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CD69 suppresses sphingosine 1-phosophate receptor-1 (S1P1) function through interaction with membrane helix 4.

Bankovich AJ, Shiow LR, Cyster JG - J. Biol. Chem. (2010)

Bottom Line: Expression of CD69 led to a reduction of S1P(1) in cell lysates, likely reflecting degradation.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.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Department of Microbiology and Immunology, University of California, San Francisco, California 94143, USA.

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.

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CD69 and S1P1 interact in mouse 3T3 fibroblast cells. Cells expressing or co-expressing the indicated constructs were lysed and subjected to immunoprecipitation with anti-Flag M2 beads. The material in the IP was then run on SDS-PAGE and Western blotted for Flag to detect the S1P receptor, and for HA to detect the C-type lectin construct and actin as a loading control. The amount of each protein in the cell lysate was compared with the amount immunoprecipitated by Flag-S1P1 with anti-Flag antibody. When equivalent amounts of Flag were immunoprecipitated, there was a 10-fold greater interaction between CD69 and S1P1 when compared with the interaction with control proteins NKRp1A and S1P3. This result is representative of two experiments.
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Figure 1: CD69 and S1P1 interact in mouse 3T3 fibroblast cells. Cells expressing or co-expressing the indicated constructs were lysed and subjected to immunoprecipitation with anti-Flag M2 beads. The material in the IP was then run on SDS-PAGE and Western blotted for Flag to detect the S1P receptor, and for HA to detect the C-type lectin construct and actin as a loading control. The amount of each protein in the cell lysate was compared with the amount immunoprecipitated by Flag-S1P1 with anti-Flag antibody. When equivalent amounts of Flag were immunoprecipitated, there was a 10-fold greater interaction between CD69 and S1P1 when compared with the interaction with control proteins NKRp1A and S1P3. This result is representative of two experiments.

Mentions: To test whether immune-specific 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. Immunoprecipitation experiments with lysates from cotransduced cells revealed that S1P1 co-immunoprecipitated CD69 but not NKRp1A, whereas S1P3 co-immunoprecipitated little of either lectin molecule (Fig. 1). These results suggest a lack of immune-specific molecule requirement for the CD69-S1P1 interaction.


CD69 suppresses sphingosine 1-phosophate receptor-1 (S1P1) function through interaction with membrane helix 4.

Bankovich AJ, Shiow LR, Cyster JG - J. Biol. Chem. (2010)

CD69 and S1P1 interact in mouse 3T3 fibroblast cells. Cells expressing or co-expressing the indicated constructs were lysed and subjected to immunoprecipitation with anti-Flag M2 beads. The material in the IP was then run on SDS-PAGE and Western blotted for Flag to detect the S1P receptor, and for HA to detect the C-type lectin construct and actin as a loading control. The amount of each protein in the cell lysate was compared with the amount immunoprecipitated by Flag-S1P1 with anti-Flag antibody. When equivalent amounts of Flag were immunoprecipitated, there was a 10-fold greater interaction between CD69 and S1P1 when compared with the interaction with control proteins NKRp1A and S1P3. This result is representative of two experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2903414&req=5

Figure 1: CD69 and S1P1 interact in mouse 3T3 fibroblast cells. Cells expressing or co-expressing the indicated constructs were lysed and subjected to immunoprecipitation with anti-Flag M2 beads. The material in the IP was then run on SDS-PAGE and Western blotted for Flag to detect the S1P receptor, and for HA to detect the C-type lectin construct and actin as a loading control. The amount of each protein in the cell lysate was compared with the amount immunoprecipitated by Flag-S1P1 with anti-Flag antibody. When equivalent amounts of Flag were immunoprecipitated, there was a 10-fold greater interaction between CD69 and S1P1 when compared with the interaction with control proteins NKRp1A and S1P3. This result is representative of two experiments.
Mentions: To test whether immune-specific 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. Immunoprecipitation experiments with lysates from cotransduced cells revealed that S1P1 co-immunoprecipitated CD69 but not NKRp1A, whereas S1P3 co-immunoprecipitated little of either lectin molecule (Fig. 1). These results suggest a lack of immune-specific molecule requirement for the CD69-S1P1 interaction.

Bottom Line: Expression of CD69 led to a reduction of S1P(1) in cell lysates, likely reflecting degradation.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.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Department of Microbiology and Immunology, University of California, San Francisco, California 94143, USA.

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.

Show MeSH