Mouse Beta Chains Research Articles

Regulation of cell type-specific mouse Fc epsilon RI beta-chain gene expression by GATA-1 via four GATA motifs in the promoter.

The FcR beta-chain, a subunit of two related multisubunit receptor complexes, the FcepsilonRI and FcgammaRIII, amplifies the mast cell response and is necessary for the cell surface expression of FcepsilonRI in mouse. The transient reporter assay indicated that -69/+4 region is required for cell type-specific transcriptional regulation of mouse beta-chain gene. EMSA using Abs against transcription factors or competitive oligonucleotides demonstrated that -58/-40 region (containing overlapping three GATA-1 sites, -53/-48, -46/-51, and -42/-47) and -31/-26 region (containing one GATA-1 site) are recognized by GATA-1. The promoter activity of beta-chain was decreased by nucleotide replacements of the GATA-1 sites in mouse mast cell line PT18. Furthermore, exogenously produced GATA-1 up-regulated the promoter activity in CV-1 cells, which are negative in the beta-chain production and the up-regulation was apparently suppressed by GATA-1 site mutations. These results indicate that cell type-specific transcription of mouse beta-chain gene is regulated by GATA-1.

Identification of a Cys Motif in the Common β Chain of the Interleukin 3, Granulocyte-Macrophage Colony-stimulating Factor, and Interleukin 5 Receptors Essential for Disulfide-linked Receptor Heterodimerization and Activation of All Three Receptors

The human interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors undergo covalent dimerization of the respective specific alpha chains with the common beta subunit (betac) in the presence of the cognate ligand. We have now performed alanine substitutions of individual Cys residues in betac to identify the Cys residues involved and their contribution to activation of the IL-3, GM-CSF, and IL-5 receptors. We found that substitution of Cys-86, Cys-91, and Cys-96 in betac but not of Cys-100 or Cys-234 abrogated disulfide-linked IL-3 receptor dimerization. However, although Cys-86 and Cys-91 betac mutants retained their ability to form non-disulfide-linked dimers with IL-3Ralpha, substitution of Cys-96 eliminated this interaction. Binding studies demonstrated that all betac mutants with the exception of C96A supported high affinity binding of IL-3 and GM-CSF. In receptor activation experiments, we found that betac mutants C86A, C91A, and C96A but not C100A or C234A abolished phosphorylation of betac in response to IL-3, GM-CSF, or IL-5. These data show that although Cys-96 is important for the structural integrity of betac, Cys-86 and Cys-91 participate in disulfide-linked receptor heterodimerization and that this linkage is essential for tyrosine phosphorylation of betac. Sequence alignment of betac with other cytokine receptor signaling subunits in light of these data shows that Cys-86 and Cys-91 represent a motif restricted to human and mouse beta chains, suggesting a unique mechanism of activation utilized by the IL-3, GM-CSF, and IL-5 receptors.

Molecular cloning of rat C4b binding protein alpha- and beta-chains: structural and functional relationships among human, bovine, rabbit, mouse, and rat proteins.

The C4b binding protein (C4BP) functions as a regulator of the complement system by interacting with the activated form of the fourth complement component, C4b. Human C4BP also interacts with the anticoagulant protein S and the serum amyloid P component (SAP). It is composed of seven identical 70-kDa alpha-chains and one 45-kDa beta-chain. The alpha-chain contains a binding site for C4b, whereas the beta-chain contains the protein S binding site. Recent studies have shown rabbit and bovine plasma to lack a C4BP-protein S complex, and the mouse beta-chain gene to have evolved into a pseudogene. Using a gel filtration chromatography system in combination with Western blotting, we detected a complex between C4BP and protein S in rat plasma, similar to the complex known in human plasma. Using purified rat C4BP and SAP we were unable to detect any complex between the two proteins, but rat C4BP was able to form a complex with human SAP. Rat cDNA clones encoding the C4BP alpha- and beta-chains were isolated from a rat liver cDNA library. The rat alpha-chain cDNA predicted a mature polypeptide chain of 545 amino acid residues, whereas the beta-chain cDNA predicted a mature polypeptide of 243 amino acid residues. The overall amino acid sequence identities between the rat alpha-chain and the mouse, human, rabbit, and bovine alpha-chains were 64, 60, 59, and 52%, respectively. The identities between the rat beta-chain and the human and bovine beta-chains were 68 and 57%, respectively. The rat represents the first non-primate species in which the C4BP-protein S interaction has been found to be conserved.

Expression of the Human α2 Integrin Subunit in Mouse Melanoma Cells Confers the Ability to Undergo Collagen-Directed Adhesion, Migration, and Matrix Reorganization

Previous studies have shown that cultured human and other mammalian cell require the alpha2 beta1 integrin receptor to reorganize and contract 3-dimensional extracellular matrix lattices containing collagen type I. This function is of prime importance for the later phases of wound healing, in which fibroblasts reorganize and contract extracellular matrix components newly deposited in the granulation tissue. It is also known that highly aggressive human melanoma cells have acquired this function, possibly enhancing their invasive potential. To further study alpha2 beta1-mediated functions, we expressed the human alpha2 and beta1 chains of integrins in mouse melanoma cells (BULT). The parental cell line was unable to exert known alpha2 beta1-mediated functions: The cells did not adhere, spread, or migrate on collagen type I, and they did not reorganize 3-dimensional collagen I lattices. Transfection of the human alpha2 chain was sufficient to confer not only specific adhesion to collagen type I in static adhesion assays, but also the ability to exert complex functions such as migration on collagen and efficient reorganization of collagen-I-containing matrices. Coexpression of the human beta1 chain did not further enhance these functions in BULT melanoma cells. This was underscored by the observation that alpha1-specific monoclonal antibodies were able to completely block the newly introduced functions, whereas beta1-specific antibodies had no such effect. Moreover, in transfectants expressing both the human alpha2 and beta1 chains, the human alpha2 chain did not preferentially associate with the human beta1 chain as compared with mouse beta-chains. This may indicate that the molecular structures guiding the physical association of the human alpha2 chain with beta chains are extremely highly conserved between the species.

Characterization of critical residues in the cytoplasmic domain of the human interleukin‐5 receptor α chain required for growth signal transduction

Interleukin (IL)-5 binds to a cell surface receptor composed of two polypeptide chains, alpha and beta, both belonging to the hemopoietic cytokine receptor family. Mouse cells expressing common mouse beta chain (AIC2B) that were transfected with human IL-5 receptor (R)alpha cDNA proliferated in response to picomolar concentrations of human IL-5, indicating that a functional receptor was reconstituted. We show that in these cells, human (h)IL-5 as well as mouse (m)IL-3 induce tyrosine phosphorylation of beta chain and JAK 2 kinase. Phosphorylated beta receptor was co-precipitated with anti-JAK 2 antibodies, suggesting that both molecules were physically associated. IL-5 and IL-3 also induce cytosolic DNA binding activity as measured by an electrophoretic mobility shift assay using the interferon-gamma responsive region of human Fc gamma 1 gene DNA element. A deletion mutant of hIL-5R alpha lacking the cytoplasmic part could bind hIL-5 normally in association with the beta chain, but was unable to transmit a biological signal. The cytoplasmic domain was also indispensable for tyrosine phosphorylation and activation of DNA binding proteins. A membrane-proximal proline-rich element of the hIL-5R alpha cytoplasmic domain that is conserved among different members of the hemopoietic cytokine receptor family was essential for biological activity. Point mutations in this motif also knocked out IL-5-inducible JAK 2 phosphorylation.

Symmetric interspecies hybrids of mouse and human hemoglobin: molecular basis of their abnormal oxygen affinity.

Interspecies hybrids of HbA and Hb from mouse C57BL/10 [alpha 2M beta 2H and alpha 2H beta 2M (H = human, M = mouse)], representing 19 and 27 sequence differences per alpha beta dimers (as compared with human alpha beta dimer) have been generated in vitro. The efficiency of the assembly of the interspecies hybrids by the alloplex intermediate pathway is about twofold higher than the low-pH-mediated subunit approach. The interspecies hybrids exhibit a cooperative O2 binding. The intrinsic O2 affinity of mouse Hb is slightly lower than HbA, while the 2,3-diphosphoglycerate (DPG) effect is comparable. Interestingly, the interspecies hybrid alpha 2M beta 2H has high O2 affinity (compared to either human or mouse Hb), while the interspecies hybrid alpha 2H beta 2M exhibits a very low O2 affinity. These results suggest that the mouse beta chain generates a tetramer with very low oxygen affinity. However, the complementarity of the mouse alpha and beta chains generates a set of unique interactions that compensate for the low-oxygen-affinity propensity of the mouse beta chain. DPG binds the tetramer in the central cavity formed by the two beta subunits, hence the DPG effects on the interspecies hybrids should be as in the parent molecule. However, the results of the present study demonstrate that the DPG binding pocket is influenced by the nature of the alpha chain present in the tetramer. The mouse alpha chain reduces considerably the DPG right shift of the O2 affinity of the human beta-chain containing hybrid.(ABSTRACT TRUNCATED AT 250 WORDS)

Transfected human B lymphoblastoid cells express the mouse Ad beta-chain in association with DR alpha.

The human EBV-transformed cell line TOM was transfected with plasmid DNA containing a complete genomic copy of the murine class II Ad beta gene. Drug resistant transfectant clones showed cell surface expression of the Ad beta gene product, as detected by flow microfluorimetry when using a mouse monoclonal antibody (MKD6) specific for Ad beta. Immunoprecipitation and two-dimensional gel electrophoretic analysis of the Ad beta-containing molecules from the transfected cells revealed that the mouse beta-chain was expressed in noncovalent association with the human DR alpha-chain rather than with DQ alpha, the human A alpha homologue. The implications of this unexpected pairing for our understanding of the processes controlling the association of class II alpha and beta gene products and of the roles of Ia molecules in normal and pathologic immune function are discussed.

Globin synthesis in mouse erythroleukemia cells in vitro: a switch in beta chains due to inducing agent

Friend erythroleukemia cells, originally derived from DBA/2 mice, differentiate when cultured with inducing agents. Studies of the different effects of inducing agents on clone 745 have revealed that both dimethyl sulfoxide (DMSO) and hemin produce benzidine-positive cells. Butyric acid produced mature but benzidine-negative cells in this clone. All agents induced globin synthesis above the 0.1% of protein synthesis found in uninduced cells. DMSO induction stimulated globin synthesis 9%, hemin 2%, and butyric acid 3%. Total beta/alpha ratios were approximately unity with all agents. Although the inducing agents all stimulated total globin synthesis in Friend cells, the relative rates of synthesis of the two mouse beta chains were affected differently by the various agents. Hemin markedly increased the proportion of beta minor. For example, DBA/2 mouse reticulocytes synthesized 20% beta minor and 80% beta major. DMSO induction of clone 745 caused 20%-33% synthesis of beta minor. In contrast, hemin increased the proportion of beta minor to 64%-69%. Thus the Friend erythroleukemia cell system provides an in vitro approach to the study of the regulation of globin-chain switching.

Hemoglobin Beta chain structural variation in mice: evolutionary and functional implications.

The Hbb(d) allele at the hemoglobin beta chain locus of Mus musculus is composed of two linked genes, coding for structurally different beta chains, betadmin and betadmaj. Mus caroli has only one beta chain, which combines structural features of both betadmin and betadmaj and thus may be a Lepore type of beta chain. Sequence data suggest that selection may have been important in the evolution of the mouse beta chains.