Identification of a cysteine-rich receptor for fibroblast growth factors

1992 ◽  
Vol 12 (12) ◽  
pp. 5600-5609
Author(s):  
L W Burrus ◽  
M E Zuber ◽  
B A Lueddecke ◽  
B B Olwin
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Integral Membrane Protein ◽  
Fibroblast Growth ◽  
Fgf Receptor ◽  
Ovary Cells ◽  
Multiple Receptors ◽  
High Degree

The fibroblast growth factor (FGF) family consists of seven members whose activities are thought to be mediated by multiple receptors. Here we describe the cDNA cloning, expression, and characterization of a cysteine-rich FGF receptor (CFR) that is distinct from previously identified FGF receptors. The deduced amino acid sequence for CFR suggests that it is an integral membrane protein containing a large extracellular domain comprising 16 cysteine-rich repeated units and an intracellular domain of 13 amino acids. No reported sequences exhibit significant homologies to either the repeated extracellular motif or to the entire CFR amino acid sequence. Several CFR transcripts are present in embryonic chick tissue, suggesting that CFR undergoes alternate mRNA splicing or that related genes are present. Chinese hamster ovary cells transfected with the CFR cDNA express a 150-kDa polypeptide that binds FGF-1, FGF-2, and FGF-4 but does not bind several non-FGF family members. The high degree of evolutionary conservation among vertebrate CFRs and its ability to bind three different FGFs with high affinity suggest that this unique receptor plays an important role in FGF biology.

scholarly journals Identification of a cysteine-rich receptor for fibroblast growth factors.

1992 ◽  
Vol 12 (12) ◽  
pp. 5600-5609 ◽  
Author(s):  
L W Burrus ◽  
M E Zuber ◽  
B A Lueddecke ◽  
B B Olwin
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Integral Membrane Protein ◽  
Fibroblast Growth ◽  
Fgf Receptor ◽  
Ovary Cells ◽  
Multiple Receptors ◽  
High Degree

The fibroblast growth factor (FGF) family consists of seven members whose activities are thought to be mediated by multiple receptors. Here we describe the cDNA cloning, expression, and characterization of a cysteine-rich FGF receptor (CFR) that is distinct from previously identified FGF receptors. The deduced amino acid sequence for CFR suggests that it is an integral membrane protein containing a large extracellular domain comprising 16 cysteine-rich repeated units and an intracellular domain of 13 amino acids. No reported sequences exhibit significant homologies to either the repeated extracellular motif or to the entire CFR amino acid sequence. Several CFR transcripts are present in embryonic chick tissue, suggesting that CFR undergoes alternate mRNA splicing or that related genes are present. Chinese hamster ovary cells transfected with the CFR cDNA express a 150-kDa polypeptide that binds FGF-1, FGF-2, and FGF-4 but does not bind several non-FGF family members. The high degree of evolutionary conservation among vertebrate CFRs and its ability to bind three different FGFs with high affinity suggest that this unique receptor plays an important role in FGF biology.

N-terminal residues in murine P-selectin glycoprotein ligand-1 required for binding to murine P-selectin

Blood ◽  
2003 ◽  
Vol 101 (2) ◽  
pp. 552-559 ◽  
Author(s):  
Lijun Xia ◽  
Vishwanath Ramachandran ◽  
J. Michael McDaniel ◽  
Kiem N. Nguyen ◽  
Richard D. Cummings ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Fluid Phase ◽  
Terminal Region ◽  
Wild Type ◽  
Ovary Cells ◽  
N Terminus

P-selectin binds to the N-terminal region of human P-selectin glycoprotein ligand-1 (PSGL-1). For optimal binding, this region requires sulfation on 3 tyrosines and specific core-2O-glycosylation on a threonine. P-selectin is also thought to bind to the N terminus of murine PSGL-1, although it has a very different amino acid sequence than human PSGL-1. Murine PSGL-1 has potential sites for sulfation at Tyr13 and Tyr15 and for O-glycosylation at Thr14 and Thr17. We expressed murine PSGL-1 or constructs with substitutions of these residues in transfected Chinese hamster ovary cells that coexpressed the glycosyltransferases required for binding to P-selectin. The cells were assayed for binding to fluid-phase P-selectin and for tethering and rolling on P-selectin under flow. In both assays, substitution of Tyr13 or Thr17 markedly diminished, but did not eliminate, binding to P-selectin. In contrast, substitution of Tyr15 or Thr14 did not affect binding. Substitution of all 4 residues eliminated binding. Treatment of cells with chlorate, an inhibitor of sulfation, markedly reduced binding of wild-type PSGL-1 to P-selectin but did not further decrease binding of PSGL-1 with substitutions of both tyrosines. These data suggest that sulfation of Tyr13 andO-glycosylation of Thr17 are necessary for murine PSGL-1 to bind optimally to P-selectin. Because it uses only one tyrosine, murine PSGL-1 may rely more on other peptide components andO-glycosylation to bind to P-selectin than does human PSGL-1.

scholarly journals Peroxisome proliferator-induced acyl-CoA thioesterase from rat liver cytosol: molecular cloning and functional expression in Chinese hamster ovary cells

1997 ◽  
Vol 323 (2) ◽  
pp. 525-531 ◽  
Author(s):  
Susanna T. ENGBERG ◽  
Toshifumi AOYAMA ◽  
Stefan E. H. ALEXSON ◽  
Takashi HASHIMOTO ◽  
L. Thomas SVENSSON
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Rat Liver ◽  
Chinese Hamster Ovary ◽  
Specific Activity ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cell Protein ◽  
Peroxisome Proliferator ◽  
Ovary Cells

We have isolated and cloned a cDNA that codes for one of the peroxisome proliferator-induced acyl-CoA thioesterases of rat liver. The deduced amino acid sequence corresponds to the major induced isoform in cytosol. Analysis and comparison of the deduced amino acid sequence with the established consensus sequences suggested that this enzyme represents a novel kind of esterase with an incomplete lipase serine active site motif. Analyses of mRNA and its expression indicated that the enzyme is significantly expressed in liver only after peroxisome proliferator treatment, but isoenzymes are constitutively expressed at high levels in testis and brain. The reported cDNA sequence is highly homologous to the recently cloned brain acyl-CoA thioesterase [Broustas, Larkins, Uhler and Hajra (1996) J. Biol. Chem. 271, 10470–10476], but subtle differences throughout the sequence, and distinct differences close to the resulting C-termini, suggest that they are different enzymes, regulated in different manners. A full-length cDNA clone was expressed in Chinese hamster ovary cells and the expressed enzyme was characterized. The palmitoyl-CoA hydrolysing activity (Vmax) was induced approx. 9-fold to 1 μmol/min per mg of cell protein, which was estimated to correspond to a specific activity of 250 μmol/min per mg of cDNA-expressed enzyme. Both the specific activity and the acyl-CoA chain length specificity were very similar to those of the purified rat liver enzyme.

Spontaneous splicing mutations at the dihydrofolate reductase locus in Chinese hamster ovary cells

1986 ◽  
Vol 6 (6) ◽  
pp. 1926-1935
Author(s):  
P J Mitchell ◽  
G Urlaub ◽  
L Chasin
Keyword(s):  
Amino Acid ◽  
Dihydrofolate Reductase ◽  
Splice Site ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Splice Sites ◽  
Ovary Cells ◽  
Splicing Mutations ◽  
Intron 4

We isolated and characterized three spontaneous mutants of Chinese hamster ovary cells that were deficient in dihydrofolate reductase activity. All three mutants contained no detectable enzyme activity and produced dihydrofolate reductase mRNA species that were shorter than those of the wild type by about 120 bases. Six exons are normally represented in this mRNA; exon 5 was missing in all three mutant mRNAs. Nuclease S1 analysis of the three mutants indicated that during the processing of the mutant RNA, exon 4 was spliced to exon 6. The three mutant genes were cloned, and the regions around exons 4 and 5 were sequenced. In one mutant, the GT dinucleotide at the 5' end of intron 5 had changed to CT. In a second mutant, the first base in exon 5 had changed from G to T. In a revertant of this mutant, this base was further mutated to A, a return to a purine. Approximately 25% of the mRNA molecules in the revertant were spliced correctly to produce an enzyme with one presumed amino acid change. In the third mutant, the AG at the 3' end of intron 4 had changed to AA. A mutation that partially reversed the mutant phenotype had changed the dinucleotide at the 5' end of intron 4 from GT to AT. The splicing pattern in this revertant was consistent with the use of cryptic donor and acceptor splice sites close to the original sites to produce an mRNA with three base changes and a protein with two amino acid changes. These mutations argue against a scanning model for the selection of splice site pairs and suggest that only a single splice site need be inactivated to bring about efficient exon skipping (a regulatory mechanism for some genes). The fact that all three mutants analyzed exhibited exon 5 splicing mutations indicates that these splice sites are hot spots for spontaneous mutation.

scholarly journals Molecular Cloning of a Functional Allatostatin Gut/Brain Receptor and an Allatostatin Preprohormone from the SilkwormBombyx mori

2001 ◽  
Vol 276 (50) ◽  
pp. 47052-47060 ◽  
Author(s):  
Thomas Secher ◽  
Camilla Lenz ◽  
Giuseppe Cazzamali ◽  
Gunnar Sørensen ◽  
Michael Williamson ◽  
...  
Keyword(s):  
Amino Acid ◽  
Molecular Cloning ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Peptide Hormone ◽  
Functional Expression ◽  
Bar Gene ◽  
Ovary Cells ◽  
Intron Phasing ◽  
Insect Gut

The cockroach-type or A-type allatostatins are inhibitory insect neuropeptides with the C-terminal sequence Tyr/Phe-X-Phe-Gly-Leu-NH2. Here, we have cloned an A-type allatostatin receptor from the silkwormBombyx mori(BAR). BAR is 361 amino acid residues long, has seven transmembrane domains, shows 60% amino acid residue identity with the firstDrosophilaallatostatin receptor (DAR-1), and 48% identity with the secondDrosophilaallatostatin receptor (DAR-2). The BAR gene has two introns and three exons. These two introns coincide with and have the same intron phasing as two introns in the DAR-1 and DAR-2 genes, showing that the three receptors are not only structurally but also evolutionarily related. Furthermore, we have cloned aBombyxallatostatin preprohormone that contains eight different A-type allatostatins. Chinese hamster ovary cells permanently transfected with BAR DNA react on the addition of 4 × 10−9mBombyxA-type allatostatins with a second messenger cascade (measured as bioluminescence), showing that BAR is a functional A-type allatostatin receptor. Southern blots suggest thatBombyxhas at least one other BAR-related gene in addition to the BAR gene described in this paper. Northern blots and quantitative reverse transcriptase-polymerase chain reaction of different larval tissues show that BAR mRNA is mainly expressed in the gut and to a much lesser extent in the brain. To our knowledge, this is the first report on the molecular cloning and functional expression of an insect gut/brain peptide hormone receptor.

scholarly journals Human PIG-U and Yeast Cdc91p Are the Fifth Subunit of GPI Transamidase That Attaches GPI-Anchors to Proteins

2003 ◽  
Vol 14 (5) ◽  
pp. 1780-1789 ◽  
Author(s):  
Yeongjin Hong ◽  
Kazuhito Ohishi ◽  
Ji Young Kang ◽  
Satoshi Tanaka ◽  
Norimitsu Inoue ◽  
...  
Keyword(s):  
Amino Acid ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Amino Acid Identity ◽  
Complementation Group ◽  
Long Chain Fatty Acid ◽  
Ovary Cells ◽  
Eukaryotic Proteins ◽  
Gpi Transamidase

Many eukaryotic proteins are anchored to the cell surface via glycosylphosphatidylinositol (GPI), which is posttranslationally attached to the carboxyl-terminus by GPI transamidase. The mammalian GPI transamidase is a complex of at least four subunits, GPI8, GAA1, PIG-S, and PIG-T. Here, we report Chinese hamster ovary cells representing a new complementation group of GPI-anchored protein-deficient mutants, class U. The class U cells accumulated mature and immature GPI and did not have in vitro GPI transamidase activity. We cloned the gene responsible, termed PIG-U, that encoded a 435-amino-acid hydrophobic protein. The GPI transamidase complex affinity-purified from cells expressing epitope-tagged-GPI8 contained PIG-U and four other known components. Cells lacking PIG-U formed complexes of the four other components normally but had no ability to cleave the GPI attachment signal peptide. Saccharomyces cerevisiae Cdc91p, with 28% amino acid identity to PIG-U, partially restored GPI-anchored proteins on the surface of class U cells. PIG-U and Cdc91p have a functionally important short region with similarity to a region conserved in long-chain fatty acid elongases. Taken together, PIG-U and the yeast orthologue Cdc91p are the fifth component of GPI transamidase that may be involved in the recognition of either the GPI attachment signal or the lipid portion of GPI.

Epitope-specific antibody-induced cleavage of angiotensin-converting enzyme from the cell surface

2002 ◽  
Vol 362 (3) ◽  
pp. 585-595 ◽  
Author(s):  
Irina V. BALYASNIKOVA ◽  
Eric H. KARRAN ◽  
Ronald F. ALBRECHT ◽  
Sergei M. DANILOV
Keyword(s):  
Transmembrane Domain ◽  
Umbilical Vein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Integral Membrane Protein ◽  
Converting Enzyme ◽  
Ovary Cells ◽  
Angiotensin I Converting Enzyme ◽  
Hydrophobic Transmembrane Domain ◽  
Umbilical Vein Endothelial Cells

Angiotensin I-converting enzyme (ACE; CD143, EC 3.4.15.1) is a type-1 integral membrane protein that can also be released into extracellular fluids (such as plasma, and seminal and cerebrospinal fluids) as a soluble enzyme following cleavage mediated by an unidentified protease(s), referred to as ACE secretase, in a process known as ‘shedding'. The effects of monoclonal antibodies (mAbs) to eight different epitopes on the N-terminal domain of ACE on shedding was investigated using Chinese hamster ovary cells (CHO cells) expressing an ACE transgene and using human umbilical vein endothelial cells. Antibody-induced shedding of ACE was strongly epitope-specific: most of the antibodies increased the shedding by 20–40%, mAbs 9B9 and 3A5 increased the shedding by 270 and 410% respectively, whereas binding of mAb 3G8 decreased ACE shedding by 36%. The ACE released following mAb treatment lacked a hydrophobic transmembrane domain anchor. The antibody-induced shedding was completely inhibited at 4°C and by zinc chelation using 1,10-phenanthroline, suggesting involvement of a metalloprotease in this process. A hydroxamate-based metalloprotease inhibitor (batimastat, BB-94) was 15 times more efficacious in inhibiting mAb-induced ACE shedding than basal (constitutive) ACE release. Treatment of CHO-ACE cells with BB-94 more effectively prevented elevation in antibody-dependent (but not basal) ACE release induced by 3,4-dichloroisocoumarin and iodoacetamide. These data suggest that different secretases might be responsible for ACE release under basal compared with antibody-induced shedding. Further experiments with more than 40 protease inhibitors suggest that calpains, furin and the proteasome may participate in this process.

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