ABSTRACT
In addition to membrane immunoglobulin (mIg), the B-cell antigen receptor contains Ig-alpha/Ig-beta heterodimers that link mIg to intracellular signaling molecules. To compare the ability of the cytoplasmic domains of Ig-alpha and Ig-beta to transduce signals in B- and T-cells, we constructed chimeric genes encoding the extracellular and transmembrane domains of human CD8 alpha and the cytoplasmic domain of murine Ig-alpha (CD8/Ig-alpha) or Ig-beta (CD8/Ig-beta). In murine B-cell hybridoma LK 35.2 cells, antibody-mediated cross-linking of mIg, CD8/Ig-alpha, or CD8/Ig-beta induced similar increases in intracellular calcium levels and protein tyrosine phosphorylation. Substitution of alanine for the conserved leucine, but not the conserved isoleucine, residue within the putative activation motif of CD8/Ig-beta destroyed signaling ability. In murine T-cell hybridoma DO-11.10 cells, cross-linking of the T-cell antigen receptor, CD8/Ig-alpha, or CD8/Ig-beta stimulated equivalent protein tyrosine phosphorylation and interleukin-2 production. Thus, the cytoplasmic domains of Ig-alpha and Ig-beta are equally capable of initiating early signaling events downstream of B- and T-cell antigen receptors as well as evoking a complete biological effector response in lymphocytes.
Subject(s)
Antigens, CD , B-Lymphocytes/immunology , Lymphocyte Activation , Receptors, Antigen, B-Cell/immunology , T-Lymphocytes/immunology , Animals , Base Sequence , CD79 Antigens , CD8 Antigens/immunology , Cross-Linking Reagents , DNA Primers , Humans , Immunoglobulins/genetics , Immunoglobulins/immunology , Leucine/genetics , Membrane Glycoproteins/genetics , Membrane Glycoproteins/immunology , Mice , Molecular Sequence Data , Mutation , Phosphorylation , Receptors, Antigen, B-Cell/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Signal Transduction , Tyrosine/metabolismABSTRACT
The ability of benzyloxycarbonyl-(125I)Tyr-Ala-CHN2 to label cysteine proteinases in a variety of human tissues was investigated. The inhibitor bound only to cathepsin B in tissues homogenized at pH 5.0. When liver was autolysed at pH 4.0 for up to 4 h, the inhibitor also bound to a protein of Mr 25,000. This was identified immunologically and chromatographically as cathepsin L. Both cathepsins B and L were found primarily in kidney, liver and spleen. In spleen, an additional protein of Mr 25,000 was also labelled. This protein could not be precipitated by antibodies to any of cathepsins B, H and L. This protein has tentatively been identified as human cathepsin S by its tissue distribution, chromatographic properties and molecular size. This work clearly shows that peptidyldiazomethanes are specific probes for cysteine proteinases, and that benzyloxycarbonyl-(125I)Tyr-Ala-CHN2 binds to three such enzymes in human tissues.