Immunoisolation of TCR Signaling Complexes from Jurkat T Leukemic Cells

Abstract

The formation of multimolecular assemblies of signaling molecules at the plasma membrane is key to triggering signaling cascades following activation of transmembrane receptors at the plasma membrane. We have developed a method to immunoisolate activated T cell receptors (TCRs) and associated signaling molecules in plasma membrane subdomains from Jurkat T leukemic cells. The immunoisolation procedure for the signaling complexes uses magnetic beads, which are coupled to TCR-activating antibodies. Following mechanical cellular disruption using nitrogen cavitation, conjugates of these beads with Jurkat cells are formed and isolated. TCR-signaling complexes within plasma membrane fragments associate with the antibody-coupled beads and are retrieved along with the beads using a magnet. We found that the immunoisolated plasma membrane fragments are highly enriched for activated TCRs and associated signaling proteins. This isolation procedure allows a detailed and precise biochemical analysis of the assembly of signaling proteins in plasma membrane subdomains.

A novel antiapoptotic mechanism based on interference of Fas signaling by CD44 variant isoforms

“…This is an alternative procedure to the standard co-immunoprecipitations for examining interactions between membrane proteins. 29 The advantage of this procedure is that it is detergent (Figure 5d). These data confirm that only CD44v, but not CD44s, interacted with Fas and that CD44v6 and v9 strongly interacted with Fas, whereas CD44v3 did so at lesser intensity.…”

Signal Transduction in T Helper Cells: CD4 Coreceptors Exert Complex Regulatory Effects on T Cell Activation and Function

“…Formation of signalosomes is aided by compartmentalization of the plasma membrane into detergent-insoluble, sphingolipid/cholesterol-enriched micro-domains, which promote the recruitment of signal transduction molecules to the TCR signaling machine upon TCR engagement (Harder and Kuhn, 2001;Leitenberg et al, 2001). These areas of the T cell surface are also known as lipid "rafts".…”

“…These areas of the T cell surface are also known as lipid "rafts". Palmitoylation constitutively embeds several components such as Lck, Fyn, and LAT into these lipid micro-domains, whereas others such as ZAP-70 relocalize into rafts upon TCR engagement (Harder and Kuhn, 2001;Werlen et al, 2000).…”

The CD44 standard/ezrin complex regulates Fas-mediated apoptosis in Jurkat cells

“…This alternative procedure to ''standard'' co-immunoprecipitations offers the advantage that it is detergent-free, and therefore provides an excellent method to examine protein interactions among membrane proteins found in lipid rafts [31]. To study the interaction between CD44 and ezrin, we targeted CD44 in Jurkat neo, Jurkat CD44s, Jurkat CD44v2-10, v3, v6, v9 and Jurkat CD44sDcyt cells by cavitation bomb extractions, followed by immunoblotting with anti-ezrin antibody ( Fig.…”

“…Cells were intensively washed in H-Buffer, resuspended in H-Buffer+ (H-Buffer with 6.6 lM Pervanadate and 1 mini tablet of a protease inhibitor cocktail (Roche, Penzberg, Germany) for 10 ml of solution. The number of cells coated with beads was counted under the microscope and approximately 40 · 10 5 cells per sample were disrupted in a nitrogen cavitation bomb at À196°C and 600 psi (pounds per square inch) [31]. After disruption of the membranes, the samples were washed and retrieved four times and finally resuspended in 2· SDS sample buffer.…”