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C Gerard

Publications and source records attributed to C Gerard.

At least 109 records · Page 6Linked to original sources
PubMed1993

Polarized distribution of gamma interferon-stimulated MHC antigens and transferrin receptors in a clonal cell line isolated from Fisher rat thyroid (FRT cells).

The characteristics of a polarized epithelial cell line and dynamics of an endogenous polarized plasma membrane constituent were studied by use of an subclone, FRT-9, from the Fisher rat thyroid cell line, FRT. Transmission electron microscopy (conventional, freeze-fracture), determination of transepithelial electrical parameters and immuno-fluorescence study, were used to establish polarity and demonstrated the basolateral distribution of transferrin receptors and the major histocompatibility complex antigens (constitutive Class I or gamma interferon-induced Class II).

Animals↗
PubMed1992

Structural diversity in the extracellular faces of peptidergic G-protein-coupled receptors. Molecular cloning of the mouse C5a anaphylatoxin receptor.

The mouse C5a receptor gene was isolated using the human C5a receptor cDNA probe recently described (Gerard, N. P., and C. Gerard. 1991. Nature 349:614). By analogy with the human gene, the mouse homolog contains two exons with the 5' untranslated region and initiating methionine codon present in exon 1 and the remainder of the molecule in exon 2. Generation of an expressible cDNA for the mouse C5a receptor was accomplished using the polymerase chain reaction and a sense oligodeoxynucleotide primer which included an initiation codon just 5' to the sequence encoding the N-linked glycosylation site. When transfected into human 293 kidney epithelial cells the cloned cDNA directs expression of a binding site for human C5a anaphylatoxin with a binding constant of 2.5 +/- 0.3 nM; the human C5a receptor expressed under identical conditions has a Kd of 1.7 +/- 0.2 nM. Overall, the deduced amino acid sequences of the receptors are 65% identical given the analogous gene structures. Alignment of the sequences as seven transmembrane segment receptors reveals that the greatest structural diversity (approximately 70%) exists in the putative extracellular domains. In contrast, species differences among other members of this family of seven membrane-spanning receptors is generally only 10 to 20%, even for receptors whose ligands are relatively small and not expected to interact with sites on the extracellular surfaces. A high degree of structural identify is observed for the C5a receptors in the transmembrane segments and in all but one of the loops predicted to exist in the cytoplasm. Inasmuch as critical structures responsible for high affinity binding of the 74 amino acid polypeptide to both C5a receptors involve features conserved between species, these data provide the starting point for mutagenesis studies to determine the nature of the binding and activation sites for the chemotactic receptors. Additionally, these data provide a reagent for immunologic and molecular genetic studies on the role of C5a receptors in inflammatory models.

Amino Acid Sequence↗
PubMed1992

The human leukocyte platelet-activating factor receptor. cDNA cloning, cell surface expression, and construction of a novel epitope-bearing analog.

A human myeloid transcript of approximately 4 kilobases was cloned as a cDNA from an expression library based on homology with the guinea pig cDNA recently described by Honda et al. (Honda, Z-I., Nakamura, M., Miki, I., Minami, M., Watanabe, T., Seyama, Y., Okado, H., Tok, H., Ito, K., Miyamato, T., and Shimizu, T. (1991) Nature 349, 342-346) as a receptor for platelet-activating factor (PAF). The cloned DNA confers high affinity binding sites for platelet-activating factor when transfected into COS-7 cells and has binding and desensitization properties similar to the human leukocyte receptor. Southern analysis using this cDNA indicates that the PAF receptor gene is present as a single copy in the human genome. The deduced protein sequence predicts seven hydrophobic regions for the PAF receptor, characteristic of the rhodopsin gene family, and is 83% identical to the deduced protein sequence of the corresponding guinea pig molecule. A modified human PAF receptor cDNA was constructed by inserting an additional 30 nucleotides after the 5'-ATG, encoding the amino acid sequence MDYKDDDDKEF, which is specifically recognized by a monoclonal antibody. The modified cDNA encodes a functional PAF receptor and is detected by antibody on the membrane of transfected COS-7 cells. The use of this construct supports the structural model for the rhodopsin-like superfamily of receptors which places the NH2-terminal sequence on the extracellular side of the membrane, and should additionally be useful for affinity purification of the receptor protein.

Amino Acid Sequence↗
PubMed1992

Insulin like growth factor I is an autocrine regulator of human colon cancer cell differentiation and growth.

The polyanionic compound suramin triggers enterocyte-like differentiation of the human colic adenocarcinoma cell clone HT29-D4. We now demonstrate that suramin interferes with the binding of IGF-I to its receptor at the surface of HT29-D4 cells. Half-maximum inhibition of 125I-IGF-I binding was obtained in the presence of 25 micrograms/ml suramin. Moreover, the drug was able to dissociate 125I-IGF previously bound to its cell surface receptor. Affinity labeling HT29-D4 cells were cultured in the presence of 10 micrograms/ml of alpha-IR3, a monoclonal antibody directed against the binding site of IGF-I, an inhibition of cell proliferation and a stimulation of cell differentiation was observed. After 10 days of treatment with alpha-IR3, HT29-D4 cells formed a regular monolayer of enterocyte-like cells exhibiting an apical brush border and tight junctions delimiting two domains of the plasma membrane (apical and basolateral). Furthermore, we show that IGF-I significantly increased the initial rate of glucose uptake by HT29-D4 cells, while we have previously shown that suramin decreased glucose consumption. From these data we conclude that IGF-I secreted by the cells themselves, stimulates proliferation of HT29-D4 cells via an autocrine mechanism. Blockade of this stimulation by suramin or by a specific monoclonal antibody inhibits cell growth, glucose uptake and triggers the process of enterocytic differentiation.

Biological Transport↗
PubMed1992

Functional high efficiency expression of cloned leukocyte chemoattractant receptor cDNAs.

Human kidney 293 TSA cells were transfected by a calcium phosphate method with human formylpeptide and C5a receptor cDNAs with high efficiency. Formylpeptide receptor positive transfectants expressed a total of 968,000 +/- 34,000 receptors per cell with two affinity states (Kds of ca. 0.43 nM and 39 nM), which in the presence of 100 microM GTP gamma S decreased by ca. 4-fold the number of high-affinity sites. The ligand binding pharmacology of cloned and expressed formylpeptide receptors were indistinguishable from endogenous receptors on human neutrophils. Expressed formylpeptide and C5a receptors were functionally active in mobilizing intracellular calcium via a pertussis toxin sensitive mechanism with an ED50 for formylpeptide of ca. 0.5-1.0 nM. This expression system, in which receptor expression can be monitored by flow cytometric methods and in which intracellular calcium responses are measurable, unlike in the more popular COS-7 cell expression system, will provide a useful basis for the analysis of chemoattractant receptor structure-function relationships.

Base Sequence↗
PubMed1992

Mapping of genes for the human C5a receptor (C5AR), human FMLP receptor (FPR), and two FMLP receptor homologue orphan receptors (FPRH1, FPRH2) to chromosome 19.

The genes encoding receptors for the chemotactic ligands C5a (C5AR) and FMLP (FPR) were mapped using a panel of somatic cell hybrids to chromosome 19. Because the hybridization pattern on Southern analysis suggested an intron structure or related genes in the case of FPR, genomic clones were characterized. Two structural homologues of the FMLP receptor, clones 81 (FPRH1) and 82 (FPRH2), were identified, which similarly map to chromosome 19. The structural homologues do not recognize the ligand FMLP, but are likely chemotactic receptors.

Amino Acid Sequence↗
PubMed1992

Exon 16 del: a novel form of human neutral endopeptidase (CALLA).

The enzyme neutral metalloendopeptidase (E.C. 3.4.24.11), also known as the common acute lymphocytic leukemia antigen, neutral endopeptidase, or enkephalinase, functions as an inactivator of a wide variety of signaling oligopeptides such as substance P, neurokinin A, enkephalins, endothelin, atrial natriuretic factor, and formyl chemotactic peptides. A cDNA clone isolated from a human lung library encodes a fragment of neutral metalloendopeptidase containing an internal 81 base pair deletion when compared with the human placental cDNA for this enzyme. Comparison of the deleted cDNA sequence with the intron-exon structure recently determined as the common acute lymphocytic leukemia antigen reveals that the 81 base pairs corresponds precisely with exon 16. RNA analysis using splice junction oligonucleotides indicates that the 16 del form constitutes a minor but significant fraction of the RNA species present in human lung. Expression of constructs containing "wild type" and "exon 16 del" neutral endopeptidases in COS-7 cells reveals that deletion of this 27 amino acid segment reduces enzymatic activity toward the synthetic substrate glutaryl-alanyl-alanyl-phenyl-alanyl-4-methoxy-2-naphthylamide to barely detectable levels.

Amino Acid Sequence↗
PubMed1991

Receptor class desensitization of leukocyte chemoattractant receptors.

To better define their regulation, formylpeptide and C5a chemoattractant receptor cDNAs were transiently expressed with high efficiency (approximately 35-54%) in human kidney cells. As in neutrophils, both receptors were active in elevating intracellular calcium (ED50 approximately 0.5-1 nM). Agonist-specific desensitization for calcium elevation was observed for both chemoattractant receptors at doses of approximately 1 nM. Heterologous desensitization of formylpeptide, C5a, and alpha 1-adrenergic receptors required high doses of phorbol ester (100 nM phorbol 12-myristate 13-acetate). To further study the phenomenon of desensitization, formylpeptide and C5a receptor cDNAs were cotransfected resulting in approximately 80% of receptor-positive cells expressing both receptors. These cells also possessed endogenous alpha 1-adrenergic receptors. Interestingly, chemoattractant receptors were cross-desensitized by pretreatment with low doses of either C5a or formylmethionylleucylphenylalanine (10 nM) but not by the alpha 1-adrenergic agonist norepinephrine (up to 10 microM). Neither chemoattractant desensitized alpha 1-adrenergic receptors. This phenomenon was reproduced in human neutrophils. These data suggest a previously uncharacterized mechanism of receptor regulation, which is intermediate between homologous and heterologous desensitization. Class desensitization of chemoattractant receptors is less selective than homologous desensitization but is far more efficient and specific than heterologous desensitization. Receptor class desensitization may affect functional classes of receptors via modification of either the receptor or the shared guanine nucleotide-binding regulatory protein.

Adenoviruses, Human↗
PubMed1991

Human substance P receptor (NK-1): organization of the gene, chromosome localization, and functional expression of cDNA clones.

The gene for the human substance P receptor (NK-1) was cloned using cDNA probes made by the polymerase chain reaction from primers based on the rat sequence. The gene spans 45-60 kb and is contained in five exons, with introns interrupting at sites homologous to those in the NK-2 receptor gene. Analysis of restriction digests of genomic DNA from mouse/human cell hybrids indicates the NK-1 receptor is a single-copy gene located on human chromosome 2. Polymerase chain reaction using primers based on the 5' and 3' ends of the coding sequence was used to generate full-length cDNAs from human lung and from IM9 lymphoblast cells. When transfected into COS-7 cells, the NK-1 receptor binds 125I-BHSP with a Kd of 0.35 +/- 0.07 nM and mediates substance P induced phosphatidylinositol metabolism. The receptor is selective for substance P; the relative affinity for neurokinin A and neurokinin B is 100- and 500-fold lower, respectively. Human IM9 lymphoblast cells express relatively high levels of the NK-1 receptor, and Northern blot analysis indicates modulation of mRNA levels by glucocorticoids and growth factors, suggesting that this cell line may be useful as a model for studying the control of NK-1 receptor gene expression.

Amino Acid Sequence↗
PubMed1991

A serine esterase released by human alveolar macrophages is closely related to liver microsomal carboxylesterases.

We identified a 60-kDa diisopropylfluorophosphate-(DFP) reactive protein in human bronchoalveolar lavage fluid, at a yield of 50-100 pmol/lavage. The protein is associated with the cell-free lavage fluid sediment, which consists mainly of surfactant. [3H]DFP labeling is inhibited by heating to 56 degrees C, 2 mM phenylmethylsulfonylfluoride and 1 mM bis(4-nitrophenyl)-phosphate. An identical 60-kDa [3H]DFP-reactive protein is present in the insoluble fraction of alveolar macrophage-conditioned culture medium and in total membrane preparations of alveolar macrophages. The [3H]DFP-labeled protein was purified approximately 30-fold from lavage fluid sediment by size-exclusion (Sephacryl S-200) and ion-exchange (Mono-Q) chromatography. Cyanogen bromide treatment of the partially purified protein produced a major labeled peptide of 14 kDa with an NH2-terminal sequence 90% identical to a region of form 1 rabbit liver microsomal carboxylesterase. Esterase activity in unlabeled starting material, detected using p-nitrophenyl valerate as substrate, copurified with the [3H]DFP-labeled enzyme. Degenerate oligonucleotide primers were designed based on the partial amino acid sequence and on a highly conserved region of known liver carboxylesterase sequences. Polymerase chain reaction using these primers and reverse-transcribed human alveolar macrophage mRNA yielded a 354-base pair product which was then used to screen a human alveolar macrophage cDNA library. A complete esterase sequence was obtained from two incomplete, overlapping clones, and is virtually identical to human liver carboxylesterase partial sequences. Northern blot analysis demonstrated a single approximately 1.7-kilobase transcript in human monocytes and alveolar macrophages, with much higher levels in the latter. These data indicate that human alveolar macrophages both contain and release a serine esterase that is apparently identical to liver microsomal carboxylesterase. Its enzymatic profile suggests it is a major component of alveolar macrophage-nonspecific esterase activity. We hypothesize that it acts as a detoxication enzyme in the lung.

Adult↗
PubMed1991

Molecular characterization of the interleukin-8 receptor.

Recently a rabbit cDNA (F3R) was characterized as binding and causing calcium mobilization induced by the formyl-methionine-leucine-phenylalanine peptide (fMLP). In the study reported here, cloned DNAs were isolated from rabbit genomic DNA by PCR based on the sequence of F3R. The cloned DNAs have several differences in the DNA sequence compared to the reported F3R sequence that alter the predicted protein sequence. COS-7 cells transfected with these clones in a mammalian expression vector bind human IL-8 with high affinity, but do not bind fMLP. We therefore believe that the cDNAs isolated encode the rabbit IL-8 receptor.

Amino Acid Sequence↗
PubMed1991

The chemotactic receptor for human C5a anaphylatoxin.

Host defence and inflammatory responses are controlled and amplified by receptor-mediated events often initiated by a chemotactic factor that directs the approach of phagocytic cells. Complement receptors CR1 and CR3 are responsible for the phagocytic and adhesive properties of neutrophils, whereas the C5a receptor mediates the pro-inflammatory and chemotactic actions of the complement anaphylatoxin C5a. In addition to stimulating chemotaxis, granule enzyme release and superoxide anion production, this receptor stimulates upregulation of expression and activity of the adhesion molecule MAC-1, and of CR1, and a decrease in cell-surface glycoprotein 100MEL-14 on neutrophils. In vivo, the C5a receptor may participate in anaphylactoid and septic shock. The human C5a receptor was cloned from U937 and HL-60 cells and identified by high affinity binding when expressed in COS-7 cells. The deduced amino-acid sequence of the receptor reveals the expected motifs befitting its interaction with cellular GTP-binding proteins.

Amino Acid Sequence↗