Abstract
Lymphocyte egress from lymph nodes requires the G-protein-coupled sphingosine 1-phosphate receptor-1 (S1P1). The activation antigen CD69 associates with and inhibits the function of S1P1, inhibiting egress. Here we undertook biochemical characterization of the requirements for S1P1-CD69 complex formation. Domain swapping experiments between CD69 and the related type II transmembrane protein, NKRp1A, identified a requirement for the transmembrane and membrane proximal domains for specific interaction. Mutagenesis of S1P1 showed a lack of requirement for N-linked glycosylation, tyrosine sulfation, or desensitization motifs but identified a requirement for transmembrane helix 4. Expression of CD69 led to a reduction of S1P1 in cell lysates, likely reflecting degradation. Unexpectedly, the S1P1-CD69 complex exhibited a much longer half-life for binding of S1P than S1P1 alone. In contrast to wild-type CD69, a non-S1P1 binding mutant of CD69 failed to inhibit T cell egress from lymph nodes. These findings identify an integral membrane interaction between CD69 and S1P1 and suggest that CD69 induces an S1P1 conformation that shares some properties of the ligand-bound state, thereby facilitating S1P1 internalization and degradation.
Highlights
CD69 is a type II transmembrane protein of the C-type lectin family and the Cd69 gene is encoded within the NK C-type lectin cluster [7,8,9]
These data suggested that the presence of sphingosine 1-phosphate receptor-1 (S1P1) keeps the low amounts of CD69 produced in naïve T cells from reaching the cell surface
Mapping CD69 Interaction Sites—To test whether immunespecific molecules were required for the CD69-S1P1 interaction we transduced 3T3 fibroblasts with retroviruses expressing HA-tagged CD69 or the related type II transmembrane protein, human NKRp1A, and Flag-tagged S1P1 or S1P3
Summary
CD69 is a type II transmembrane protein of the C-type lectin family and the Cd69 gene is encoded within the NK C-type lectin cluster [7,8,9]. All these constructs were expressed at similar levels on the surface of cells lacking FLAG-S1P1 as assessed by flow cytometry (supplemental Fig. S1).
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