An ER Retrieval-and-Release System Reveals Ligand-Independent Basal Signaling by Cell Surface TCRs

Abstract The T cell receptor (TCR) machinery comprises the ligand-binding TCRαβ and the CD3 signaling dimers. Individual subunits are synthesized in the endoplasmic reticulum (ER) and only the fully assembled TCRs reach the cell surface. We engineered a system to retained CD3ε is in the ER. This allowed only partial TCR assembly, while continuous retrieval of CD3ε from the Golgi back to the ER enabled complete assembly. Synchronous TCR release by biotin addition caused a transient increase in surface TCR abundance that gradually returned to steady-state levels. Mathematical modeling revealed that TCR levels were controlled by feedback mechanisms. Notably, surface-localized, but not ER-localized, TCRs spontaneously generated ligand-independent basal signaling. Phosphoproteomic analyses revealed that this signal is similar, but not identical, to ligand-induced TCR activation signals, with distinct phosphorylation patterns already evident at the CD3 subunits. Our findings establish the TCR retrieval-and-release system as a tool to investigate ligand-independent basal TCR signaling.