Isolation of newly expressed surface T cell antigen receptor complexes by serial precipitation with anti-TCR antibodies and immobilized streptavidin

Abstract

Expression of the multisubunit T cell antigen receptor (TCR) complex is an enigmatic process requiring coordinated regulation of at least six different gene products (α, β, γ, δ, ε, and ζ ), the ordered pairing of partner chains within the endoplasmic reticulum (ER), and intracellular transport of complete, but not incomplete, TCR complexes to the cell surface. Movement of nascent TCR glycoproteins from the ER to the Golgi compartment is easily studied using various lectins and/or glycosidases specific for oligosaccharide modifications that occur within the Golgi system. In contrast, cell surface transport of TCR complexes is relatively difficult to assess, since this requires physical separation of intracellular complexes from surface TCR complexes. In the current report we describe a method for the isolation of newly transported surface TCR complexes which utilizes metabolic and surface labeling techniques in conjunction with serial precipitation methods. Specifically, we describe the use of anti-TCR antibodies and immobilized streptavidin to isolate nascent TCRα proteins localized on the plasma membrane. This technique is rapid, specific, and provides a novel approach for studying the intracellular transport of nascent immune receptor molecules to the cell surface.