title: Basal and antigen-induced exposure of the proline-rich sequence in CD3ε.
abstract: The CD3ε cytoplasmic tail contains a conserved proline-rich sequence (PRS) that influences TCR-CD3 expression and signaling. Although the PRS can bind the SH3.1 domain of the cytosolic adapter Nck, whether the PRS is constitutively available for Nck binding or instead represents a cryptic motif that is exposed via conformational change upon TCR-CD3 engagement (CD3Δc) is currently unresolved. Furthermore, the extent to which a cis-acting CD3ε basic amino acid-rich stretch (BRS), with its unique phosphoinositide-binding capability, might impact PRS accessibility is not clear. In this study, we found that freshly harvested primary thymocytes expressed low to moderate basal levels of Nck-accessible PRS ("open-CD3"), although most TCR-CD3 complexes were inaccessible to Nck ("closed-CD3"). Ag presentation in vivo induced open-CD3, accounting for half of the basal level found in thymocytes from MHC(+) mice. Additional stimulation with either anti-CD3 Abs or peptide-MHC ligands further elevated open-CD3 above basal levels, consistent with a model wherein antigenic engagement induces maximum PRS exposure. We also found that the open-CD3 conformation induced by APCs outlasted the time of ligand occupancy, marking receptors that had been engaged. Finally, CD3ε BRS-phosphoinositide interactions played no role in either adoption of the initial closed-CD3 conformation or induction of open-CD3 by Ab stimulation. Thus, a basal level of open-CD3 is succeeded by a higher, induced level upon TCR-CD3 engagement, involving CD3Δc and prolonged accessibility of the CD3ε PRS to Nck.
authors.affiliation: Department of Immunology, College of Medicine, Mayo Clinic, Rochester, MN 55905, USA.
authors.forename: Javier
authors.initials: J
authors.lastname: de la Cruz
authors.forename: Travis
authors.initials: T
authors.lastname: Kruger
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Parks
authors.forename: Robert L
authors.initials: RL
authors.lastname: Silge
authors.forename: Nicolai S C
authors.initials: NS
authors.lastname: van Oers
authors.forename: Immanuel F
authors.initials: IF
authors.lastname: Luescher
authors.forename: Adam G
authors.initials: AG
authors.lastname: Schrum
authors.forename: Diana
authors.initials: D
authors.lastname: Gil
mesh_terms: D020816:Amino Acid Motifs / Q000276:immunology; D000818:Animals; D000938:Antigen-Presenting Cells / Q000276:immunology* / Q000378:metabolism*; D017252:CD3 Complex / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D045744:Cell Line, Tumor; D018984:Epitopes, T-Lymphocyte / Q000502:physiology; D006825:Hybridomas; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008822:Mice, Transgenic; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011392:Proline / Q000276:immunology / Q000378:metabolism*; D017260:Receptor-CD3 Complex, Antigen, T-Cell / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism
chemical_list: D017252:CD3 Complex; C485468:Cd3e protein, mouse; D018984:Epitopes, T-Lymphocyte; D010446:Peptide Fragments; D017260:Receptor-CD3 Complex, Antigen, T-Cell; D011392:Proline