Biomedical subjects

R D Ye

Publications and source records attributed to R D Ye.

67 records · Page 4Linked to original sources

PubMed1993

Reconstitution of recombinant N-formyl chemotactic peptide receptor with G protein.

A recombinant human neutrophil N-formyl peptide receptor (rFPR) expressed in transfected mouse fibroblasts (TX2 cells) was analyzed for its ability to couple physically with the heterotrimeric G protein, Gi. Immunoprecipitation of photoaffinity-labeled rFPR and endogenous neutrophil formyl peptide receptor (nFPR) with an anti-FPR peptide antibody demonstrated that the receptors were identical in both size and extent of glycosylation. Coupling of rFPR with endogenous TX2 Gi was demonstrated by coimmunoprecipitation of the two proteins with an anti-Gi antibody. Moreover, rFPR was able to form a physical complex with purified Gi in a soluble reconstitution system. We observed similar affinities of rFPR and nFPR for Gi. This report provides the first direct evidence that rFPR associates physically with Gi and provides a foundation for analysis of the G protein coupling capacity of mutant rFPRs.

Animals↗

PubMed1992

Absence of G(i) proteins in the Sf9 insect cell. Characterization of the uncoupled recombinant N-formyl peptide receptor.

We investigated the interaction of the N-formyl peptide receptor (NFPR) with G proteins in infected Sf9 insect cells expressing the recombinant NFPR. Recombinant receptor expression of up to 27 pmol/mg protein was achieved in these cells. The receptor was recognized by an antiserum raised against an NFPR carboxyl-terminal peptide, and displayed specific and saturable binding of the formyl peptide ligand fMet-Leu-[3H]Phe. Scatchard analysis of the binding data yielded a dissociation constant of approximately 62 nM, a binding affinity of 60- to 120-fold lower than that of the high affinity sites in neutrophils and in transfected mammalian cell lines expressing the NFPR. That this low binding affinity was due to a lack of receptor coupling to G protein was suggested by the failure of guanine nucleotides to regulate receptor affinity and by the lack of formyl peptide-stimulated GTPase activity in these cells. Furthermore, immunoblotting with an anti-G(i) antibody and ADP-ribosylation experiments indicated that the approximately 40-kDa G(i) alpha subunit, which couples to the NFPR in neutrophils, is not present in Sf9 cell membranes. Thus, the current study provides for the first time evidence that a major G protein is absent in the Sf9 insect cells. Potential applications of the Sf9 system for in vitro reconstitution of the NFPR-G protein interaction are discussed.

Adenosine Diphosphate Ribose↗

PubMed1992

Isolation of a cDNA that encodes a novel granulocyte N-formyl peptide receptor.

A cDNA of 1650 base pairs was isolated by screening an HL-60 granulocyte library with an N-formyl peptide receptor (NFPR) cDNA probe under low stringency conditions. The cDNA encodes a protein of 351 amino acids tentatively named FPR2, with a calculated molecular weight of 39 kDa. Sequence analysis revealed that FPR2 is 69% identical in sequence to the human NFPR and shares extensive homology to several other chemoattractant receptors. FPR2 expressed in transfected cells mediated formyl peptide-stimulated calcium mobilization at micromolar concentrations of ligand. FPR2 messenger is detected in granulocytic HL-60 cells, but not in undifferentiated HL-60 cells. These findings suggest that FPR2 is a novel receptor for formyl peptide ligand and a new member of the chemoattractant receptor gene family.

Amino Acid Sequence↗

PubMed1991

Characterization of a human cDNA that encodes a functional receptor for platelet activating factor.

We have cloned a cDNA for the platelet activating factor (PAF) receptor by screening an HL-60 granulocyte cDNA library with degenerate oligonucleotide probes based on conserved sequences of rhodopsin-type receptors. The 342-amino acid receptor contains 7 putative transmembrane domains, but lacks sites for N-linked glycosylation at the N-terminus. Stably transfected fibroblasts expressing the cloned cDNA responded to sub-nanomolar PAF stimulation with calcium mobilization, which could be inhibited by the PAF antagonist L-659,989. The PAF receptor message was detected as a single species of approximately 4 kb in human placenta, lung, and differentiated HL-60 granulocytes. Expression of the cloned cDNA in undifferentiated HL-60 cells devoid of endogenous PAF receptor resulted in specific and saturable binding of the PAF antagonist WEB 2086 with a dissociation constant of 30.7 nM.

Amino Acid Sequence↗

PubMed1991

Transmembrane signalling by the N-formyl peptide receptor in stably transfected fibroblasts.

We investigated the requirement for N-formyl peptide receptor-mediated transmembrane signalling in transfected mouse fibroblasts that express the receptor. Stably transfected cells displayed specific binding for N-formyl-Met-Leu-[3H]Phe with a dissociation constant of 3 nM. The cells responded to ligand stimulation with mobilization of calcium from intracellular stores. Calcium mobilization was ligand dose-dependent (EC50 = 3 nM fMet-Leu-Phe) and could be inhibited by pertussis toxin treatment. These results provide the first demonstration that expression of the single-chain N-formyl peptide receptor in mouse fibroblasts is sufficient for mediating ligand-induced early transmembrane signalling events, which do not appear to require other neutrophil-specific cellular components.

Animals↗

PubMed1991

Localization of the signal for rapid internalization of the bovine cation-independent mannose 6-phosphate/insulin-like growth factor-II receptor to amino acids 24-29 of the cytoplasmic tail.

The signal for the rapid internalization of the cation-independent mannose 6-phosphate/insulin-like growth factor-II receptor has been previously localized to the inner half of the 163-amino acid cytoplasmic tail, including tyrosine 24 and tyrosine 26 (Lobel, P., Fujimoto, K., Ye, R. D., Griffiths, G., and Kornfeld, S. (1989) Cell 57, 787-796). To define this signal more precisely, we generated a series of truncation and substitution mutants and analyzed them for their ability to mediate the endocytosis of extracellular lysosomal enzymes. Mutant receptors containing cytoplasmic domains of 29 or more amino acids functioned normally in endocytosis whereas a mutant receptor with a 28-amino acid cytoplasmic domain was internalized extremely slowly. Alanine scanning of the region between amino acids 19 and 30 identified tyrosine 26 and valine 29 as the most important residues for rapid receptor internalization. Tyrosine 24 and lysine 28 also contributed to the signal while the other amino acids were not critical. The tyrosine residues could be substituted with phenylalanines with no loss of activity, indicating the requirement for an aromatic residue in these positions rather than tyrosine specifically. Conservative substitutions of arginine or histidine for lysine 28 also preserved the internalization signal. These results indicate that the sequence Tyr-Lys-Tyr-Ser-Lys-Val serves as the internalization signal for the mannose 6-phosphate/insulin-like growth factor-II receptor. The crucial elements of this sequence are present in the cytoplasmic tails of a number of other membrane receptors and proteins known to undergo rapid internalization.

Amino Acid Sequence↗

PubMed1989

Mutations in the cytoplasmic domain of the 275 kd mannose 6-phosphate receptor differentially alter lysosomal enzyme sorting and endocytosis.

The cation-independent mannose 6-phosphate receptor (Cl-MPR) sorts newly synthesized lysosomal enzymes in the Golgi and endocytoses extracellular lysosomal enzymes. To determine the role of the 163 amino acid cytoplasmic domain of the Cl-MPR in these functions, receptor-deficient mouse L cells were transfected with normal bovine Cl-MPR cDNA or cDNAs mutated in the cytoplasmic domain. The normal Cl-MPR functioned in sorting and endocytosis. Mutant receptors with 40 and 89 residues deleted from the carboxyl terminus of the cytoplasmic tail functioned normally in endocytosis, but were partially impaired in sorting. Mutant receptors with larger deletions leaving only 7 and 20 residues of the cytoplasmic tail were defective in endocytosis and sorting. A mutant receptor containing alanine instead of tyrosine residues at positions 24 and 26 was defective in endocytosis, and partially impaired in sorting. Receptors deficient in endocytosis accumulated at the cell surface. These results indicate that the cytoplasmic domain of the Cl-MPR contains different signals for rapid endocytosis and efficient lysosomal enzyme sorting.

Amino Acid Sequence↗

PubMed1989

Structure of the gene for human plasminogen activator inhibitor-2. The nearest mammalian homologue of chicken ovalbumin.

Plasminogen activator inhibitor-2 (PAI-2) can regulate the formation of plasmin by inhibiting urokinase and tissue plasminogen activator. PAI-2 is induced in monocytes and endothelium by inflammatory mediators, and it is made in the placenta during pregnancy. PAI-2 is a member of the serine protease inhibitor gene family, and it is particularly similar to chicken ovalbumin. Like ovalbumin, PAI-2 is secreted without cleavage of a signal peptide. To determine the structure of the PAI-2 gene, two bacteriophage lambda human genomic DNA libraries were screened with PAI-2 cDNA probes. Characterization of three positive clones shows that the human PAI-2 gene spans 16.5 kilobases and has eight exons. The 5'-untranslated sequence of the PAI-2 mRNA is 77 base pairs in length as suggested by primer extension and S1 nuclease mapping. The eukaryotic consensus sequence TATAAAA is found 22 base pairs 5' of the proposed cap site. The PAI-2 gene is on chromosome 18q21-23 as determined by hybridization to flow-sorted chromosomes and by in situ hybridization. There appear to be two common PAI-2 alleles that differ by six nucleotides in exons 1, 4, and 8. The structure of the PAI-2 gene is quite different from that of PAI-1 although these two inhibitors have common target protease specificity. In contrast, the structure of the PAI-2 gene is very similar to that of the chicken ovalbumin gene. When protein sequences are aligned to obtain maximal identity, six of the seven intron positions in the PAI-2 gene are identical to those in the chicken ovalbumin gene. We conclude that PAI-2 is the closest mammalian homologue of avian ovalbumin.

Amino Acid Sequence↗

PubMed1989

Use of yeast artificial chromosome clones for mapping and walking within human chromosome segment 18q21.3.

Well-characterized large genomic clones obtained from yeast artificial chromosome (YAC) libraries provide the framework to localize genes and approach genetic disease. We developed universally applicable approaches to establish authenticity, localize and orient internal genes, map restriction sites, and rescue the distal ends of large human genomic DNA inserts. We selected human chromosome segment 18q21.3 as a model system. Molecular cloning of this segment was initiated by characterizing three plasminogen activator inhibitor type 2 (PAI-2) clones [290, 180, and 60 kilobases (kb)] isolated from a YAC library. Comparison of YAC and bacteriophage lambda genomic DNA clones confirmed the fidelity of the PAI-2 locus. Detailed rare cutting restriction maps were generated by ramped contour-clamped homogeneous electric field electrophoresis. The PAI-2 locus was located and oriented within the YACs, which span a distance 70 kb 5' to 220 kb 3' of PAI-2. Moreover, both left and right ends of the YAC genomic DNA inserts were rescued by amplifying circularized cloning sites with an inverted form of the polymerase chain reaction. These unique terminal genomic DNA fragments were used to rescreen the YAC library and isolate overlapping clones that extend the map. These approaches will enable neighboring loci to be definitively linked and establish the feasibility of using YAC technology to clone and map chromosomal segments.

Base Sequence↗

PubMed1988

Mammalian protein secretion without signal peptide removal. Biosynthesis of plasminogen activator inhibitor-2 in U-937 cells.

Plasminogen activator inhibitor-2 (PAI-2) is a serine protease inhibitor that regulates plasmin generation by inhibiting urokinase and tissue plasminogen activator. The primary structure of PAI-2 suggests that it may be secreted without cleavage of a single peptide. To confirm this hypothesis we have studied the glycosylation and secretion of PAI-2 in human monocytic U-937 cells by metabolic labeling, immunoprecipitation, glycosidase digestion, and protein sequencing. PAI-2 is variably glycosylated on asparagine residues to yield intracellular intermediates with zero, one, two, or three high mannose-type oligosaccharide units. Secretion of the N-glycosylated species began by 1 h of chase and the secreted molecules contained both complex-type N-linked and O-linked oligosaccharides. Enzymatically deglycosylated PAI-2 had an electrophoretic mobility identical to that of the nonglycosylated precursor and also to that of PAI-2 synthesized in vitro in a rabbit reticulocyte lysate from synthetic mRNA derived from full length PAI-2 cDNA. The amino-terminal protein sequence of secreted PAI-2 began with the initiator methionine residue. These results indicate that PAI-2 is glycosylated and secreted efficiently without the cleavage of a signal peptide. PAI-2 shares this property with its nearest homologue in the serine protease inhibitor family, chicken ovalbumin, and appears to be the first well characterized example of this phenomenon among natural mammalian proteins.

Animals↗

PubMed1987

Human thrombomodulin: complete cDNA sequence and chromosome localization of the gene.

A human umbilical vein endothelial cell cDNA library in lambda gt11 was screened for expression of thrombomodulin antigens with affinity-purified rabbit polyclonal anti-thrombomodulin immunoglobulin G (IgG) and mouse monoclonal anti-human thrombomodulin IgG. Among 7 million recombinant clones screened, 12 were recognized by both antibodies. Two of these, lambda HTm10 and lambda HTm12, were shown to encode thrombomodulin by comparison of the amino acid sequence deduced from the nucleotide sequence to the amino acid sequence determined directly from tryptic peptides of thrombomodulin. Thrombomodulin mRNA was estimated to be 3.7 kilobases in length by Northern blot analysis of endothelial cell and placental poly(A)+ RNA. Thrombomodulin mRNA was not detected in human brain, HepG2 hepatoma cells, or the monocytic U937 cell line. Additional cDNA clones were selected by hybridization with the 1.2-kilobase insert of lambda HTm10. One isolate, lambda HTm15, contained a 3693 base pair cDNA insert with an apparent 5'-noncoding region of 146 base pairs, an open reading frame of 1725 base pairs, a stop codon, a 3'-noncoding region of 1779 base pairs, and a poly(A) tail of 40 base pairs. The cDNA sequence encodes a 60.3-kDa protein of 575 amino acids. The predicted protein sequence includes a signal peptide of approximately 21 amino acids, an amino-terminal ligand-binding domain of approximately 223 amino acids, an epidermal growth factor (EGF) homology region of 236 amino acids, a serine/threonine-rich segment of 34 amino acids, a membrane-spanning domain of 23 amino acids, and a cytoplasmic tail of 38 amino acids. The EGF-homology region consists of six tandemly repeated EGF-like domains.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1987

cDNA cloning and expression in Escherichia coli of a plasminogen activator inhibitor from human placenta.

Two nearly full-length cDNAs for placental plasminogen activator inhibitor (PAI) have been isolated from a human placenta lambda gt11 cDNA library. One positive (lambda PAI-75.1) expressed a protein that could adsorb and purify anti-PAI antibodies. The expressed protein inhibited the activity of human urokinase in a fibrin autography assay, and formed a 79-kDa (reduced) covalent complex with 125I-urokinase that could be immunoprecipitated with anti-PAI. The cDNA insert of the longer isolate (lambda PAI-75.15) consisted of 1909 base pairs, including a 5'-noncoding region of 55 base pairs, an open reading frame of 1245 base pairs, a stop codon, a 3'-noncoding region of 581 base pairs, and a poly(A) tail. The size of the mRNA was estimated to be 2.0 kilobases by Northern blot analysis. The translated amino acid sequence consisted of 415 amino acids, corresponding to a 46.6-kDa protein. The sequence was related to members of the serpin gene family, particularly ovalbumin and the chicken gene Y protein. Like these avian proteins, placental PAI appears to lack a cleavable NH2-terminal signal peptide. Residues 347-376 were identical to the partial sequence reported recently for a PAI isolated from the human monocytic U-937 cell line. Placental PAI mRNA was apparently expressed at low levels in human umbilical vein endothelial cells, but was not detectable in HepG2 hepatoma cells. It was present in U-937 cells and was inducible at least 10-fold by phorbol 12-myristate 13-acetate. Thus placental PAI is a unique member of the serpin gene family, distinct from endothelial-type PAI. It is probably identical to monocyte-macrophage PAI.

Amino Acid Sequence↗

PubMedDate not supplied

Strategies for positioning fluorescent probes and crosslinkers on formyl peptide ligands.

Chemoattractant receptors represent a major subset of the G-protein coupled receptor (GPCR) family. One of the best characterized, the N-formyl peptide receptor (FPR), participates in host defense responses of neutrophils. The features of the ligand which regulate its interaction with the FPR are well-known. By manipulating these features we have developed new ligands to probe structural and mechanistic aspects of the peptide-receptor interaction. Three ligand groups have been developed: 1) ligands containing a Lys residue located in positions 2 through 7 that can be conjugated to FITC (N-formyl-Met1-Lys2-Phe3-Phe4, N-formyl-Met1-Leu2-Lys3-Phe4, N-formyl-Met1-Leu2-Phe3-Lys4, N-formyl-Met1-Leu2-Phe3-Phe4-Lys5, N-formyl-nLeu1-Leu2-Phe3-nLeu4-Tyr5-Lys6 and N-formyl-Met1-Leu2-Phe3-Phe4-Gly5-Gly6-Lys7; 2) fluorescent pentapeptide ligands (N-formyl-Met-X-Phe-Phe-Lys(FITC) where X = Leu, Ala, Val or Gly); and 3) small crosslinking ligands where the photoaffinity crosslinker 4-azidosalicylic acid (ASA) was conjugated to Lys in positions 3 and 4 and p-benzoyl-phenylalanine (Bpa) was located in position 2 in N-formyl-Met1-Bpa2-Phe3-Tyr4. The peptides were characterized according to activity and affinity in human neutrophils and cell lines transfected with FPR. All of the peptides were agonists, with parallel affinity and activity. In the first group, the peptide activity decreases as Lys is placed closer to the N-formyl group and the activity is improved by 1-3 orders of magnitude by conjugation with FITC. In the second group, the dissociation rate of the peptide from the receptor increases as position 2 is replaced by aliphatic amino acids with smaller alkyl groups. In the third group, crosslinking ligands remain biologically active, display nM affinity and covalently label the FPR.

Animals↗