Differential effects of the allosteric enhancer (2-amino-4,5-dimethyl-trienyl)[3-(trifluoromethyl) phenyl]methanone (PD81,723) on agonist and antagonist binding and function at the human wild-type and a mutant (T277A) adenosine A1 receptor11Abbreviations: CCPA, 2-chloro-N6-cyclopentyladenosine; DPCPX, 1,3-dipropyl-8-cyclopentylxanthine; CPA, N6-cyclopentyladenosine, R-PIA, N6-[-(R)-1-methyl-2-phenylethyl]adenosine; NECA, 5′-(N-ethyl)-carboxamidoadenosine; N-0840, N6-cyclopentyl-9-methyladenine; theophylline, 1,3-dimethylxanthine; PD81,723, (2-amino-4,5-dimethyl-trienyl)[3-(trifluoromethyl) phenyl]methanone; BCA, bicinchoninic acid; BCS, bovine calf serum; cAMP, adenosine 3′,5′-cyclic monophosphate; DMEM, Dulbecco’s modified Eagle’s medium; CHO, Chinese hamster ovary; and wt, wild-type.

A methotrexate-resistant Chinese hamster ovary cell line deficient in methotrexate uptake has been complemented to methotrexate sensitivity by transfection with DNA isolated from either wild-type Chinese hamster ovary or human G2 cells. Primary and secondary transfectants regained the ability to take up methotrexate in a manner similar to that of wild-type cells, and in the case of those transfected with human DNA, to contain human-specific DNA sequences. The complementation by DNA-mediated gene transfer of this methotrexate-resistant phenotype provides a basis for the cloning of a gene involved in methotrexate uptake.

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Mutation at autosomal loci of Chinese hamster ovary cells: involvement of a high-frequency event silencing two linked alleles

Molecular and Cellular Biology ◽

10.1128/mcb.3.7.1172-1181.1983 ◽

1983 ◽

Vol 3 (7) ◽

pp. 1172-1181

Author(s):

W E Bradley

Keyword(s):

Cell Lines ◽

Chinese Hamster Ovary ◽

High Frequency ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Low Frequency ◽

Class Ii ◽

Class I ◽

Wild Type ◽

Ovary Cells

Two classes of cell lines heterozygous at the galactokinase (glk) locus have been isolated from Chinese hamster ovary cells. Class I, selected by plating nonmutagenized wild-type cells at low density in medium containing 2-deoxygalactose at a partially selective concentration, underwent subsequent mutation to the glk-/- genotype at a low frequency (approximately 10(-6) per cell), which was increased by mutagenesis. Class II heterozygotes, isolated by sib selection from mutagenized wild-type cells, had a higher spontaneous frequency of mutation to the homozygous state (approximately 10(-4) per cell), which was not affected by mutagenesis. About half of the glk-/- mutants derived from a class II heterozygote, but not the heterozygote itself, were functionally hemizygous at the syntenic thymidine kinase (tk) locus. Similarly, a tk+/- heterozygote with characteristics analogous to the class II glk+/- cell lines underwent high-frequency mutation to tk-/-, and most of these mutants, but not the tk+/- heterozygote, were functionally hemizygous at the glk locus. A model is proposed, similar to that for the mutational events at the adenine phosphoribosyl transferase locus (W. E. C. Bradley and D. Letovanec, Somatic Cell Genet. 8:51-66, 1982), of two different events, high and low frequency, being responsible for mutation at either of the linked loci tk and glk. The low-frequency event may be a point mutation, but the high-frequency event, in many instances, involves coordinated inactivation of a portion of a chromosome carrying the two linked alleles. Class II heterozygotes would be generated as a result of a low-frequency event at one allele, and class I heterozygotes would be generated by a high-frequency event. Supporting this model was the demonstration that all class I glk+/- lines examined were functionally hemizygous at tk.

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Expression of human Mn SOD in Chinese hamster ovary cells confers protection from oxidant injury

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1993.264.6.l598 ◽

1993 ◽

Vol 264 (6) ◽

pp. L598-L605

Author(s):

B. Warner ◽

R. Papes ◽

M. Heile ◽

D. Spitz ◽

J. Wispe

Keyword(s):

Chinese Hamster Ovary ◽

Cho Cells ◽

Chinese Hamster Ovary Cells ◽

Lethal Dose ◽

Chinese Hamster ◽

Eukaryotic Expression ◽

Wild Type ◽

Oxidant Injury ◽

Sod Activity ◽

Recombinant Cho Cells

Manganese superoxide dismutase (Mn SOD) is an important component of antioxidant defense in aerobic cells because of its location in the mitochondria, a significant source of oxygen radicals and an important target of oxidant injury. To test the hypothesis that increased mitochondrial Mn SOD protects from oxidant injury, Chinese hamster ovary (CHO) cells were transfected with a eukaryotic expression vector containing the human Mn SOD cDNA. In recombinant CHO cells, Mn SOD activity was increased threefold over wild-type controls. Acute survival during paraquat exposure (0–500 microM) was significantly improved in CHO cells expressing human Mn SOD, with 71% of recombinant CHO cells surviving at the 50% lethal dose (LD50) for wild-type CHO controls. Cell growth following exposure to paraquat (100 microM) was also significantly improved in recombinant CHO cells. CHO cells expressing human Mn SOD continued to grow and divide after paraquat exposure, whereas growth of wild-type CHO cells was negligible. Protection against oxidant-induced injury was directly related to increased Mn SOD, occurring in the absence of changes in other antioxidant enzymes including catalase, Cu,Zn SOD, and glutathione associated cellular antioxidant mechanisms. We conclude that increased expression of human Mn SOD in vitro directly confers protection against oxidant injury.

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Kinetics of endosome acidification in mutant and wild-type Chinese hamster ovary cells.

The Journal of Cell Biology ◽

10.1083/jcb.105.6.2713 ◽

1987 ◽

Vol 105 (6) ◽

pp. 2713-2721 ◽

Cited By ~ 64

Author(s):

D J Yamashiro ◽

F R Maxfield

Keyword(s):

Chinese Hamster Ovary ◽

Cho Cells ◽

Ph Value ◽

Ph Regulation ◽

Chinese Hamster Ovary Cells ◽

Mutant Cell ◽

Chinese Hamster ◽

Wild Type ◽

Cell Biol ◽

Endocytic Compartments

Acidification of endocytic compartments is necessary for the proper sorting and processing of many ligands and their receptors. Robbins and co-workers have obtained Chinese hamster ovary (CHO) cell mutants that are pleiotropically defective in endocytosis and deficient in ATP-dependent acidification of endosomes isolated by density centrifugation (Robbins, A. R., S. S. Peng, and J. L. Marshall. 1983. J. Cell Biol. 96:1064-1071; Robbins, A. R., C. Oliver, J. L. Bateman, S. S. Krag, C. J. Galloway, and I. Mellman. 1984. J. Cell Biol. 99:1296-1308). In this and the following paper (Yamashiro, D. J., and F. R. Maxfield. 1987. J. Cell Biol. 105:2723-2733) we describe detailed studies of endosome acidification in the mutant and wild-type CHO cells. Here we describe a new microspectrofluorometry method based on changes in fluorescein fluorescence when all cellular compartments are equilibrated to the same pH value. Using this method we measured the pH of endocytic compartments during the first minutes of endocytosis. We found in wild-type CHO cells that after 3 min, fluorescein-labeled dextran (F-Dex) was in endosomes having an average pH of 6.3. By 10 min, both F-Dex and fluorescein-labeled alpha 2-macroglobulin (F-alpha 2M) had reached acidic endosomes having an average pH of 6.0 or below. In contrast, endosome acidification in the CHO mutants DTG 1-5-4 and DTF 1-5-1 was markedly slowed. The average endosomal pH after 5 min was 6.7 in both mutant cell lines. At least 15 min was required for F-Dex and F-alpha 2M to reach an average pH of 6.0 in DTG 1-5-4. Acidification of early endocytic compartments is defective in the CHO mutants DTG 1-5-4 and DTF 1-5-1, but pH regulation of later compartments on both the recycling pathway and lysosomal pathway is nearly normal. The properties of the mutant cells suggest that proper functioning of pH regulatory mechanisms in early endocytic compartments is critical for many pH-mediated processes of endocytosis.

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A Paradigm for Peptide Hormone-GPCR Analyses

Molecules ◽

10.3390/molecules25184272 ◽

2020 ◽

Vol 25 (18) ◽

pp. 4272

Author(s):

Fred Naider ◽

Jeffrey M. Becker

Keyword(s):

Conformational Dynamics ◽

Peptide Hormone ◽

Yeast Saccharomyces Cerevisiae ◽

Peptide Analogs ◽

C Terminus ◽

Peptide Interactions ◽

Agonist And Antagonist ◽

Antagonist Binding ◽

And Function ◽

G Protein Coupled

Work from our laboratories over the last 35 years that has focused on Ste2p, a G protein-coupled receptor (GPCR), and its tridecapeptide ligand α-factor is reviewed. Our work utilized the yeast Saccharomyces cerevisiae as a model system for understanding peptide-GPCR interactions. It explored the structure and function of synthetic α-factor analogs and biosynthetic receptor domains, as well as designed mutations of Ste2p. The results and conclusions are described using the nuclear magnetic resonance interrogation of synthetic Ste2p transmembrane domains (TMs), the fluorescence interrogation of agonist and antagonist binding, the biochemical crosslinking of peptide analogs to Ste2p, and the phenotypes of receptor mutants. We identified the ligand-binding domain in Ste2p, the functional assemblies of TMs, unexpected and interesting ligand analogs; gained insights into the bound α-factor structure; and unraveled the function and structures of various Ste2p domains, including the N-terminus, TMs, loops connecting the TMs, and the C-terminus. Our studies showed interactions between specific residues of Ste2p in an active state, but not resting state, and the effect of ligand activation on the dimerization of Ste2p. We show that, using a battery of different biochemical and genetic approaches, deep insight can be gained into the structure and conformational dynamics of GPCR-peptide interactions in the absence of a crystal structure.

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Unusual forms of low density lipoprotein receptors in hamster cell mutants with defects in the receptor structural gene.

The Journal of Cell Biology ◽

10.1083/jcb.102.5.1567 ◽

1986 ◽

Vol 102 (5) ◽

pp. 1567-1575 ◽

Cited By ~ 35

Author(s):

K F Kozarsky ◽

H A Brush ◽

M Krieger

Keyword(s):

Chinese Hamster Ovary ◽

Chinese Hamster Ovary Cells ◽

Low Density Lipoprotein ◽

Chinese Hamster ◽

Ldl Receptor ◽

Density Lipoprotein ◽

Low Density ◽

Wild Type ◽

Ovary Cells ◽

Ldl Receptors

The structure and processing of low density lipoprotein (LDL) receptors in wild-type and LDL receptor-deficient mutant Chinese hamster ovary cells was examined using polyclonal anti-receptor antibodies. As previously reported for human LDL receptors, the LDL receptors in wild-type Chinese hamster ovary cells were synthesized as precursors which were extensively processed by glycosylation to a mature form. In the course of normal receptor turnover, an apparently unglycosylated portion of the cysteine-rich N-terminal LDL binding domain of the receptor is proteolytically removed. The LDL receptor-deficient mutants fall into four complementation groups, ldlA, ldlB, ldlC, and ldlD; results of the analysis of ldlB, ldlC, and ldlD mutants are described in the accompanying paper (Kingsley, D. M., K. F. Kozarsky, M. Segal, and M. Krieger, 1986, J. Cell. Biol, 102:1576-1585). Analysis of ldlA cells has identified three classes of mutant alleles at the ldlA locus: null alleles, alleles that code for normally processed receptors that cannot bind LDL, and alleles that code for abnormally processed receptors. The abnormally processed receptors were continually converted to novel unstable intracellular intermediates. We also identified a compound-heterozygous mutant and a heterozygous revertant which indicate that the ldlA locus is diploid. In conjunction with other genetic and biochemical data, the finding of multiple mutant forms of the LDL receptor in ldlA mutants, some of which appeared together in the same cell, confirm that the ldlA locus is the structural gene for the LDL receptor.

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