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At least 163 records · Page 9Linked to original sources
PubMed1993

Comparison of COS cell transfected AT1A and AT1B angiotensin II receptors and angiotensin II receptor isoforms in rat tissues using isoelectric focusing.

Rat adrenal AT1A and AT1B receptors from transfected COS-7 cells were labelled with 125I-Angiotensin II, solubilised, and run on isoelectric focusing gels. Receptors from rat tissues were treated similarly. COS-7 cell-expressed AT1A and AT1B receptors each produced a single peak of specific radioactivity at pI 6.8. Rat liver and rat ovary tissue preparations gave peaks at pI 6.8 and 6.5, respectively. In contrast, rat adrenal tissue preparations gave four peaks at pI 7.0, 6.8, 6.5, and 6.3. The additional isoforms found in the rat adrenal tissue preparations may represent post-translationally modified or novel receptors.

Adrenal Glands↗
PubMed2000

COS cells expression cloning of tyrosine-phosphorylated proteins by immunocytochemistry.

Tyrosine phosphorylation is an important post-translational modification of proteins, essential in many aspects of the cell economy, particularly in signal transduction pathways. Despite the importance of protein tyrosine phosphorylation, the approaches available for molecular cloning remain limited. We have developed a COS cell-based eukaryotic expression cloning procedure for phosphotyrosine-containing proteins by immunocytochemistry of cell monolayers. The approach takes advantage of the low basal levels of tyrosine phosphorylated, robust transient expression, availability of specific antibodies against tyrosine-phosphorylated residues, and rescue of episomal DNA after immunocytochemistry. The technique is validated by cloning the rat proto-oncogene c-fgr in its tyrosine-phosphorylated form out of a rat kidney cDNA library containing over 10(6) primary recombinants. This technique set the grounds for expression cloning of tyrosine-phosphorylated proteins in eukaryotic cells, and it is anticipated that further modifications and refinements will allow the identification of protein tyrosine phosphatase substrates.

Animals↗
PubMed2006

Physostigmine modulation of acetylcholine currents in COS cells transfected with mouse muscle nicotinic receptor.

Physostigmine (Phy), a reversible inhibitor of acetylcholine (ACh) esterase (AChE), may also act as a low potency agonist and a modulator of the nicotinic receptor. The actions of Phy on mouse muscle nicotinic receptors in the COS-7 cell line were studied by the patch-clamp technique. Currents were recorded in the whole-cell mode 3-7 days after cell transfection by plasmids coding alphabetagammadelta combination of receptor subunits. The application of ACh to cells clamped at -10 mV produced inward currents which displayed desensitization. The application of Phy in concentrations up to 1 x 10(-3) M did not give reliable specific whole-cell membrane responses. The application of Phy in concentrations of 10(-6)-10(-4) M together with ACh modulated the amplitude; accelerated desensitization of currents induced by ACh and increased the final extent of desensitization in a concentration-dependent manner. This finding is in contrast to the suppression and slowing down of desensitization by Phy and 1-methyl-galanthamine observed in Torpedo receptors.

Acetylcholine↗
PubMed1986

Secretion of rat serum albumin by COS cells transfected with a spliced cDNA gene. A system to study protein sorting.

Certain structural features of secreted proteins may function as "sorting signals" to direct the various steps required in the secretory pathway. In order to identify and study the function of these signals we have cloned a complete cDNA gene encoding rat serum albumin (RSA) and expressed this gene in COS-1 cells via an SV40-plasmid shuttle vector. The gene was constructed by splicing together a segment of genomic DNA and three cDNA fragments excised from recombinant plasmids. DNA endonuclease digestion and ligation at restriction sites common to overlapping regions of these four RSA DNA fragments assured the maintenance of the translation reading frame during the construction of this gene. COS-1 cells transfected with the recombinant vector containing the full-length RSA gene (pSV2rsa) synthesize and secrete RSA immunoreactive material into the culture medium. This mammalian expression system provides a means to study the signals and processes involved in intracellular transport of secreted proteins.

Animals↗
PubMed1997

Characterization of Rev function using subgenomic and genomic constructs in T and COS cells.

The effect of the human immunodeficiency type 1 (HIV-1) Rev protein on the splicing and cytoplasmic accumulation of HIV-1 RNAs was investigated in COS and T cells. Subgenomic and genomic constructs were used which expressed varying levels of complexity in their potential RNA constituents. Using all constructs, in both cell types, an inhibitory effect of Rev on the level of fully spliced HIV-1 RNAs could be demonstrated. An increase in the nuclear level of unspliced pre-mRNA was seen in the presence of Rev with genomic constructs. Thus, the inhibitory effect on splicing was not merely due to enhancement of nuclear export of the pre-mRNA with these constructs. In both cell types, a positive effect of Rev on the cytoplasmic accumulation of HIV-1 RNAs could also be seen. However, in T cells, the Rev-dependent RNAs were still capable of accumulating at a reduced level in the cytoplasmic fraction in the absence of Rev. The identity of the cell type, construct, and RNA species impacted on the phenotypic manifestation of Rev function.

Animals↗
PubMed1995

Binding of human factor VIIa to tissue factor induces cytosolic Ca2+ signals in J82 cells, transfected COS-1 cells, Madin-Darby canine kidney cells and in human endothelial cells induced to synthesize tissue factor.

Tissue factor (TF) is the most potent trigger of blood clotting known. It activates factor VII (FVII) thereby initiating a cascade of proteolytic reactions resulting in thrombin production. The cloning of TF revealed its structural characteristics to be those of a receptor related to the class 2 cytokine receptor superfamily, but until now no intracellular signal has been discovered related to binding of the ligand (FVIIa) to the putative receptor. We have studied possible intracellular signaling effects of the FVIIa-TF interaction by measuring cytosolic free Ca2+ in single fura-2-loaded cells and found that 200 nM FVIIa caused Ca2+ transients in about 30% of human umbilical vein endothelial cells treated with interleukin-1 beta to express TF, compared to below 5% in uninduced cells. A gradual increase of the basal Ca2+ level was also caused by binding of FVIIa. In the human bladder carcinoma cell line J82, which has a high constitutive TF activity, similar results were found. An antibody neutralizing TF activity decreased the response rate to control levels. COS-1 cells which do not make TF did not respond to FVIIa as opposed to COS-1 cells expressing TF after transfection with a human TF cDNA construct. The canine kidney cell line MDCK, a constitutive TF producer, responded especially well; up to 100% of the cells examined showed Ca2+ oscillations which were dose dependent with regard to frequency, latency, maximal amplitude, and recruitment of responding cells. The frequency was reduced by inhibition of Ca2+ influx with 100 microM LaCl3. In confluent MDCK cells the Ca2+ oscillations were synchronous, constituting the first evidence of a synchronous cytosolic Ca2+ oscillator generated by global application of agonist. Thus, TF mediates a cytosolic Ca2+ signal upon interaction with its ligand FVIIa, thereby suggesting a more complex biological role for TF.

Animals↗
PubMed2004

A small heat-shock protein, p26, from the crustacean Artemia protects mammalian cells (Cos-1) against oxidative damage.

A small heat-shock protein (p26) purified from stress-resistant embryos of the crustacean, Artemia franciscana, was introduced into cultured cells of green monkey kidney (Cos-1) using the BioPORTER delivery system. Cells containing p26 exhibited impressive resistance to hydrogen peroxide compared to controls. Introduction of the disaccharide trehalose did not provide protection against oxidative damage, but enhanced substantially the protective performance of p26 when both were present. These studies extend previous research on the protective role played by p26 in cells exposed to various forms of stress, presumably through its ability to function as a molecular chaperone.

Animals↗
PubMed1990

Genes for Plasmodium falciparum surface antigens cloned by expression in COS cells.

Two genes encoding membrane antigens of Plasmodium falciparum were isolated by transient expression in mammalian cells and selection with human immune sera from African adults exposed to P. falciparum malaria. COS-7 cells were transfected with a plasmid expression library constructed from P. falciparum genomic DNA, and cells expressing reactive malaria antigens on their surface were enriched by adherence to antibody-coated dishes. One of the genes isolated is distinctive in that it does not contain repeat sequences typical of many malarial genes cloned by immunoscreening of bacterial expression libraries. The second gene apparently encodes a polymorphic version of the P. falciparum merozoite surface antigen Ag513, since the two sequences are identical in the 5' and 3' coding regions but diverge completely in the center. The COS-7 expression system provides an alternate means for cloning genes encoding malarial membrane antigens by using those antibodies in complex immune sera that bind membrane-associated, nondenatured molecules.

Amino Acid Sequence↗
PubMed2001

Coexpression of the CUG-binding protein reduces DM protein kinase expression in COS cells.

Myotonic dystrophy (DM) is the most common form of adult onset muscular dystrophy. Patients have a large CTG repeat expansion in the 3' untranslated region of the DMPK gene, which encodes DM protein kinase. RNA trans-dominant models, which hypothesize that the expanded CUG trinucleotide repeat on DMPK mRNA sequesters a factor or disrupts the RNA metabolism of the DMPK mRNA itself and other mRNAs in a trans dominant manner, have been proposed. A candidate for the sequestered factor, termed CUG-binding protein (CUG-BP), exists in several alternatively spliced isoforms. We found a human isoform with a twelve base insertion (deduced amino acids Leu-Tyr-Leu-Gln) and an isoform with a three base insertion (deduced amino acid Ala) insertion. In order to elucidate the effects of CUG-BP on DMPK expression, we introduced CUG-BP and DMPK cDNA transiently into COS-7 cells. Cotransfection of CUG-BP did not significantly affect the expression of either wild type or mutant DMPK at the mRNA level. On the other hand, cotransfection of CUG-BP significantly affected the expression of both the wild type and mutant DMPKs at the protein level. This reduction was remarkable when the mutant DMPK construct was used.

Animals↗
PubMed1998

Molecular and functional characteristics of MAP-2a: ability of MAP-2a versus MAP-2b to induce stable microtubules in COS cells.

Microtubule-associated protein-2 (MAP-2) is a prominent cytoskeletal protein in the mammalian nervous system. Two high-molecular-weight (HMW) MAP-2 isoforms, MAP-2a and MAP-2b, are developmentally regulated. MAP-2b is expressed through the life of the neuron, while MAP-2a expression coincides with the time of synaptic formation. MAP-2a and MAP-2b differ in size by approximately 10 kD. Attempts to differentiate MAP-2a from MAP-2b led to the identification of additional exons; exons 7A, 8, 13, and 16. The focus of the present study was to define the complete molecular composition of MAP-2a that was prerequisite for investigating the functional characteristic of the MAP-2a protein. Detailed examination of rat brain mRNA by Northern blot analysis and RT-PCR showed that MAP-2a contains only exon 8 in addition to the exons found in the MAP-2b transcript. Exons 7A, 13, and 16 are not present in the MAP-2a transcript. Antibody generated to exon 8 expressed protein, immunoprecipitated a HMW protein from adult rat brain that co-migrated with MAP-2a and was immunopositive with other MAP-2 antibodies. Comparative transfections of full-length MAP-2a and MAP-2b cDNA into COS-7 cells demonstrated that MAP-2a influenced the microtubule network differently than MAP-2b by inducing rapid and stable microtubule bundle formation even in the presence of nocodazole.

Animals↗
PubMed1993

Palmitoylation of bovine opsin and its cysteine mutants in COS cells.

Previously, bovine rhodopsin has been shown to be palmitoylated at cysteine residues 322 and 323. Here we report on palmitoylation of bovine opsin in COS-1 cells following expression of the synthetic wild-type opsin gene and several of its cysteine mutants in the presence of [3H]palmitic acid. Two moles of palmitic acid are introduced per wild-type opsin molecule in thioester linkages. Palmitoylation is abolished when both Cys-322 and Cys-323 are replaced by serine residues. Replacement of Cys-322 by serine prevents palmitoylation at Cys-323, whereas replacement of the latter with serine allows palmitoylation at Cys-322. Opsin mutants that evidently do not contain a Cys-110/Cys-187 disulfide bond and presumably remain in the endoplasmic reticulum are not palmitoylated. Replacement of Cys-140 or Cys-185 reduces the extent of palmitoylation of the opsin. Lack of palmitoylation at Cys-322 and/or Cys-323 does not affect 11-cis-retinal binding, absorption maximum or extinction coefficient of the chromophore, the bleaching behavior of the chromophore, or the light-dependent binding and activation of transducin. Mutants containing serine substitutions at Cys-140 or Cys-323 showed reduced light-dependent phosphorylation by rhodopsin kinase.

Amino Acid Sequence↗
PubMed1993

Selective impairment of the synthesis of basic fibroblast growth factor binding domains of heparan sulphate in a COS cell mutant defective in N-sulphotransferase.

N-Sulphation is a key step in the overall sulphation of heparan sulphate. We have isolated a COS cell-derived mutant, CM-15, that is impaired in its ability to bind to basic fibroblast growth factor (bFGF) and has a 2- to 3-fold reduction in N-sulphotransferase activity [Ishihara et al., (1992a) Anal. Biochem., 206, 400-407]. We now provide structural evidence that CM-15 is selectively impaired in the synthesis of highly sulphated regions or 'blocks' that display high-affinity binding to bFGF; these are completely N-sulphated blocks of decasaccharide or greater length that are enriched in O-sulphate groups. The synthesis of sulphated blocks that did not show high affinity to the growth factor was relatively unimpaired in the mutant cells; this included fully N-sulphated octamer (or smaller) blocks and, unexpectedly, decasaccharide or larger blocks that were poorly O-sulphated. In the latter fraction, the failure to form high-affinity binding regions was the result of a failure to stimulate O-sulphation rather than N-sulphation in CM-15 cells. In agreement with other studies, disaccharide analysis of the wild-type-derived sulphated blocks suggested that 2-O-sulphation of iduronate residues in the polymer was a necessary element to produce a high-affinity binding sequence once N-sulphation was completed in the decasaccharide or larger fraction. These results suggest that a selective reduction in both N- and O-sulphation in the larger blocks produced by CM-15 cells is a consequence of the reduction of N-sulphotransferase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1995

C-reactive protein binds to Fc gamma RI in transfected COS cells.

C-Reactive protein (CRP) is an acute phase serum protein in man that binds to certain bacterial polysaccharides and to components exposed on damaged cells. CRP is bound by receptors on phagocytic cells and functions as an opsonin for its ligands. Interactions of CRP with a specific CRP receptor (CRP-R) and with the high affinity receptor for IgG, Fc gamma RI, on monocytic cells have previously been demonstrated. It was not possible to fully characterize CRP binding to Fc gamma RI in these studies, since cells and cell lines expressing Fc gamma RI also have the CRP-R. In the present study we examined the interaction of CRP with Fc gamma RI in COS-7 cells transfected with a cDNA encoding this receptor. Expression of Fc gamma RI and specific CRP binding to transfected cells were demonstrated by flow cytometry. By two-color analysis, the cell population binding CRP was the same as the population that bound the Fc gamma RI-specific mAb 10.1 and 32.2 CRP inhibited the binding of radiolabeled IgG1 and IgG4 by up to 60%. A CRP molecule that was mutated in the amino acid sequence homologous to the IgG sequence proposed to interact with Fc gamma RI failed to bind to transfected cells, but retained the ability to bind to the CRP-R on monocytic cells. These studies confirm the binding of CRP to Fc gamma RI and identify a site on CRP that is essential for this binding.

Amino Acid Sequence↗
PubMed1992

Amino- and carboxy-terminal deletion mutants of Gs alpha are localized to the particulate fraction of transfected COS cells.

To elucidate the structural basis for membrane attachment of the alpha subunit of the stimulatory G protein (Gs alpha), mutant Gs alpha cDNAs with deletions of amino acid residues in the amino and/or carboxy termini were transiently expressed in COS-7 cells. The particulate and soluble fractions prepared from these cells were analyzed by immunoblot using peptide specific antibodies to monitor distribution of the expressed proteins. Transfection of mutant forms of Gs alpha with either 26 amino terminal residues deleted (delta 3-28) or with 59 amino terminal residues deleted (delta 1-59) resulted in immunoreactive proteins which localized primarily to the particulate fraction. Similarly, mutants with 10 (delta 385-394), 32 (delta 353-384), or 42 (delta 353-394) amino acid residues deleted from the carboxy terminus also localized to the particulate fraction, as did a mutant form of Gs alpha lacking amino acid residues at both the amino and carboxy termini (delta 3-28)/(delta 353-384). Mutant and wild type forms of Gs alpha demonstrated a similar degree of tightness in their binding to membranes as demonstrated by treatment with 2.5 M NaCl or 6 M urea, but some mutant forms were relatively resistant compared with wild type Gs alpha to solubilization by 15 mM NaOH or 1% sodium cholate. We conclude that: (a) deletion of significant portions of the amino and/or carboxyl terminus of Gs alpha is still compatible with protein expression; (b) deletion of these regions is insufficient to cause cytosolic localization of the expressed protein. The basis of Gs alpha membrane targeting remains to be elucidated.

Amino Acid Sequence↗
PubMed1995

Characterization of chicken kit tyrosine kinase receptor in Cos cell transfectants and in chicken brain.

The Kit tyrosine kinase (Kit) encoded by the c-kit proto-oncogene is a receptor for stem cell factor (SCF). Kit proteins of mice and humans are expressed in various kinds of hematopoietic progenitor cells and are essential for the growth of these cells. Wild-type chKit (chKit+) and a mutant chKit (chKit42) that contained an amino acid change from Asp777 to Asn corresponding to that in Kit of the W42 mutant mice were produced in Cos-1 cells transfected with expression plasmids containing the chicken c-kit cDNA, and characterized using two kinds of anti-chKit antisera. The W42 mutant Kit has previously been shown to be defective for kinase activity. The chKit+ of 145 kilodalton (kDa) and 130 kDa with varying degrees of N-linked glycosylation were detected. Western blot analysis using an anti-phosphotyrosine monoclonal antibody showed that autophosphorylation of chKit+ was greatly enhanced upon chicken SCF induction. The chKit+ did not respond to mouse SCF. The kinase activity of chKit42 was abolished by the amino acid substitution, indicating the Asp777 residue was essential for the activity. In addition, 145 kDa chKit conjugated with sialic acid residue(s) was detected in chicken brain by immunoprecipitation using the antisera. An in vitro kinase assay showed the kinase activity of this protein. These structural and functional similarities of chKit to mammalian Kit proteins shown in this study implicate a possible role of chKit in chicken hematopoietic system.

Animals↗