Human leukocytes contain a large pool of free forms of CD18.

Monoclonal antibodies to CD18, the common chain of leukocyte integrins, recognize in various types of human lymphoid and myeloid cells under the conditions of nonreducing Western blotting three species of CD18 of mol. wt. 96, 87, and 78 kDa, respectively. Using a unique monoclonal antibody MEM-148 reacts exclusively with free CD18 molecules, but not with leukocyte integrin heterodimers. We demonstrate that only the upper one (96 kDa) is present on the cell surface within the CD11/CD18 integrin heterodimers, while the lower ones (87 and 78 kDa) are found intracellularly as free molecules unassociated with CD11 chains or other molecules. These intracellular free CD18 chains may in part represent biosynthetic precursors; alternatively, these species may represent an intracellular source of the recently observed free, proteolytically truncated CD18 chains expressed on the surface of activated myeloid cells.

Phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG), a novel ubiquitously expressed transmembrane adaptor protein, binds the protein tyrosine kinase csk and is involved in regulation of T cell activation.

According to a recently proposed hypothesis, initiation of signal transduction via immunoreceptors depends on interactions of the engaged immunoreceptor with glycosphingolipid-enriched membrane microdomains (GEMs). In this study, we describe a novel GEM-associated transmembrane adaptor protein, termed phosphoprotein associated with GEMs (PAG). PAG comprises a short extracellular domain of 16 amino acids and a 397-amino acid cytoplasmic tail containing ten tyrosine residues that are likely phosphorylated by Src family kinases. In lymphoid cell lines and in resting peripheral blood alpha/beta T cells, PAG is expressed as a constitutively tyrosine-phosphorylated protein and binds the major negative regulator of Src kinases, the tyrosine kinase Csk. After activation of peripheral blood alpha/beta T cells, PAG becomes rapidly dephosphorylated and dissociates from Csk. Expression of PAG in COS cells results in recruitment of endogenous Csk, altered Src kinase activity, and impaired phosphorylation of Src-specific substrates. Moreover, overexpression of PAG in Jurkat cells downregulates T cell receptor-mediated activation of the transcription factor nuclear factor of activated T cells. These findings collectively suggest that in the absence of external stimuli, the PAG-Csk complex transmits negative regulatory signals and thus may help to keep resting T cells in a quiescent state.