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PubMed1995

Secretion of Alzheimer beta/A4 protein (1-40) and intracellular retention of beta/A4 protein (1-42) in transfected COS cells.

The amyloid beta/A4 protein (beta/A4) of Alzheimer disease is heterogeneous. A beta 1-40 (Asp1 to Val40) is rather soluble, whereas A beta 1-42 (Asp1 to Ala42) is more insoluble and can be the seed of deposition of A beta 1-40. Although beta/A4 itself could not be expressed transiently in COS cells by cDNA transfection, it was expressed after directly adding a signal sequence to its N-terminal end. The expressed A beta 1-40 was secreted, whereas A beta 1-42 was hardly secreted. A beta 1-40 secretion was not inhibited by metabolic inhibitors such as brefeldin A. The normally produced A beta 1-40 could not be retained intracellularly, whereas the abnormally generated A beta 1-42 might be accumulated intracellularly, resulting in cellular toxicity.

Amino Acid Sequence↗
PubMed1995

Polarity orientation and assembly process of microtubule bundles in nocodazole-treated, MAP2c-transfected COS cells.

Microtubule bundles reminiscent of those found in neuronal processes are formed in fibroblasts and Sf9 cells that are transfected with the microtubule-associated proteins tau, MAP2, or MAP2c. To analyze the assembly process of these bundles and its relation to the microtubule polarity, we depolymerized the bundles formed in MAP2c-transfected COS cells using nocodazole, and observed the process of assembly of microtubule bundles after removal of the drug in cells microinjected with rhodamine-labeled tubulin. Within minutes of its removal, numerous short microtubule fragments were observed throughout the cytoplasm. These short fragments were randomly oriented and were already bundled. Somewhat longer, but still short bundles, were then found in the peripheral cytoplasm. These bundles became the primordium of the larger bundles, and gradually grew in length and width. The polarity orientation of microtubules in the reformed bundle as determined by "hook" procedure using electron microscope was uniform with the plus end distal to the cell nucleus. The results suggest that some mechanism(s) exists to orient the polarity of microtubules, which are not in direct continuity with the centrosome, during the formation of large bundles. The observed process presents a useful model system for studying the organization of microtubules that are not directly associated with the centrosomes, such as those observed in axons.

Animals↗
PubMed1994

The role of the cytoplasmic domains of individual subunits of the acetylcholine receptor in 43 kDa protein-induced clustering in COS cells.

The 43 kDa protein, a cytoplasmic peripheral membrane protein, is closely associated with the acetylcholine receptor (AChR) at the neuromuscular junction, where it is thought to anchor the receptor in the postsynaptic membrane. We have used the 43 kDa protein-induced clustering of AChRs that occurs when both proteins are transiently expressed in COS cells to investigate which parts of the AChR might interact with the 43 kDa protein. By constructing chimeric subunits, we showed that the cytoplasmic domains of neither the epsilon nor delta subunits are required for 43 kDa protein-induced clustering. Systematic mutational analysis of the long cytoplasmic loops of the alpha and beta subunits showed that most of the loops can be altered without affecting the ability of the AChR to be clustered; in each case, however, one or more sequences could not be tested, because mutation in these regions prevented AChR assembly. Our results suggest either that these regions are involved in clustering or that the 43 kDa protein can interact with multiple, alternative sites on the cytoplasmic surface of the AChR. Our experiments also show that the postulated sites of tyrosine phosphorylation in the beta subunit and of serine phosphorylation in the alpha subunit can be mutated without affecting 43 kDa protein-induced AChR clustering.

Amino Acid Sequence↗
PubMed1988

Role of N-linked oligosaccharides attached to human renin expressed in COS cells.

One or both of two putative N-glycosylation sites (at asparagine-5 and -75) of human renin was eliminated by amino acid replacement of the asparagine residue with an alanine residue using site-directed mutagenesis. The three glycosylation-deficient renins (Asn-5, Asn-75, Asn-5 and -75 mutants) were expressed in COS cells and secreted into the conditioned media. The secreted amounts of the three mutants were different from one another, although the mutant and wild-type renins had practically the same specific activity. An Asn-5 and -75 mutant which did not contain any glycosylation sites was unstable in the medium, suggesting that the N-linked oligosaccharides play an important role in stabilization of human renin.

Amino Acid Sequence↗
PubMed1990

The role of carbohydrate in the function of human plasminogen: comparison of the protein obtained from molecular cloning and expression in Escherichia coli and COS cells.

A cDNA library was constructed in the phage lambda gt11 from human liver mRNA enriched for plasminogen mRNA by chromatography on Sepharose 4B. A full-length cDNA clone of human plasminogen was isolated. The 2.7 kb cDNA encoded the entire plasminogen molecule, a signal peptide sequence and two start codons with a 5'-untranslated region of about 80 base pairs. In the 3'-non coding region of 280 base pairs a consensus signal AATAAA was found at a distance of 46 base pairs upstream of the poly(A) tail. The plasminogen cDNA was subcloned in the eukaryotic expression vector p91023 (B), and human plasminogen was expressed in monkey kidney (COS m6) cells and in Escherichia coli. The recombinant molecule obtained from COS cells has physicochemical and biological properties similar to native human plasminogen I, indicating that it has folded in a manner similar to plasminogen synthesized by liver. By contrast, plasminogen expressed in E. coli could not be activated and showed biological properties which are very different from glycosylated forms of plasminogen. However, the non-glycosylated plasminogen was bound by lysine-Sepharose and reacted with a conformation dependent monoclonal antibody to kringles 1 to 3. These data suggest that the protein has properly folded kringle domains. Our studies suggest that the carbohydrate domains may play an important role in the function of the plasminogen molecule.

Animals↗
PubMed1986

Expression of biologically active human interleukin 1 subpeptides by transfected simian COS cells.

"Interleukin 1" (IL-1) is a term used to describe the family of macrophage-derived proteins that mediate many immune and inflammatory reactions. We have previously described the molecular cloning and sequencing of the cDNA encoding the predominant (neutral) form of human IL-1, which has been designated IL-1 beta. We report here that transfection of simian COS cells with this cDNA clone results in the transcription of IL-1 mRNA and the synthesis of antibody-neutralizable intracellular IL-1 biological activity. In addition, selective deletion of regions of the IL-1 cDNA judged not to be essential for function, on the basis of conserved sequence homology, resulted in localization of a "core" region responsible for a majority of the biological activity. These results permit mapping the active site of IL-1 to a peptide of 6970 molecular weight located within the carboxyl third (between Met-136 and Gln-197) of the IL-1 precursor.

Animals↗
PubMed1991

Regulation of endoplasmic reticulum stress proteins in COS cells transfected with immunoglobulin mu heavy chain cDNA.

The mechanism by which endoplasmic reticulum (ER) stress proteins are induced by the accumulation of incompletely assembled or malfolded proteins in the ER is poorly understood. The 78-kDa glucose-regulated protein (BiP), one of the ER stress proteins, has often been detected in stable complexes with these accumulated proteins. We have transfected COS cells with an immunoglobulin (Ig) mu heavy chain expression plasmid. Expressed mu-chain accumulated in the cells and formed stable complexes with BiP. As a result, the synthesis of three ER stress proteins, BiP, the 94-kDa glucose-regulated protein (GRP94/ERp99), and ERp72, was increased as were their mRNA levels. In addition, the degradation rate of BiP was increased, possibly because of its interaction with mu-chain. Cotransfection of the mu-chain plasmid with an Ig lambda light chain expression plasmid resulted in the appearance of mu-chain in the media in a covalent complex with lambda-chain. An intracellular consequence of this was a reduction in the levels of BiP.mu-chain complex, and a diminished stimulation in the synthesis of the ER stress proteins. These results suggest that the BiP.mu-chain complex in the ER may be involved in the signaling pathway for the induction of ER stress proteins and may represent one regulatory mechanism operating in differentiating B-lymphocytes.

Cell Line↗
PubMed1990

Human peptidylglycine alpha-amidating monooxygenase: cDNA, cloning and functional expression of a truncated form in COS cells.

We have identified a cDNA encoding human peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) with a total length of 3748 bp by screening of a human thyroid carcinoma lambda gt11 library using two heterologous oligonucleotides to conserved regions which derived from frog skin and bovine pituitary PAM sequences. Furthermore we have identified a sequence which differs in a 321 bp deletion. COS cells transfected with a truncated form of this cDNA (lacking the putative carboxyl-terminal transmembrane domain) generated a functional PAM that showed a 20-fold increase of the activity compared to the control and was visualized by immunoblotting.

Amino Acid Sequence↗
PubMed1995

Comparison of A4 and A2a binding sites in striatum and COS cells transfected with adenosine A2a receptors.

A putative A4 adenosine receptor is characterized by a distinct structure activity profile of compounds in competition for [3H]2-phenylaminoadenosine ([3H]CV 1808) binding sites on rat brain membranes assayed at 4 degrees C. We now confirm that A4 binding sites can be demonstrated on ice-cold membranes of rat striatum and demonstrate a similar binding site on COS cells transfected with rat A2a adenosine receptors (COS/A2a). The characteristic A4 potency order is: CV 1808 > [1R-(1 alpha, 2 alpha, 3 beta, 5 beta)]-3-(2,6-diamino-N2-(3-carbethoxyphenyl)-9 H-purin-9-yl)-5'-(N-ethylcarbamoyl)-1,2-cyclopentanediol (CGS 22988) >> 5'-N-ethylcarboxamidoadenosine (NECA) > or = 2-[4-(2- carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680); 9-chloro-2-(2-furyl)[1,2,4]-triaolo[1,5-c]-quinazolin-5-a min e (CGS 15943) only partially inhibits binding at 1 microM. If [3H]CGS 21680 is used for ice-cold assays, or if either [3H]CV 1808 or [3H]CGS 21680 are used for assays at 21 degrees C, the potency order of competing compounds changes markedly and becomes characteristic of A2a adenosine receptor binding sites; CGS 15943 > or = CGS 21680 congruent to NECA > CGS 22988 > or = CV 1808. Binding of [3H]CGS 21680, but not [3H]CV 1808, is significantly enhanced by the pore-forming antibiotic, alamethicin. Guanosine 5'-O-(3-thiotriphosphate) decreases the binding of both radioligands to striatal membranes at 21 degrees C significantly more than to membranes on ice.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗
PubMed1988

Characterization of N-linked oligosaccharides attached to human renin expressed in COS cells.

To study the role of N-linked oligosaccharides attached to human renin, we generated three kinds of glycosylation-deficient renins in which one or both of two putative N-glycosylation sites was eliminated by amino acid replacement using site-directed mutagenesis. Examination of the three mutant renins (Asn-5 to Ala, Asn-75 to Ala, and both Asn-5 and -75 to Ala) expressed in COS cells demonstrated that both putative sites were certainly glycosylated with heterologous N-linked oligosaccharides. Moreover, the oligosaccharide chain attached at Asn-5 was different from that attached at Asn-75 in its molecular size. In addition, the secreted amount of the three mutant renins were different from one another, although the mutant and wild-type renins had practically the same specific activity. Our results suggest that the N-linked oligosaccharides have no effect on the enzymatic activity, but play an important role in stable secretion of human renin.

Cell Line↗
PubMed1988

Functional characterization of Asp-317 mutant of human renin expressed in COS cells.

Renin is an unique aspartyl (acid) protease with optimal activity at neutral pH. It has been suggested that Ala-317 of human renin contributes to neutral optimum pH of the enzyme [(1984) FEBS Lett. 174, 102-111]. The hypothesis was verified by the characterization of mutant renin in which Ala-317 was replaced with Asp by a site-directed mutagenesis. Wild-type and mutant renins, which were expressed in COS cells, exhibited different pH-activity profiles and optimum pH of the mutant enzyme was lower than that of the wild-type enzyme. This result suggests that Ala-317 of human renin plays an important role in the determination of optimum pH of the enzyme.

Alanine↗
PubMed1985

Nucleotide sequences responsible for the thermal inducibility of the Drosophila small heat-shock protein genes in monkey COS cells.

The promoter regions of the Drosophila melanogaster small heat-shock protein genes have been analysed in order to localize those sequences responsible for their heat-shock transcriptional inducibility. Different lengths of the 5' DNA sequences of these four genes were each fused individually to the Herpes simplex virus thymidine kinase (HSV-tk) transcription unit. These hybrid genes were constructed in a simian virus 40 recombinant vector for transfection in permissive monkey COS cells and tested for their heat-shock inducibility. The hsp22/HSV-tk and hsp26/HSV-tk fusion genes were found to be heat-inducible at 43 degrees C, giving rise to correctly initiated transcripts, but transcriptionally quiescent at 37 degrees C (control temperature). The hsp23 and hsp27 fusion gene constructs are, however, not heat-shock-inducible; no transcripts being detectable from hsp27/HSV-tk constructs at either temperature and all hsp23/HSV-tk clones being faithfully but constitutively expressed at low levels at both temperatures. By testing a series of 5' deletion mutants in hsp22/HSV-tk, a homologous sequence located adjacent to the TATA box in both the hsp22 and hsp26 genes was identified as being responsible for their heat-shock activation. This control element corresponds to the Pelham "consensus sequence", previously described for the Drosophila hsp70 genes. The possible modes of transcriptional induction of all four genes are discussed.

Animals↗
PubMed1996

Multiple mechanisms for the phosphorylation of C-terminal regulatory sites in rabbit muscle glycogen synthase expressed in COS cells.

Glycogen synthase can be inactivated by sequential phosphorylation at the C-terminal residues Ser652 (site 4), Ser648 (site 3c), Ser644 (site 3b) and Ser640 (site 3a) catalysed by glycogen synthase kinase-3. In vitro, glycogen synthase kinase-3 action requires that glycogen synthase has first been phosphorylated at Ser656 (site 5) by casein kinase II. Recently we demonstrated that inactivation is linked only to phosphorylation at site 3a and site 3b, and that, in COS cells, modification of these sites can occur by alternative mechanisms independent of any C-terminal phosphorylations [Skurat and Roach (1995) J. Biol. Chem. 270, 12491-12497]. To address these mechanisms multiple Ser-->Ala mutations were introduced in glycogen synthase such that only site 3a or site 3b remained intact. Additional mutation of Arg637-->Gln eliminated phosphorylation of site 3a, indicating that Arg637 may be important for recognition of site 3a by its corresponding protein kinase(s). Similarly, additional mutation of Pro645-->Ala eliminated phosphorylation of site 3b, indicating a possible involvement of 'proline-directed' protein kinase(s). Mutation of Arg637 alone did not activate glycogen synthase as expected from the loss of phosphorylation at site 3a. Rather, mutation of both Arg637 and the Ser-->Ala substitution at site 3b was required for substantial activation. The results suggest that sites 3a and 3b can be phosphorylated independently of one another by distinct protein kinases. However, phosphorylation of site 3b can potentiate phosphorylation of site 3a, by an enzyme such as glycogen synthase kinase-3.

Amino Acid Sequence↗
PubMed1991

Expression of two forms of human dopamine-beta-hydroxylase in COS cells.

We previously reported four different cDNA clones encoding human dopamine-beta-hydroxylase (Kobayashi et al., Nucl. Acids Res., 17 (1989) 1089-1102). These clones were different in a 3' untranslated region (types A and B) and/or in 6 nucleotides in mRNAs. The difference at nucleotide 910 caused an amino acid change between Ala (A) and Ser (S) at amino acid residue 304 (DBH/A and DBH/S). We succeeded in expressing both of DBH/A and DBH/S of type A cDNAs in COS cell. Both of the expressed proteins showed enzyme activities and immunoreactivities. The two proteins had similar kinetic constants, but had different homospecific activities (activities per enzyme protein); the homospecific activity of human DBH/S was low, approximately one thirteenth that of human DBH/A.

Animals↗
PubMed1995

Intracellular targeting of lysosomal cathepsin D in COS cells.

Cathepsin D carries a mannose 6-phosphate sorting signal which is recognized by a specific mannose 6-phosphate receptor, presumably at the site of the trans Golgi network, which segregates cathepsin D from the secretory proteins, and results in targeting of the enzyme to the acidic prelysosomal compartments and lysosomes in mammalian cells. Recent evidence implies that another sorting signal resides within the polypeptide backbone of the precursor cathepsin D. To evaluate the role of the propeptide region of cathepsin D in mannose 6-phosphate receptor-independent targeting to lysosomes, we prepared a deletion mutant of rat cathepsin D lacking the propeptide portion and analyzed its intracellular targeting mechanism after transfection of the mutant cDNA as well as the wild-type cDNA into COS cells. The glycosylated mutant protein was retained intracellularly, and extracellular release of mutant protein was not observed after a 48 h chase. A cell fractionation experiment demonstrated that in the cells expressing the wild-type cathepsin D, the processed form of 44 kDa cathepsin D was recovered in the dense lysosomal fraction. In contrast, in the cells expressing the mutant protein, virtually all of the cell-associated cathepsin D was present in the light fraction which was enriched in the marker enzyme NADPH cytochrome c reductase, and this molecular form of cathepsin D was not observed in the dense lysosomal fraction. An immunofluorescence study revealed that the deletion mutant protein was accumulated within the endoplasmic reticulum, unlike the wild-type protein. These results suggest that the mutant cathepsin D is not correctly recognized by the intracellular sorting system in the endoplasmic reticulum, implying that the propeptide region of cathepsin D is essential for the export of cathepsin D from the endoplasmic reticulum.

Amino Acid Sequence↗
PubMed1984

Expression of the chicken c-src gene in COS cells.

The cellular homolog of the Rous sarcoma virus transforming gene (v-src) was cloned into a plasmid containing the simian virus 40 origin of replication and transcriptional signals. This recombinant plasmid, designated pSVOHCS11 , directs the synthesis of relatively high levels of c-src mRNA and c-src protein ( pp60c -src), when the plasmid is studied 48 to 72 h after calcium phosphate-mediated DNA transfection of COS (monkey) cells. The level of c-src mRNA synthesis is 50-fold higher than the amount of c-src RNA produced in uninfected chicken embryo fibroblasts. Furthermore, the level of pp60c -src expressed in pSVOHCS11 -transfected COS cells is approximately the same as that of pp60v -src in Rous sarcoma virus-transformed cells. Using this recombinant plasmid, we demonstrated that c-src mRNA contains sequences which map 3' to the previously identified c-src-v-src regions of homology. In view of the small amount of c-src mRNA and protein that can be isolated from uninfected cells, this transient expression system offers a convenient source of material for further analyses of the c-src gene product.

Animals↗
PubMed1993

Pharmacology of GABA rho 1 and GABA alpha/beta receptors expressed in Xenopus oocytes and COS cells.

1. The rho 1 protein, which we previously cloned from retina, assembles as a homooligomer that transduces the binding of gamma-aminobutyric acid (GABA) into robust chloride currents. However, its insensitivity to bicuculline, pentobarbitone and benzodiazepines, all potent agents at typical GABAA receptors, suggested that it may react atypically to other GABA agonists and antagonists. 2. cDNAs for the rho 1 and the alpha 5 beta 1 receptors for GABA were expressed as homo- and heterooligomers, respectively, in Xenopus oocytes. The selectivities of the respective receptors for various agonists were investigated using concentration-response experiments in voltage clamped cells. 3. The most potent agonists at the rho 1 receptor were trans-4-aminocrotonic acid (TACA) > GABA > muscimol; at the alpha 5 beta 1 receptor the rank order was muscimol > GABA > 4,5,6,7-tetrahydroisoxazole[4,5-c]pyridine-3-ol (THIP). The most specific agonists were cis-(2-(aminomethyl)-cyclopropyl-carboxylic acid (CAMP) and THIP for the rho 1 and the alpha 5 beta 1 receptors, respectively. 4. Comparing GABA, TACA and cis-aminocrotonic acid (CACA) at rho 1 receptors expressed in COS cells gave results almost indistinguishable from those found at oocytes; the pharmacology of rho 1 seems independent of the expression system. 5. Agonists THIP, piperidine-4-sulphonic acid (P4S), and isoguvacine, whose C-C-C-N chains are constrained by rings into a folded conformation and were potent at the alpha 5 beta 1 receptor, were among the weakest at the rho 1 receptor. However CACA and CAMP, which align better with the extended than the folded conformation, were weakest at the alpha 5 beta 1 receptor but moderately potent at the pl receptor. These findings suggest that the rho l receptor recognizes agonists in the extended conformation, in contrast to GABAA receptors, which are believed to recognize agonists in the partially folded conformation.6. In contrast to the alpha 5 beta 1 receptor, gradations in maximum responses were apparent in the rho l receptor,suggesting various degrees of partial agonism. In particular, imidazole-4-acetic acid (I4AA), whose maximum response was only 3% of GABA's maximum, had an apparent Kd for activating the rho l receptor of 16 microM; but it had an apparent Kd for competitively blocking the receptor of 0.64 microM. This difference suggests that steric constraints in the activated (open channel) receptor are tighter than in the resting receptor.7. Hill coefficients approached 2 at the rho l receptor, but were closer to unity at the alpha 5 beta 1 receptor. Thus,the rho l receptor displayed higher cooperativity.8. Unlike typical GABAA receptors, the rho l receptor was insensitive to the competitive antagonists bicuculline, SR95531, securinine, and (+)-tubocurarine.

Animals↗
PubMed1990

Formation of amyloid-like fibrils in COS cells overexpressing part of the Alzheimer amyloid protein precursor.

A pathological hallmark of Alzheimer's disease is the deposition of amyloid fibrils in the brain. The principal component of the amyloid fibril is beta/A4 protein, which is derived from a large membrane-bound glycoprotein, Alzheimer amyloid protein precursor (APP). Although the deposition of amyloid is thought to result from the aberrant processing of APP, the detailed molecular mechanisms of amyloidogenesis remain unclear. A C-terminal fragment of APP which spans the beta/A4 and cytoplasmic domains has a tendency to self-aggregate. In an attempt to establish a cultured-cell model for amyloid fibril formation, we have transfected COS-1 cells with complementary DNA encoding the C-terminal 100 residues of APP. In the perinuclear regions of a small population of DNA-transfected cells, we observed inclusion-like deposits which showed a strong immunohistochemical reaction towards an anti-C-terminal APP antibody or an anti-beta/A4 amyloid core-specific antibody. Electron microscope observations of the inclusion-carrying cells revealed an accumulation of amyloid-like fibrils of 8-22 nm diameter near and on the nuclear membrane. The fibrils showed a beaded or helical structure, and reacted positively with the anti-C-terminus antibody by immunoelectron microscopy. These results suggest that the formation of amyloid fibrils is an inherent characteristic of the C-terminal peptide of APP. The present system provides a suitable model for the molecular dissection of the process of brain amyloidogenesis.

Alzheimer Disease↗