Subject(s)
Membrane Proteins/genetics , Tacrolimus Binding Protein 1A/genetics , Transcription, Genetic , beta-Galactosidase/genetics , 3T3 Cells , Animals , Cell Line , Escherichia coli/enzymology , Escherichia coli/genetics , Flow Cytometry/methods , Luminescent Measurements , Mammals , Membrane Proteins/biosynthesis , Membrane Proteins/chemistry , Mice , Molecular Conformation , Mutagenesis , Protein Structure, Secondary , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Sequence Deletion , Tacrolimus Binding Protein 1A/biosynthesis , Tacrolimus Binding Protein 1A/chemistry , Transfection/methods , beta-Galactosidase/chemistryABSTRACT
We present a method for monitoring receptor dimerization at the membrane of live cells. Chimeric proteins containing the epidermal growth factor (EGF) receptor extracellular and transmembrane domains fused to weakly complementing beta-galactosidase (beta-gal) deletion mutants were expressed in cells in culture. Treatment of the cells with EGF-like compounds for as little as 15 s resulted in chimeric receptor dimerization detectable as beta-gal enzymatic activity. The dose response of chimeric receptors was ligand specific. beta-galactosidase complementation was reversible upon removal of ligand and could be reinduced. Antibodies that block ligand binding inhibited receptor dimerization and beta-gal complementation. These results demonstrate that beta-gal complementation provides a rapid, simple, and sensitive assay for protein interactions and for detecting and monitoring the kinetics of receptor dimerization.
Subject(s)
Epidermal Growth Factor/pharmacology , ErbB Receptors/metabolism , Animals , Cell Membrane/metabolism , Dimerization , ErbB Receptors/drug effects , ErbB Receptors/genetics , Genetic Complementation Test , Humans , Mice , Protein Binding , Recombinant Fusion Proteins/metabolism , beta-Galactosidase/genetics , beta-Galactosidase/metabolismSubject(s)
Genes , Genetic Complementation Test/methods , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , beta-Galactosidase/genetics , beta-Galactosidase/metabolism , Animals , Cells, Cultured , Dimerization , ErbB Receptors/genetics , ErbB Receptors/metabolism , Gene Expression , Genetic Complementation Test/trends , Humans , Models, Molecular , Protein Binding/genetics , Protein Structure, Tertiary/genetics , Protein Structure, Tertiary/physiology , Sensitivity and Specificity , Signal Transduction/geneticsABSTRACT
Experiments with somatic cell hybrids and stable heterokaryons have demonstrated that differentiated cells exhibit a remarkable capacity to change. Heterokaryons have been particularly useful in determining the extent to which the differentiated state of a cell is plastic. Cell fate can be altered by a change in the balance of positive and negative trans-acting regulators. Although a single regulator may be sufficient in certain environments to trigger a change in cell fate, that regulator may be ineffective in other cell contexts where it encounters a different composition of regulators.
Subject(s)
Gene Expression Regulation, Developmental , Hybrid Cells/cytology , Hybrid Cells/metabolism , Animals , Cell Differentiation/genetics , Genes, Regulator , Humans , Mice , Muscle Fibers, Skeletal/cytology , Muscles/cytology , Muscles/metabolism , Myogenic Regulatory Factors/genetics , Organ Specificity/genetics , Rats , Transcriptional ActivationABSTRACT
Continuous regulation is required to maintain a given cell state or to allow it to change in response to the environment. Studies of the mechanisms underlying such regulation have often been hindered by the inability to control gene expression at will. Among the inducible systems available for regulating gene expression in eukaryotes, the tetracycline (tet) regulatable system has distinct advantages. It is highly specific, non-toxic and non-eukaryotic, and consequently does not have pleiotropic effects on host cell genes. Previously this system also had drawbacks, as it did not extinguish gene expression completely, precluding the study of toxic or growth-inhibitory gene products. We report here the development of a facile reversible tetracycline-inducible retroviral system (designated RetroTet-ART) in which activators and repressors together are expressed in cells. Gene expression can now be actively repressed in the absence of tet and induced in the presence of tet, as we have engineered distinct dimerization domains that allow co-expression of homodimeric tet-regulated transactivators and transrepressors in the same cells, without the formation of non-functional heterodimers. Using this system, we show that growth arrest by the cell cycle inhibitor p16 is reversible and dependent on its continuous expression.
Subject(s)
Cell Division/physiology , Cyclin-Dependent Kinase Inhibitor p16/drug effects , Tetracycline/pharmacology , 3T3 Cells , Animals , Cells, Cultured , Cyclin-Dependent Kinase Inhibitor p16/physiology , Dimerization , Gene Expression Regulation/drug effects , Mice , Mice, Inbred C3H , Retroviridae/geneticsABSTRACT
Antibodies to the acetylcholine receptor (AChR), which are diagnostic of the human autoimmune disease myasthenia gravis, block AChR function and increase the rate of AChR degradation leading to impaired neuromuscular transmission. Steroids are frequently used to alleviate symptoms of muscle fatigue and weakness in patients with myasthenia gravis because of their well-documented immunosuppressive effects. We show here that the steroid dexamethasone significantly increases total surface AChRs on cultured human muscle exposed to myasthenia gravis sera. Our results suggest that the clinical improvement observed in myasthenic patients treated with steroids is due not only to an effect on the immune system but also to a direct effect on muscle. We propose that the identification and development of pharmacologic agents that augment receptors and other proteins that are reduced by human genetic or autoimmune disease will have broad therapeutic applications.
Subject(s)
Autoantibodies/immunology , Dexamethasone/pharmacology , Muscles/physiology , Myasthenia Gravis/immunology , Receptors, Cholinergic/biosynthesis , Cell Membrane/physiology , Cells, Cultured , Clone Cells , Humans , Kinetics , Muscles/drug effects , Receptors, Cholinergic/drug effects , Receptors, Cholinergic/immunologyABSTRACT
In multinucleated heterokaryons formed from the fusion of differentiated muscle cells to either hepatocytes or fibroblasts, muscle-specific gene expression is activated, liver-specific gene expression is repressed, and there are changes in the location of the Golgi apparatus. An understanding of the regulatory mechanisms that underlie this plasticity is of particular interest given the stability of the differentiated state in vivo. We have now investigated whether MyoD or myogenin, regulators of muscle-specific gene expression that have a helix-loop-helix motif, can induce the phenotypic conversion observed in heterokaryons. When these regulators were stably or transiently introduced into fibroblasts or hepatocytes by microinjection, transfection or retroviral infection with complementary DNA in expression vectors, fibroblasts expressed muscle-specific genes, whereas hepatocytes did not. However, fusion of hepatocytes stably expressing MyoD to fibroblasts resulted in activation in the heterokaryon of muscle-specific genes of both cell types. These results imply that other regulators, present in fibroblasts but not in hepatocytes, are necessary for the activation of muscle-specific genes, and indicate that the differentiated state of a cell is dictated by its history and a dynamic interaction among the proteins that it contains.
Subject(s)
Fibroblasts/metabolism , Gene Expression Regulation , Liver/metabolism , Muscle Proteins/biosynthesis , Muscle Proteins/physiology , Actins/biosynthesis , Cell Differentiation , Cell Line , Genetic Vectors , Humans , Microinjections , Muscle Proteins/genetics , Muscles/cytology , Muscles/metabolism , MyoD Protein , Myogenin , Myosins/biosynthesis , Protein Conformation , Stem Cells/cytology , TransfectionABSTRACT
Major changes in cytoarchitecture and gene expression were induced in short-term heterokaryons. When human hepatocytes were fused with mouse muscle cells, the hepatocyte Golgi apparatus changed from its usual polar location to a uniformly circumnuclear location typical of striated muscle. Human liver albumin ceased to be expressed, and expression of the human muscle cell-surface antigen 5.1H11 was induced without DNA replication or cell division. Coexpression of liver and muscle proteins was rarely observed. These novel findings provide insight into the regulation of gene expression and the targeting and localization of organelles with a central role in cell polarity, intracellular transport, and secretion.