title: CD69 suppresses sphingosine 1-phosophate receptor-1 (S1P1) function through interaction with membrane helix 4.
abstract: Lymphocyte egress from lymph nodes requires the G-protein-coupled sphingosine 1-phosphate receptor-1 (S1P(1)). The activation antigen CD69 associates with and inhibits the function of S1P(1), inhibiting egress. Here we undertook biochemical characterization of the requirements for S1P(1)-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 S1P(1) 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 S1P(1) in cell lysates, likely reflecting degradation. Unexpectedly, the S1P(1)-CD69 complex exhibited a much longer half-life for binding of S1P than S1P(1) alone. In contrast to wild-type CD69, a non-S1P(1) binding mutant of CD69 failed to inhibit T cell egress from lymph nodes. These findings identify an integral membrane interaction between CD69 and S1P(1) and suggest that CD69 induces an S1P(1) conformation that shares some properties of the ligand-bound state, thereby facilitating S1P(1) internalization and degradation.
authors.affiliation: Howard Hughes Medical Institute and Department of Microbiology and Immunology, University of California, San Francisco, California 94143, USA.
authors.forename: Alexander J
authors.initials: AJ
authors.lastname: Bankovich
authors.forename: Lawrence R
authors.initials: LR
authors.lastname: Shiow
authors.forename: Jason G
authors.initials: JG
authors.lastname: Cyster
mesh_terms: D016475:3T3 Cells; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism*; D000945:Antigens, Differentiation, T-Lymphocyte / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002634:Chemotaxis, Leukocyte; D006207:Half-Life; D006801:Humans; D047468:Immunoprecipitation; D037181:Lectins, C-Type / Q000378:metabolism*; D008214:Lymphocytes / Q000166:cytology / Q000378:metabolism; D008246:Lysophospholipids / Q000378:metabolism; D051379:Mice; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D049349:Receptors, Lysosphingolipid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013110:Sphingosine / Q000031:analogs & derivatives / Q000378:metabolism; D000099192:CD69 Antigens
chemical_list: D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D037181:Lectins, C-Type; D008246:Lysophospholipids; D050505:Mutant Proteins; D049349:Receptors, Lysosphingolipid; D013110:Sphingosine; D000099192:CD69 Antigens; C060506:sphingosine 1-phosphate
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