the histone content of the soluble chromatin were investigated and compared with that of higher eukaryotes, represented by calf thymus. The chromatin is present as nucleosome filaments which resemble the calf thymus array in that they show a more regular arrangement than those described for Trypanosoma. SDS-polyacrylamide gel electrophoresis and protein characterization revealed that the four core histones described in Giardia are in the same range of divergence with the histones from other lower eukaryotes. In addition, G. lamblia presented an H1 histone with electrophoretic mobility resembling the H1 of higher eukaryotes, in spite of the fact that H1 has a different molecular mass in calf thymus. Giardia also presents a basic protein which was identified as an HU-like DNA-binding protein usually present in eubacteria, indicating a chimaeric composition for the DNA-binding protein set in this species. Finally, the phylogenetic analysis of selected core histone protein sequences place Giardia divergence before Trypanosoma, despite the fact that Trypanosoma branch shows an acceleration in the evolutionary rate pointing to an unusual evolutionary behavior in this lineage.

Cloning of linoleate diol synthase reveals homology with prostaglandin H synthases.

Linoleate diol synthase is a homotetrameric ferric hemeprotein, which catalyzes dioxygenation of linoleic acid to (8R)-hydroperoxylinoleate and isomerization of the hydroperoxide to (7S,8S)-dihydroxylinoleate. Ferryl intermediates and a tyrosyl radical are formed in the reaction. Linoleate diol synthase was digested with endoproteinase Lys-C, and internal peptides were sequenced. The sequence information was used for reverse transcription-polymerase chain reaction analysis, and a cDNA probe was obtained. Northern blot analysis of linoleate diol synthase suggested a 3.7-kilobase pair (kb) mRNA. A full-length clone of the linoleate diol synthase gene was obtained by screening of a genomic lambda-ZAP II library of the fungus Gaeumannomyces graminis. The 5'-untranslated region contained CAAT- and TATA-like boxes. The gene contained three short introns and spanned over 3.2-kb. The deduced open reading frame consisted of 2.9-kb, which corresponded to 978 amino acids and a molecular subunit mass of 108,000. Data base analysis with the gapped BLAST algorithm showed that 391 residues of linoleate diol synthase was 23-24% identical and 36-37% positive with the catalytic domain of mammalian prostaglandin H (PGH) synthase-2. Based on homology with PGH synthases, the proximal heme ligand of linoleate diol synthase was tentatively identified as His-379 and the important tyrosine for catalysis as residue 376 (apparent consensus EFNXXXYXWH). The distal heme ligand was tentatively identified as His-203 (apparent consensus THXXFXT). We conclude from catalytic and structural similarities that linoleate diol synthase and PGH synthases likely share common ancestry and may belong to a gene family of fatty acid heme dioxygenases.

SHP-2 binds to Tyr763 and Tyr1009 in the PDGF beta-receptor and mediates PDGF-induced activation of the Ras/MAP kinase pathway and chemotaxis.

Activation of the beta-receptor for platelet-derived growth factor (PDGF) by its ligand leads to autophosphorylation on a number of tyrosine residues. Here we show that Tyr763 in the kinase insert region is a novel autophosphorylation site, which after phosphorylation binds the protein tyrosine phosphatase SHP-2. SHP-2 has also previously been shown to bind to phosphorylated Tyr1009 in the PDGF beta-receptor. Porcine aortic endothelial (PAE) cells transfected with a PDGF beta-receptor in which Tyr763 and Tyr1009 were mutated to phenylalanine residues failed to associate with SHP-2 after ligand stimulation. Moreover, PDGF-BB-induced Ras GTP-loading and Erk2 activation were severely compromised in the receptor mutant. Whereas the mitogenic response to PDGF-BB remained at the same level as in cells expressing wild-type PDGF beta-receptor, chemotaxis induced by PDGF-BB was significantly decreased in the case of the Y763F/Y1009F mutant cells, suggesting an important role for SHP-2 in chemotactic signaling.

Role of immunoglobulin-like domains 2-4 of the platelet-derived growth factor alpha-receptor in ligand-receptor complex assembly.

Platelet-derived growth factor (PDGF) is a dimeric protein that exerts its effects through tyrosine kinase alpha- and beta-receptors. The extracellular part of each receptor is composed of five Ig-like domains. Recombinant forms of alpha-receptor domains 1-4 (alphaRD1-4), 1-3 (alphaRD1-3), and 1 and 2 (alphaRD1-2) were prepared after expression in Chinese hamster ovary cells and were used to study the assembly of soluble ligand-receptor complexes. When incubated with micromolar concentrations of PDGF, both alphaRD1-3 and alphaRD1-4 formed complexes of 1:2 molar composition, i.e. one dimeric PDGF molecule bound two soluble receptors. alphaRD1-3, in contrast to alphaRD1-4, formed detectable 1:1 complexes under conditions of ligand excess. alphaRD1-4 displayed an increased ability to form 1:2 complexes as compared with alphaRD1-3 under conditions of limiting concentrations of ligand. We thus conclude that Ig-like domain 4-mediated receptor-receptor interactions contribute to 1:2 PDGF.alphaRD1-4 complex formation. Since alphaRD1-4 and alphaRD1-3 were equipotent in blocking binding of subnanomolar concentrations of PDGF to cell-surface receptors, we also conclude that this effect is predominantly achieved through formation of Ig-like domain 4-independent 1:1 ligand-receptor complexes. Finally, since alphaRD1-2 bound PDGF-BB with high affinity, whereas PDGF-AA was bound only with low affinity, we conclude that Ig-like domain 3 of the PDGF alpha-receptor contains epitopes of particular importance for PDGF-AA binding and that most of the PDGF-BB-binding epitopes reside in Ig-like domains 1 and 2.

Identification of vascular endothelial growth factor receptor-1 tyrosine phosphorylation sites and binding of SH2 domain-containing molecules.

Receptor tyrosine phosphorylation is crucial for signal transduction by creating high affinity binding sites for Src homology 2 domain-containing molecules. By expressing the intracellular domain of Flt-1/vascular endothelial growth factor receptor-1 in the baculosystem, we identified two major tyrosine phosphorylation sites at Tyr-1213 and Tyr-1242 and two minor tyrosine phosphorylation sites at Tyr-1327 and Tyr-1333 in this receptor. This pattern of phosphorylation of Flt-1 was also detected in vascular endothelial growth factor-stimulated cells expressing intact Flt-1. In vitro protein binding studies using synthetic peptides and immunoblotting showed that phospholipase C-gamma binds to both Y(p)1213 and Y(p)1333, whereas Grb2 and SH2-containing tyrosine protein phosphatase (SHP-2) bind to Y(p)1213, and Nck and Crk bind to Y(p)1333 in a phosphotyrosine-dependent manner. In addition, unidentified proteins with molecular masses around 74 and 27 kDa bound to Y(p)1213 and another of 75 kDa bound to Y(p)1333 in a phosphotyrosine-dependent manner. SHP-2, phospholipase C-gamma, and Grb2 could also be shown to bind to the intact Flt-1 intracellular domain. These results indicate that a spectrum of already known as well as novel phosphotyrosine-binding molecules are involved in signal transduction by Flt-1.

Prothymosin alpha stimulates Ca2+-dependent phosphorylation of elongation factor 2 in cellular extracts.

Prothymosin alpha (PTA) stimulates in a dose-dependent manner the phosphorylation of a 105-kDa protein (p105) in cell extracts from different cell types. Protein sequencing and immunological analysis indicated that this protein is elongation factor 2 (EF-2). We propose that calcium/calmodulin-dependent protein kinase III is responsible for the PTA-dependent EF-2 phosphorylation based on the following lines of evidence: (a) Ca2+ is required for the effect; (b) calmodulin enhances the reaction, and calmodulin inhibitors block the phosphorylation; and (c) no phosphorylation is seen in cell extracts depleted of calmodulin-binding proteins. To obtain a strong phosphorylated EF-2 band, we found it necessary to add PTA to cytosolic extracts from synchronized dividing cells in various phases of the cell cycle except in mitosis. Since PTA is a nuclear protein everywhere in the cell cycle except in mitosis, when it is found in the cytoplasm, we hypothesize that, if PTA activates EF-2 phosphorylation in vivo, as present data suggest, its presence in the cytoplasm during mitosis could explain why EF-2 phosphorylation is mainly restricted to that phase of the cell cycle. Moreover, other bands in addition to EF-2 were phosphorylated in a calmodulin- and PTA-dependent manner, and several of them (in a range between 50 and 60 kDa) have similar Mr to those that conform to the holoenzyme calcium/calmodulin dependent protein kinase II, suggesting that PTA could have a more general function modulating the activity of various Ca2+/CaM-dependent enzymes along the cell cycle.

Identification of novel phosphorylation sites in hormone-sensitive lipase that are phosphorylated in response to isoproterenol and govern activation properties in vitro.

Hormone-sensitive lipase (HSL) is the rate-limiting enzyme in lipolysis. Stimulation of rat adipocytes with isoproterenol results in phosphorylation of HSL and a 50-fold increase in the rate of lipolysis. In this study, we used site-directed mutagenesis and two-dimensional phosphopeptide mapping to show that phosphorylation sites other than the previously identified Ser-563 are phosphorylated in HSL in response to isoproterenol stimulation of 32P-labeled rat adipocytes. Phosphorylation of HSL in adipocytes in response to isoproterenol and in vitro phosphorylation of HSL containing Ser --> Ala mutations in residues 563 and 565 (S563A, S565A) with protein kinase A (PKA), followed by tryptic phosphopeptide mapping resulted in two tryptic phosphopeptides. These tryptic phosphopeptides co-migrated with the phosphopeptides released by the same treatment of F654HPRRSSQGVLHMPLYSSPIVK675 phosphorylated with PKA. Analysis of the phosphorylation site mutants, S659A, S660A, and S659A,S660A disclosed that mutagenesis of both Ser-659 and Ser-660 was necessary to abolish the activation of HSL toward a triolein substrate after phosphorylation with PKA. Mutation of Ser-563 to alanine did not cause significant change of activation compared with wild-type HSL. Hence, our results demonstrate that in addition to the previously identified Ser-563, two other PKA phosphorylation sites, Ser-659 and Ser-660, are present in HSL and, furthermore, that Ser-659 and Ser-660 are the major activity controlling sites in vitro.

Destabilization of peptide binding and interdomain communication by an E543K mutation in the bovine 70-kDa heat shock cognate protein, a molecular chaperone.

We have compared 70-kDa heat shock cognate protein (Hsc70) isolated from bovine brain with recombinant wild type protein and mutant E543K protein (previously studied as wild type in our laboratory). Wild type bovine and recombinant protein differ by posttranslational modification of lysine 561 but interact similarly with a short peptide (fluorescein-labeled FYQLALT) and with denatured staphylococcal nuclease-(Delta135-149). Mutation E543K results in 4. 5-fold faster release of peptide and lower stability of complexes with staphylococcal nuclease-(Delta135-149). ATP hydrolysis rates of the wild type proteins are enhanced 6-10-fold by the addition of peptide. The E543K mutant has a peptide-stimulated hydrolytic rate similar to that of wild type protein but a higher unstimulated rate, yielding a mere 2-fold enhancement. All three versions of Hsc70 possess similar ATP-dependent conformational shifts, and all show potassium ion dependence. These data support the following model: (i) in the presence of K+, Mg2+, and ATP, the peptide binding domain inhibits the ATPase; (ii) binding of peptide relieves this inhibition; and (iii) the E543K mutation significantly attenuates the inhibition by the peptide binding domain and destabilizes Hsc70-peptide complexes.

Phosphorylation of Ser465 and Ser467 in the C terminus of Smad2 mediates interaction with Smad4 and is required for transforming growth factor-beta signaling.

Members of the Smad family of intracellular signal transducers are essential for transforming growth factor-beta (TGF-beta) to exert its multifunctional effects. After activation of TGF-beta receptors, Smad2 and Smad3 become phosphorylated and form heteromeric complexes with Smad4. Thereafter, these activated Smad complexes translocate to the nucleus, where they may direct transcriptional responses. Here we report that TGF-beta mediates phosphorylation of Smad2 at two serine residues in the C terminus, i.e. Ser465 and Ser467, which are phosphorylated in an obligate order; phosphorylation of Ser465 requires that Ser467 be phosphorylated. Transfection of Smad2 with mutation of Ser465 and/or Ser467 to alanine residues into Mv1Lu cells resulted in dominant-negative inhibition of TGF-beta signaling. These Smad2 mutants were found to stably interact with an activated TGF-beta receptor complex, in contrast to wild-type Smad2, which interacts only transiently. Mutation of Ser465 and Ser467 in Smad2 abrogated complex formation of this mutant with Smad4 and blocked the nuclear accumulation not only of Smad2, but also of Smad4. Thus, heteromeric complex formation of Smad2 with Smad4 is required for nuclear translocation of Smad4. Moreover, peptides from the C terminus of Smad2 containing phosphorylated Ser465 and Ser467 were found to bind Smad4 in vitro, whereas the corresponding unphosphorylated peptides were less effective. Thus, phosphorylated Ser465 and Ser467 in Smad2 may provide a recognition site for interaction with Smad4, and phosphorylation of these sites is a key event in Smad2 activation.

cAMP-dependent phosphorylation activates phosphofructokinase from mantle tissue of the mollusc Mytilus galloprovincialis. Identification of the phosphorylated site.

Phosphofructokinase from mantle tissue of the sea mussel Mytilus galloprovincialis was phosphorylated in vitro by a protein kinase isolated from the same tissue, homologous to mammalian cAMP-dependent protein kinase; the maximal level of phosphorylation achieved was around 1 mol of Pi/mol of phosphofructokinase subunit. The covalent incorporation of phosphate leads to a notable increase in the enzyme activity assayed at near-physiological concentrations of substrates and allosteric modulators and neutral pH. Tryptic digestion of labeled phosphofructokinase released a phosphopeptide whose sequence was Lys-Asp-Ser(P)-Ile-Trp-Ile-Gln-Thr-Gly-Arg. This sequence showed high homology with the phosphopeptides from other invertebrates whose phosphofructokinase is also activated by cAMP-dependent phosphorylation.

B12-dependent ribonucleotide reductases from deeply rooted eubacteria are structurally related to the aerobic enzyme from Escherichia coli.

The ribonucleotide reductases from three ancient eubacteria, the hyperthermophilic Thermotoga maritima (TM), the radioresistant Deinococcus radiodurans (DR), and the thermophilic photosynthetic Chloroflexus aurantiacus, were found to be coenzyme-B12 (class II) enzymes, similar to the earlier described reductases from the archaebacteria Thermoplasma acidophila and Pyrococcus furiosus. Reduction of CDP by the purified TM and DR enzymes requires adenosylcobalamin and DTT. dATP is a positive allosteric effector, but stimulation of the TM enzyme only occurs close to the temperature optimum of 80-90 degrees C. The TM and DR genes were cloned by PCR from peptide sequence information. The TM gene was sequenced completely and expressed in Escherichia coli. The deduced amino acid sequences of the two eubacterial enzymes are homologous to those of the archaebacteria. They can also be aligned to the sequence of the large protein of the aerobic E. coli ribonucleotide reductase that belongs to a different class (class I), which is not dependent on B12. Structure determinations of the E. coli reductase complexed with substrate and allosteric effectors earlier demonstrated a 10-stranded beta/alpha-barrel in the active site. From the conservation of substrate- and effector-binding residues we propose that the B12-dependent class II enzymes contain a similar barrel.

Two different 8 kDa monomers are involved in the oligomeric organization of the native Echinococcus granulosus antigen B.

The present work describes the purification and characterization of antigen B (AgB), the thermostable lipoprotein from E. granulosus. Native AgB was purified to homogeneity by a new strategy involving adsorption on DEAE-Sepharose, followed by immunopurification. The purified antigen was analysed using mapped monoclonal antibodies (MoAbs) and peptide isolation by in situ digestion in gels after SDS-PAGE. Epitope mapping of 7 MoAbs using PEPSCAN, synthetic peptides and competition studies, revealed that six of them defined epitopes which clustered the N-terminal extension of a 8 kDa subunit of AgB, whilst the remaining one reacted against the stretch RGLIAEGE, corresponding to the C-terminus. The epitopes defined by the seven MoAbs were found to be present in all the subunits. Furthermore, the similarities of the peptide finger prints obtained by HPLC analysis and amino acid sequencing of tryptic peptides isolated from the 8, 16 and 24 kDa subunits, indicated that they have most if not all the amino acid sequence in common. We also found evidence that the band representing a component of an apparent molecular weight of 8 kDa in SDS-PAGE, believed to be the smallest subunit of AgB, contained at least two components, which may constitute the building blocks of the higher molecular weight subunits.

Mutation of a Src phosphorylation site in the PDGF beta-receptor leads to increased PDGF-stimulated chemotaxis but decreased mitogenesis.

Ligand induced activation of the beta-receptor for platelet-derived growth factor (PDGF) leads to activation of Src family tyrosine kinases. We have explored the possibility that the receptor itself is a substrate for Src. We show that Tyr934 in the kinase domain of the PDGF receptor is phosphorylated by Src. Cell lines expressing a beta-receptor mutant, in which Tyr934 was replaced with a phenyalanine residue, showed reduced mitogenic signaling in response to PDGF-BB. In contrast, the mutant receptor mediated increased signals for chemotaxis and actin reorganization. Whereas the motility responses of cells expressing wild-type beta-receptors were attenuated by inhibition of phosphatidylinositol 3'-kinase, those of cells expressing the mutant receptor were only slightly influenced. In contrast, PDGF-BB-induced chemotaxis of the cells with the mutant receptor was attenuated by inhibition of protein kinase C, whereas the chemotaxis of cells expressing the wild-type beta-receptor was less affected. Moreover, the PDGF-BB-stimulated tyrosine phosphorylation of phospholipase C-gamma was increased in the mutant receptor cells compared with wild-type receptor cells. In conclusion, the characteristics of the Y934F mutant suggest that the phosphorylation of Tyr934 by Src negatively modulates a signal transduction pathway leading to motility responses which involves phospholipase C-gamma, and shifts the response to increased mitogenicity.

Identification of the site in the cGMP-inhibited phosphodiesterase phosphorylated in adipocytes in response to insulin and isoproterenol.

Stimulation of rat adipocytes with insulin and isoproterenol results in serine phosphorylation and activation of the adipocyte cGMP-inhibited phosphodiesterase (cGI PDE), events believed to be important in the antilipolytic action of insulin (Degerman, E., Smith, C.J., Tornqvist, H., Vasta, V., Manganiello, V.C., and Belfrage, P. (1990) Proc. Natl. Acad. Sci. U.S.A. 87,533-537). Here we demonstrate, by two-dimensional phosphopeptide mapping, that the major phosphopeptide generated by trypsin, or trypsin followed by Asp-N protease digestion of [32P]cGI PDE phosphorylated in adipocytes in response to isoproterenol and/or insulin, in each case co-migrates with the phosphopeptide released by the same treatment of M297FRRPS(P)LPCISREQ310. This peptide was synthesized based on the deduced sequence of the cloned rat adipocyte cGI PDE and phosphorylated by cAMP-dependent protein kinase (protein kinase A). Radiosequencing of authentic and synthetic tryptic 32P-peptides showed that a single site in cGI PDE (Ser302) was phosphorylated in adipocytes incubated with isoproterenol and/or insulin. The more than additive phosphorylation and activation of cGI PDE in response to the two hormones found in this report and previously (Smith, C.J., Vasta, V., Degerman, E., Belfrage, P., and Manganiello, V.C. (1991) J. Biol. Chem. 266, 13385-13390) is proposed to reflect cross-talk between their respective signal transduction pathways at the level of the cGI PDE serine protein kinase or upstream regulatory component(s).

Structural determinants in the platelet-derived growth factor alpha-receptor implicated in modulation of chemotaxis.

Activation of the platelet-derived growth factor (PDGF) beta-receptor leads to cell growth and chemotaxis. The PDGF alpha-receptor also mediates a mitogenic signal, but fails to induce cell migration in certain cell types. To examine this difference in signal transduction, a series of point-mutated PDGF alpha-receptors were analyzed. Porcine aortic endothelial cells expressing mutant PDGF alpha-receptors, in which tyrosine residues 768, 993, or 1018 were changed to phenylalanine residues migrated toward PDGF, whereas wild-type alpha-receptors and mutant alpha-receptors changed at tyrosine residues 720, 944, or 988 failed to migrate. All mutant receptors were mitogenically active and their capacity to activate phosphatidylinositol 3'-kinase and phospholipase C-gamma was not different from that of the wild-type receptor. Tyr-768 was found to be phosphorylated in PDGF-stimulated cells; in the Y768F mutant, there was a considerable increase in phosphorylation of Ser-767. Tyr-993 was not phosphorylated, but mutation of this tyrosine residue to a phenylalanine residue resulted in increased efficiency of phosphorylation on Tyr-988. Tyr-1018 is known to be an autophosphorylation site. Phosphorylated Tyr-768 and Tyr-1018 may bind signal transduction molecules involved in negative modulation of the chemotactic signaling capacity, whereas phosphorylated Tyr-988 may mediate increased chemotaxis. Thus our data indicate that the PDGF alpha-receptor has an intrinsic ability to transduce a chemotactic signal, and that this signal is counteracted by overriding negative signals.

Molecular cloning and expression of a 58-kDa cis-Golgi and intermediate compartment protein.

An abundant 58-kDa (p58) homodimeric and hexameric microsomal membrane protein has been biochemically characterized and localized to tubulo-vesicular elements at the endoplasmic reticulum-Golgi interface and the cis-Golgi cisternae in pancreatic acinar cells (Lahtinen, U., Dahllöf, B., and Saraste, J. (1992) J. Cell Sci. 103, 321-333). Here we report the purification of p58 by two-dimensional gel electrophoresis, and the cloning and sequencing of the rat and part of the Xenopus laevis cDNAs. The rat cDNA encodes a 517-amino acid protein having a putative signal sequence, a transmembrane domain close to the C terminus and a short cytoplasmic tail. The C-terminal tail contains a double-lysine motif (KKFF), known to mediate retrieval of proteins from the Golgi back to the endoplasmic reticulum. The rat p58 sequence was found to be 89% identical with those of ERGIC-53 and MR60, two previously identified human membrane proteins. Strong homology with the frog sequence was also observed indicating high evolutionary conservation. Overexpression of c-Myc-tagged p58 resulted in accumulation of the protein both in the endoplasmic reticulum and in an apparently enlarged Golgi complex, as well as its leakage to the plasma membrane. Immunolocalization using antibodies raised against a lumenal peptide stained the total cellular pool of p58, while anti-tail peptide antibodies detected p58 only in a restricted Golgi region. This suggests that the C-terminal tail of p58 located in the endoplasmic reticulum and transport intermediates is hidden, but becomes exposed when the protein reaches the Golgi complex.

Muscarinic toxins from the black mamba Dendroaspis polylepis.

Three new toxins acting on muscarinic receptors were isolated from the venom of the black mamba Dendroaspis polylepis. They were called muscarinic toxins alpha, beta, and gamma (MT alpha, MT beta, and MT gamma). All of the toxins have four disulphide bonds and 65 or 66 amino acids. The sequences of MT alpha and MT beta were determined. The muscarinic toxins, of which about 12 have been isolated from venoms of green and black mambas, have 60-98% sequence identity with each other, and are similar to many (about 180) other snake venom components, such as alpha-neurotoxins, cardiotoxins, and fasciculins. In contrast to the alpha-neurotoxins, muscarinic toxins do not bind to nicotinic acetylcholine receptors. The binding constants of MT alpha and MT beta were determined for human muscarinic receptors of subtypes m1-m5 stably expressed in Chinese hamster ovary cells. The toxins are less selective than the earlier discovered muscarinic toxins from the green mamba Dendroaspis angusticeps. MT alpha and the muscarinic toxin MT4 from D. angusticeps differ only in a region of three amino acids (residues 31-33), which are Leu-Asn-His in MT alpha and Ile-Val-Pro in MT4. This difference causes a pronounced shift in subtype selectivity. MT alpha has high affinity to all subtypes, with Ki (inhibition constant) values of 23 nM (m1; pKi = 7.64 +/- 0.10), 44 nM (m2; pKi = 7.36 +/- 0.06), 3 nM (m3; pKi = 8.46 +/- 0.14), 5 nM (m4; pKi = 8.32 +/- 0.07), and 8 nM (m5; pKi = 8.09 +/- 0.07). MT4 has high affinity only to m1 (Ki = 62 nM) and m4 (87 nM) receptors, and low (Ki > 1 microM) affinity to m2, m3, and m5. The region at positions 31-33 evidently plays an important role in the toxin-receptor interaction. MT beta has low affinity for m1 and m2 receptors (Ki > 1 microM) and intermediate affinity for m3 (140 nM; pKi = 6.85 +/- 0.03), m4 (120 nM; pKi = 6.90 +/- 0.06), and m5 (350 nM; pKi = 6.46 +/- 0.01). The low affinity of MT beta may reflect a tendency for spontaneous inactivation.

Characterization of stable complexes involving apolipoprotein E and the amyloid beta peptide in Alzheimer's disease brain.

Genetic evidence suggests a role for apolipoprotein E (apoE) in Alzheimer's disease (AD) amyloidogenesis. Here, amyloid-associated apoE from 32 AD patients was purified and characterized. We found that brain amyloid-associated apoE apparently exists not as free molecules but as complexes with polymers of the amyloid beta peptide (A beta). Brain A beta-apoE complexes were detected irrespective of the apoE genotype, and similar complexes could be mimicked in vitro. The fine structure of purified A beta-apoE complexes was fibrillar, and immunogold labeling revealed apoE immunoreactivity along the fibrils. Thus, we conclude that A beta-apoE complexes are principal components of AD-associated brain amyloid and that the data presented here support a role for apoE in the pathogenesis of AD.