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C W Turck

Publications and source records attributed to C W Turck.

At least 91 records · Page 5Linked to original sources
PubMed1992

Tethered ligand agonist peptides. Structural requirements for thrombin receptor activation reveal mechanism of proteolytic unmasking of agonist function.

The human platelet thrombin receptor is activated when thrombin cleaves its receptor's amino-terminal extension to reveal a new amino terminus that functions as a tethered peptide ligand. Exactly how this "agonist peptide domain" remains cryptic within the uncleaved receptor and becomes functional after receptor cleavage is unknown. In this report we define the structural features of the thrombin receptor's agonist peptide domain important for receptor activation. Studies with mutant thrombin receptors have suggested that agonist peptide domain residues 2-6 contained determinants critical for receptor activation, and the synthetic peptide SFLLR-NH2 representing the 1st 5 amino-terminal residues of the agonist peptide domain was sufficient to specify agonist activity. Acetylating or removing the agonist peptide's amino-terminal ammonium group greatly attenuated agonist activity. Agonist peptide residue Phe2 was vital for agonist function; residues Leu4 and Arg5 individually played less important roles. These structure-function relationships held for both platelet activation and activation of the cloned receptor expressed in transfected mammalian cells. Our studies suggest that structures at the extreme amino terminus of the thrombin receptor's agonist peptide domain, in particular the free ammonium group of Ser1 and the phenyl ring of Phe2, are critical for receptor activation and that the agonist function of this domain is expressed when receptor proteolysis unmasks such determinants. In addition to revealing details of the thrombin receptor's proteolytic triggering mechanism, these studies open avenues to the development of drugs targeting the thrombin receptor and to further definition for the role of the thrombin receptor in cellular regulation.

Amino Acid Sequence↗
PubMed1992

Distinct phosphotyrosines on a growth factor receptor bind to specific molecules that mediate different signaling pathways.

The receptor for platelet-derived growth factor (PDGF) binds two proteins containing SH2 domains, GTPase activating protein (GAP) and phosphatidylinositol 3-kinase (PI3-kinase). The sites on the receptor that mediate this interaction were identified by using phosphotyrosine-containing peptides representing receptor sequences to block specifically binding of either PI3-kinase or GAP. These results suggested that PI3-kinase binds two phosphotyrosine residues, each located in a 5 aa motif with an essential methionine at the fourth position C-terminal to the tyrosine. Point mutations at these sites caused a selective elimination of PI3-kinase binding and loss of PDGF-stimulated DNA synthesis. Mutation of the binding site for GAP prevented the receptor from associating with or phosphorylating GAP, but had no effect on PI3-kinase binding and little effect on DNA synthesis. Therefore, GAP and PI3-kinase interact with the receptor by binding to different phosphotyrosine-containing sequence motifs.

3T3 Cells↗
PubMed1992

An acidic fibroblast growth factor protein generated by alternate splicing acts like an antagonist.

Polymerase chain reaction amplification of cDNA for acidic fibroblast growth factor in several lines of cultured human cells revealed two forms of mRNA. The novel smaller mRNA lacks the entire second coding exon of the acidic fibroblast growth factor gene, whereas the previously identified mRNA consists of three coding exons. The truncated variant of acidic fibroblast growth factor (aFGF') is only 60 amino acids long with an apparent molecular mass of 6.7 kD on sodium dodecyl sulfate gels in contrast to 18 kD for the full-length acidic fibroblast growth factor. aFGF' elicits only minimal fibroblast proliferation and antagonizes the effects of acidic fibroblast growth factor when added exogenously to or when coexpressed with aFGF in BALB/c/3T3 fibroblasts. Thus, the truncated variant of acidic fibroblast growth factor may provide fibroblasts with a unique mechanism for endogenous regulation of their responses to acidic fibroblast growth factor.

Amino Acid Sequence↗
PubMed1992

Membrane Ig cross-linking regulates phosphatidylinositol 3-kinase in B lymphocytes.

Cross-linking of the B cell AgR results in activation of mature B cells and tolerization of immature B cells. The initial signaling events stimulated by membrane immunoglobulin (mIg) cross-linking are tyrosine phosphorylation of a number of proteins. Among the targets of mIg-induced tyrosine phosphorylation are the tyrosine kinases encoded by the lyn, blk, fyn, and syk genes, the mIg-associated proteins MB-1 and Ig-beta, phospholipase C-gamma 1 and -gamma 2, as well as many unidentified proteins. In this report we show that mIg cross-linking also regulates phosphatidylinositol 3-kinase (PtdIns 3-kinase), an enzyme that phosphorylates inositol phospholipids and plays a key role in mediating the effects of tyrosine kinases on growth control in fibroblasts. Cross-linking mIg on B lymphocytes greatly increased the amount of PtdIns 3-kinase activity which could be immunoprecipitated with anti-phosphotyrosine (anti-tyr(P) antibodies. This response was observed after mIg cross-linking in mIgM- and mIgG-bearing B cell lines and after cross-linking either mIgM or mIgD in murine splenic B cells. Thus, regulation of PtdIns 3-kinase is a common feature of signaling by several different isotypes of mIg. This response was rapid and peaked 2 to 3 min after the addition of anti-Ig antibodies. The anti-Ig-stimulated increase in PtdIns 3-kinase activity associated with anti-Tyr(P) immunoprecipitates could reflect increased tyrosine phosphorylation of PtdIns 3-kinase, increased activity of the enzyme, or both. In favor of the first possibility, the tyrosine kinase inhibitor herbimycin A blocked the increase in ant-Tyr(P)-immunoprecipitated PtdIns 3-kinase activity as well as the anti-Ig-induced tyrosine phosphorylation. Moreover, this response was not secondary to phospholipase C activation but rather seemed to be a direct consequence of mIg-induced tyrosine phosphorylation. Activation of the phosphoinositide pathway by a transfected M1 muscarinic acetylcholine receptor expressed in WEHI-231 B lymphoma cells did not increase the amount of PtdIns 3-kinase activity which could be precipitated with anti-Tyr(P) antibodies. Similarly, inhibition of the phosphoinositide pathway did not abrogate the ability of mIg cross-linking to stimulate this response. Thus, mIg-induced tyrosine phosphorylation regulates PtdIns 3-kinase, an important mediator of growth control in fibroblasts and potentially an important regulatory component in B cells as well.

Animals↗
PubMed1992

A potential fusion peptide and an integrin ligand domain in a protein active in sperm-egg fusion.

The union of sperm and egg is a special membrane fusion event that gives a signal to begin development. We have hypothesized that proteins mediating cell-cell fusion events resemble viral fusion proteins and have shown that PH-30, a sperm surface protein involved in sperm-egg fusion, shares biochemical characteristics with viral fusion proteins. We report here the complementary DNA and deduced amino-acid sequences of the mature alpha and beta subunits of PH-30. Both are type-I integral membrane glycoproteins. The alpha subunit contains a putative fusion peptide typical of viral fusion proteins and the beta subunit contains a domain related to a family of soluble integrin ligands found in snake venoms. Thus, the PH-30 alpha/beta complex resembles many viral fusion proteins in both its membrane topology and its predicted binding and fusion functions.

ADAM Proteins↗
PubMed1992

Folding and trimerization of clathrin subunits at the triskelion hub.

The triskelion shape of the clathrin molecule enables it to form the polyhedral protein network that covers clathrin-coated pits and vesicles. Domains within the clathrin heavy chain that are responsible for maintaining triskelion shape and function were identified and localized. Sequences that mediate trimerization are distal to the carboxyl terminus and are adjacent to a domain that mediates both light chain binding and clathrin assembly. Structural modeling predicts that within this domain, the region of heavy chain-light chain interaction is a bundle of three or four alpha helices. These studies establish a low resolution model of clathrin subunit folding in the central portion (hub) of the triskelion, thus providing a basis for future mutagenesis experiments.

Clathrin↗
PubMed1992

Epitopes on the outer surface protein A of Borrelia burgdorferi recognized by antibodies and T cells of patients with Lyme disease.

We have characterized immunogenic epitopes of the 31-kDa outer surface protein A (OspA) protein of Borrelia burgdorferi, which is a major surface Ag of the spirochete causing Lyme disease. Full length and truncated forms of rOspA proteins were expressed in Escherichia coli, and their reactivities with antibodies and human T cell clones isolated from patients with Lyme disease were determined. The epitopes recognized by three of four OspA-reactive T cell clones are contained within the 60 COOH-terminal amino acids. Each of the four OspA-reactive T cell clones has a different HLA class II molecule involved in Ag recognition and recognizes a distinct epitope. One T cell clone promiscuously recognized an epitope in the context of different HLA-DQ molecules. In addition, the binding of a murine monoclonal anti-OspA antibody, as well as antibodies in sera of three of five patients with Lyme disease, was dependent upon the amino acids in the carboxy-terminal protion of this protein. Taken together, our results indicate that the 60 COOH-terminal amino acids of OspA contain epitopes recognized by human antibodies and T cells.

Antibodies, Bacterial↗
PubMed1991

A homolog of the armadillo protein in Drosophila (plakoglobin) associated with E-cadherin.

Three cytoplasmic proteins, called catenins, bind to the cytoplasmic tail of the epithelial cell-cell adhesion molecule E-cadherin. The complementary DNA sequence was determined for the 92-kilodalton beta catenin of Xenopus laevis. The sequence is homologous to mammalian plakoglobin, a protein of desmosomal and zonula adherens cell junctions, and to the plakoglobin homolog in Drosophila melanogaster, the product of the segment polarity gene armadillo. A monoclonal antibody to bovine plakoglobin recognizes the analogous beta catenin in the Madin-Darby canine kidney (MDCK) cell line. Armadillo plakoglobin may link E-cadherin to the underlying actin cytoskeleton at cell-cell junctions; the E-cadherin-catenin protein complex may also participate in the transmission of developmental information.

Amino Acid Sequence↗
PubMed1991

T cell and antibody reactivity with the Borrelia burgdorferi 60-kDa heat shock protein in Lyme arthritis.

The reactivity of cloned T cells and serum antibodies, obtained from patients with chronic Lyme arthritis, with expressed recombinant B. burgdorferi 60-kDa heat shock protein homologue (HSP60) was analyzed. The expressed recombinant Borrelia burgdorferi HSP60 was bound by antibodies in the sera of patients with Lyme arthritis, but not by control sera. A T cell clone (CR253), isolated from one of four patients examined, exhibited an HLA-DR2 restricted proliferative response to the expressed recombinant B. burgdorferi HSP60. This T cell clone specifically recognized the HSP60 of B. burgdorferi and did not proliferate in response to the human, mycobacterial, or Escherichia coli HSP60 homologues. The epitope recognized by this cloned T cell, located between amino acids 260 and 274, is in a region of the spirochetal HSP60 that is not conserved between bacteria and eukaryotes.

Adolescent↗
PubMed1991

Relative quantification of collagen mRNA in fibroblasts by a radioactive polymerase chain reaction technique.

A radioactive polymerase chain reaction (PCR) method has been developed for the relative quantification of the human alpha-2 chain of type I collagen [hu alpha-2(I)] in cells. cDNAs generated by reverse transcription from the total pool of cytoplasmic RNA serve as a template for polymerase chain reaction amplification of a hu alpha-2(I) cDNA primed by two sequence-specific synthetic oligonucleotides. The distinctive 390 bp hu alpha-2(I) cDNA and two Aval fragments of 220 and 170 bp are identified by agarose gel electrophoresis. alpha-32P-dCTP of defined specific activity is included in the PCR reaction and the 390 bp cDNA is excised from the electrophoresis gel to permit direct radioactive quantification of hu alpha-2(I) mRNA. The amount of hu alpha-2(I) mRNA expressed in as few as 111 fibroblasts was determined reliably. In contrast, the hu alpha-2(I) mRNA from at least 5 x 10(5) fibroblasts was required for detection by Northern blot analysis developed with the same cDNA probe radiolabelled with alpha-32P-dCTP by random priming. Human bronchoalveolar lavage (BAL) fluids of six patients with fibrosing lung diseases stimulated the level of expression of hu alpha-2(I) mRNA in cultured human fibroblasts as determined by this technique. The radioactive PCR method thus quantifies hu alpha-2(I) mRNA in fibroblasts with sufficient sensitivity to study fibroblast activation in vitro and detect fibroblast stimuli in human clinical samples.

Autoradiography↗
PubMed1991

Polymerase chain reaction amplification of messages for growth factors in cells from human bronchoalveolar lavage fluids.

Genetic messages for polypeptide growth factors were assessed in human alveolar macrophages, obtained by bronchoalveolar lavage (BAL) from normal subjects (N = 3) and from patients with pneumonia (N = 3), pulmonary lymphoma (N = 3), and idiopathic pulmonary fibrosis (N = 3). Complementary DNAs (cDNAs) were prepared by reverse transcription of the RNA extracted from alveolar macrophages before and after culture on a plastic surface. The cDNAs encoding 10 different growth factors were amplified for electrophoretic analysis by polymerase chain reaction with a pair of 3' and 5' primers specific for each factor. Alveolar macrophages from all normal subjects and patients expressed the messages for interleukin-1 beta and transforming growth factor-beta. Alveolar macrophages from some normal subjects also contained message for insulin-like growth factor-1. Alveolar macrophages from six of nine patients with lung diseases also expressed messages for one or more additional growth factors, including epidermal growth factor, transforming growth factor-alpha, interleukin-1 alpha, and platelet-derived growth factor. The polymerase chain reaction technique thus permits determination of the profile of growth factors contributed to pulmonary reactions by alveolar macrophages, which may be important in pulmonary healing and fibrosis.

Bronchoalveolar Lavage Fluid↗
PubMed1991

Tachykinin receptor cross-talk. Immunological cross-reactivity between the external domains of the substance K and substance P receptors.

In the present study, we have chemically synthesized peptides which correspond to the four putative extracellular domains of the predicted substance K (SK) receptor protein and raised specific polyclonal antibodies against these peptides. These antibodies were then tested for both structural and functional recognition of epitopes on the substance P (SP) receptor on rat AR42J pancreatic cells and human IM9 lymphoblasts, which express the SP receptor, but not the SK receptor. Antibodies directed against the first, second, and fourth external domains of the predicted SK receptor recognized a 58-kDa protein on AR42J cells. This protein has a molecular weight similar to the previously demonstrated SP receptor on both AR42J cells and IM9 cells. Furthermore, antibodies against the second and fourth extracellular domains significantly inhibited specific 125I-SP binding on both AR42J and IM9 cells, and also significantly inhibited SP-induced mobilization of [Ca2+]i on AR42J cells. These data suggest that the second and fourth extracellular domains of the SK and SP receptors may share common structural motifs for ligand binding and signaling mechanism.

Amino Acid Sequence↗
PubMed1991

Interactions of growth factor receptors with cytoplasmic signaling molecules.

The first step in the action of many growth factors is to bind to the receptors and to stimulate autophosphorylation of the receptors on tyrosine residues. The receptors then form high-affinity physical complexes with cytoplasmic signaling molecules (Fig. 8). It is not clear whether the function of the complexes is to localize signaling molecules at the plasma membrane or to position the molecules to be favored substrates of the receptor. It is also not necessarily true that each receptor molecule binds more than one signaling molecule at a time. We have shown that each of the signaling molecules that binds to the PDGF receptor recognizes a specific site in the receptor cytoplasmic domain. A phosphotyrosine on the receptor is an important determinant of the interaction with the signaling molecule. However, the specificity of the interaction is determined by the receptor sequence surrounding each phosphotyrosine, especially the sequences on the carboxy-terminal side of the tyrosine. SH2 regions of the signaling molecules appear to bind directly to the specific recognition sequences on the receptor. Thus, the intracellular protein-protein interactions that depend on SH2 domains binding to phosphotyrosine are not as random as we once believed but are part of a highly specific system of interactions between tyrosine-phosphorylated proteins and SH2-containing signaling proteins. A major role of tyrosine kinase appears to be in creating specific recognition sites that bind SH2 domains. By elucidating the specificity of these interactions, we have been able to selectively block some interactions while allowing others to occur.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

A phosphatidylinositol-3 kinase binds to platelet-derived growth factor receptors through a specific receptor sequence containing phosphotyrosine.

Platelet-derived growth factor (PDGF) stimulates autophosphorylation of the PDGF receptor and association of the receptor with several cytoplasmic molecules, including phosphatidylinositol-3 kinase (PI3 kinase). In this study we examined the association of PI3 kinase with immunoprecipitated autophosphorylated PDGF receptor in vitro. The PI3 kinase from cell lysates bound to the wild-type receptor but not to a mutant receptor that had a deletion of the kinase insert region. A protein of an apparent size of 85 kDa bound to the receptor, consistent with previous observations that a protein of this size is associated with PI3 kinase activity. In addition, 110- and 74-kDa proteins bound to the phosphorylated receptor. Dephosphorylated receptors lost the ability to bind PI3 kinase activity as well as the 85-kDa protein. A 20-amino-acid peptide composed of a sequence in the kinase insert region that included one of the autophosphorylation sites of the receptor (tyrosine 719) as well as a nearby tyrosine (Y708) blocked the binding of PI3 kinase to the receptor, but only when the peptide was phosphorylated on tyrosine residues. A scrambled version of the peptide did not block PI3 kinase binding to the receptor even when it was phosphorylated on tyrosine. These tyrosine-phosphorylated peptides did not block binding of phospholipase C-gamma or GTPase-activating protein to the receptor. In separate experiments (receptor blots), soluble radiolabeled receptor bound specifically to an 85-kDa protein present in sodium dodecyl sulfate-polyacrylamide gel electrophoresis-fractionated 3T3 cell lysates that were transferred to nitrocellulose paper. The binding was blocked by the same tyrosine-phosphorylated peptides that prevented binding of PI3 kinase activity to immobilized receptors. These findings show that the PDGF receptor binds directly to an 85-kDa protein and to a PI3 kinase activity through specific sequences in the kinase insert region. The association of a 110-kDa protein with the receptor also involve these sequences, suggesting that this protein may be a subunit of the PI3 kinase. Phosphotyrosine is an essential structure required for the interactions of these proteins with the PDGF receptor.

Amino Acid Sequence↗
PubMed1991

Isolation and partial characterization of the structures of fibroblast activating factor-related proteins from U937 cells.

Human cultured monocyte-like tumour cells of the U937 histiocyte derived line were stimulated with phorbol myristate acetate, and generated and released an 18,000 MW polypeptide fibroblast-activating factor (FAF). Based on recognition by an antiserum to a synthetic peptide representing the 17 amino-terminal amino acids of FAF, two proteins of 32,000 and 35,000 MW were identified in extracts of U937 cells. Purification of the intracellular FAF-related proteins to homogeneity allowed the generation and amino acid sequencing of nine tryptic fragments of 4-11 amino acids. Neither of the intracellular FAF-related proteins exhibited the fibroblast proliferation-stimulating activity of FAF, suggesting that they are biosynthetic precursors analogous to the inactive propeptides of interleukin-1 beta and tumour necrosis factor-alpha.

Amino Acid Sequence↗
PubMed1990

Characterization of Borrelia burgdorferi proteins reactive with antibodies in synovial fluid of a patient with Lyme arthritis.

Four Borrelia burgdorferi proteins reactive with antibodies in the synovial fluid of a patient with Lyme arthritis were characterized. Homology between amino acid sequences of immunoreactive spirochetal proteins and human proteins, including members of the Escherichia coli GroEL protein family, suggests that antigenic mimicry may play a role in the pathogenesis of Lyme arthritis.

Amino Acid Sequence↗