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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-mediated down-modulation of S1P1 is associated with protein degradation. A, flow cytometric analysis of Flag-S1P1 or Flag-S1P3 when co-transduced and sorted for low or high levels of CD69 in WEHI-231 cells. The relative amount of CD69 expression (+ or ++) was determined by expression of an IRES GFP reporter. Histogram overlays on the right show GFP reporter and hCD4 reporter expression for cells in quadrant 1 and 2 of the top left plot, indicating the relative expression of the CD69-IRES-GFP construct and the Flag-S1P1-IRES-hCD4 construct, respectively. B, co-IP of S1P1 or S1P3 with CD69 from the cells shown in the flow cytometric analysis. Densitometry readings are indicated showing the intensity of Flag and HA signal and the calculation of the ratio of HA signal to Flag signal is shown beneath the lower panel. These data are representative of two experiments using standard lysis buffer (see “Experimental Procedures”) and one experiment using 1% Triton X in place of Brij97 and Nonidet P-40 with similar results. Nonspecific bands are indicated with an asterisk.
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Figure 8: CD69-mediated down-modulation of S1P1 is associated with protein degradation. A, flow cytometric analysis of Flag-S1P1 or Flag-S1P3 when co-transduced and sorted for low or high levels of CD69 in WEHI-231 cells. The relative amount of CD69 expression (+ or ++) was determined by expression of an IRES GFP reporter. Histogram overlays on the right show GFP reporter and hCD4 reporter expression for cells in quadrant 1 and 2 of the top left plot, indicating the relative expression of the CD69-IRES-GFP construct and the Flag-S1P1-IRES-hCD4 construct, respectively. B, co-IP of S1P1 or S1P3 with CD69 from the cells shown in the flow cytometric analysis. Densitometry readings are indicated showing the intensity of Flag and HA signal and the calculation of the ratio of HA signal to Flag signal is shown beneath the lower panel. These data are representative of two experiments using standard lysis buffer (see “Experimental Procedures”) and one experiment using 1% Triton X in place of Brij97 and Nonidet P-40 with similar results. Nonspecific bands are indicated with an asterisk.

Mentions: Under conditions of co-expression, cells with high expression of CD69 down-modulate S1P1 and reciprocally cells with high expression of S1P1 down-modulate CD69 (4). To test the fate of S1P1 that has complexed with CD69 we compared Flag-S1P1-expressing cells that had been transduced with intermediate (+) or high (++) amounts of CD69. In the CD69 intermediate samples, the cells fall mostly into two mutually exclusive populations: cells with surface Flag-S1P1 staining and little CD69, and cells with CD69 surface expression and no Flag-S1P1 (Fig. 8A, HA-CD69+). Examination of reporter gene expression in these exclusive populations shows that the Flag-S1P1 surface-negative cells have higher amounts of the CD69 reporter (GFP) than the Flag-S1P1 surface-positive cells, whereas both have similar amounts of the Flag-S1P1 reporter (hCD4) (Fig. 8A, histogram plots) in agreement with previous findings (4). In cells transduced to express very high amounts of CD69 (Fig. 8A, HA-CD692+) the surface exposure of Flag-S1P1 was almost completely suppressed. By contrast, in Flag-S1P3-expressing cells, the intensity of Flag and CD69 staining was minimally affected by the presence of even very high amounts of CD69. High expression of CD69 not only reduces surface S1P1 abundance but leads to a reduction in total S1P1 protein within the cell (Fig. 8B), suggesting the protein is targeted for degradation. By contrast, although S1P3 was less abundantly expressed, the total amount was unaffected by high CD69 expression (Fig. 8B).


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-mediated down-modulation of S1P1 is associated with protein degradation. A, flow cytometric analysis of Flag-S1P1 or Flag-S1P3 when co-transduced and sorted for low or high levels of CD69 in WEHI-231 cells. The relative amount of CD69 expression (+ or ++) was determined by expression of an IRES GFP reporter. Histogram overlays on the right show GFP reporter and hCD4 reporter expression for cells in quadrant 1 and 2 of the top left plot, indicating the relative expression of the CD69-IRES-GFP construct and the Flag-S1P1-IRES-hCD4 construct, respectively. B, co-IP of S1P1 or S1P3 with CD69 from the cells shown in the flow cytometric analysis. Densitometry readings are indicated showing the intensity of Flag and HA signal and the calculation of the ratio of HA signal to Flag signal is shown beneath the lower panel. These data are representative of two experiments using standard lysis buffer (see “Experimental Procedures”) and one experiment using 1% Triton X in place of Brij97 and Nonidet P-40 with similar results. Nonspecific bands are indicated with an asterisk.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 8: CD69-mediated down-modulation of S1P1 is associated with protein degradation. A, flow cytometric analysis of Flag-S1P1 or Flag-S1P3 when co-transduced and sorted for low or high levels of CD69 in WEHI-231 cells. The relative amount of CD69 expression (+ or ++) was determined by expression of an IRES GFP reporter. Histogram overlays on the right show GFP reporter and hCD4 reporter expression for cells in quadrant 1 and 2 of the top left plot, indicating the relative expression of the CD69-IRES-GFP construct and the Flag-S1P1-IRES-hCD4 construct, respectively. B, co-IP of S1P1 or S1P3 with CD69 from the cells shown in the flow cytometric analysis. Densitometry readings are indicated showing the intensity of Flag and HA signal and the calculation of the ratio of HA signal to Flag signal is shown beneath the lower panel. These data are representative of two experiments using standard lysis buffer (see “Experimental Procedures”) and one experiment using 1% Triton X in place of Brij97 and Nonidet P-40 with similar results. Nonspecific bands are indicated with an asterisk.
Mentions: Under conditions of co-expression, cells with high expression of CD69 down-modulate S1P1 and reciprocally cells with high expression of S1P1 down-modulate CD69 (4). To test the fate of S1P1 that has complexed with CD69 we compared Flag-S1P1-expressing cells that had been transduced with intermediate (+) or high (++) amounts of CD69. In the CD69 intermediate samples, the cells fall mostly into two mutually exclusive populations: cells with surface Flag-S1P1 staining and little CD69, and cells with CD69 surface expression and no Flag-S1P1 (Fig. 8A, HA-CD69+). Examination of reporter gene expression in these exclusive populations shows that the Flag-S1P1 surface-negative cells have higher amounts of the CD69 reporter (GFP) than the Flag-S1P1 surface-positive cells, whereas both have similar amounts of the Flag-S1P1 reporter (hCD4) (Fig. 8A, histogram plots) in agreement with previous findings (4). In cells transduced to express very high amounts of CD69 (Fig. 8A, HA-CD692+) the surface exposure of Flag-S1P1 was almost completely suppressed. By contrast, in Flag-S1P3-expressing cells, the intensity of Flag and CD69 staining was minimally affected by the presence of even very high amounts of CD69. High expression of CD69 not only reduces surface S1P1 abundance but leads to a reduction in total S1P1 protein within the cell (Fig. 8B), suggesting the protein is targeted for degradation. By contrast, although S1P3 was less abundantly expressed, the total amount was unaffected by high CD69 expression (Fig. 8B).

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
Related in: MedlinePlus