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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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Interaction between CD69 and S1P1-containing mutations in the G-protein-interacting ERY motif or the cytoplasmic tail. A, flow cytometric analysis of S1P1 ERY motif mutants, EAY and ENY, or S1P3 as a control, co-transduced with CD69 and hNKRp1A in WEHI-231 cells. Cells are costained for the CD69 ectodomain and the Flag-S1P receptors and mutants as indicated. B, co-IP experiment for the S1P1 mutants or S1P3 as indicated. These data are representative of two experiments with similar results. C, flow cytometric analysis of desensitization mutants (S5A, Δ12) and S1P non-binding mutant (R120A), co-transduced with CD69 and hNKRp1A in WEHI-231 cells. Cells are co-stained for the CD69 ectodomain and the Flag-S1P receptors and mutants as indicated. Nonspecific bands are indicated with an asterisk.
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Figure 9: Interaction between CD69 and S1P1-containing mutations in the G-protein-interacting ERY motif or the cytoplasmic tail. A, flow cytometric analysis of S1P1 ERY motif mutants, EAY and ENY, or S1P3 as a control, co-transduced with CD69 and hNKRp1A in WEHI-231 cells. Cells are costained for the CD69 ectodomain and the Flag-S1P receptors and mutants as indicated. B, co-IP experiment for the S1P1 mutants or S1P3 as indicated. These data are representative of two experiments with similar results. C, flow cytometric analysis of desensitization mutants (S5A, Δ12) and S1P non-binding mutant (R120A), co-transduced with CD69 and hNKRp1A in WEHI-231 cells. Cells are co-stained for the CD69 ectodomain and the Flag-S1P receptors and mutants as indicated. Nonspecific bands are indicated with an asterisk.

Mentions: Previously we found that pertussis toxin treatment led to recovery of CD69 expression in S1P1 dominated cells, while not allowing recovery of S1P1 expression in CD69-dominated cells (4), suggesting different requirements for down-modulation of the complex depending on which partner is more abundantly expressed. By mutating the ERY motif of S1P1 (to EAY or ENY), we found that an intact G-protein-coupling motif (25) is needed for down-modulation of the complex, irrespective of whether CD69 or S1P1 is dominant (Fig. 9A). That is, rather than observing evidence for mutually exclusive surface expression of Flag-S1P1 and CD69 (as seen with wild-type S1P1 in Fig. 9A) the molecules were coexpressed on the cell surface (Fig. 9A, EAY and ENY). As expected, cells expressing NKRp1A as a control showed no evidence of Flag-S1P1 modulation (Fig. 9A, NKRp1A-stained plots). Despite the effect of the EAY and ENY mutations on modulation, these mutations did not disrupt the ability of CD69 and S1P1 to interact in the co-immunoprecipitation assay (Fig. 9B). Disruption of a desensitization motif in the C terminus of S1P1 by mutating 5 serines to alanines (S5A) (22) prevented S1P1 from mediating efficient down-modulation of CD69 without blocking CD69 down-modulation of S1P1 (Fig. 9C). That is, the cells expressing high amounts of mutant Flag-S1P1 were mostly co-expressing surface CD69 (Fig. 9C, S5A) in contrast to the suppression of CD69 expression on the cells expressing high amounts of wild-type Flag-S1P1 (Fig. 9C). This suggests a requirement for this serine-rich motif in S1P1-mediated modulation of CD69, but not in CD69-mediated modulation of S1P1. Deletion of the last 12 amino acids from the S1P1 C terminus, a region that contains further residues involved in ligand-mediated desensitization (23) did not affect down-modulation of the CD69-S1P1 complex (Fig. 9C, Δ12). Notably, mutation of S1P1 residue Arg-120 to Ala, a mutation that disrupts S1P binding (24), prevented efficient down-modulation of the CD69-S1P1 complex (Fig. 9C, R120A). This suggests that acquisition of a ligand-bound conformation might be necessary for efficient modulation of the CD69-S1P1 complex, both in cells dominated by CD69 and in cells dominated by S1P1.


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)

Interaction between CD69 and S1P1-containing mutations in the G-protein-interacting ERY motif or the cytoplasmic tail. A, flow cytometric analysis of S1P1 ERY motif mutants, EAY and ENY, or S1P3 as a control, co-transduced with CD69 and hNKRp1A in WEHI-231 cells. Cells are costained for the CD69 ectodomain and the Flag-S1P receptors and mutants as indicated. B, co-IP experiment for the S1P1 mutants or S1P3 as indicated. These data are representative of two experiments with similar results. C, flow cytometric analysis of desensitization mutants (S5A, Δ12) and S1P non-binding mutant (R120A), co-transduced with CD69 and hNKRp1A in WEHI-231 cells. Cells are co-stained for the CD69 ectodomain and the Flag-S1P receptors and mutants as indicated. Nonspecific bands are indicated with an asterisk.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 9: Interaction between CD69 and S1P1-containing mutations in the G-protein-interacting ERY motif or the cytoplasmic tail. A, flow cytometric analysis of S1P1 ERY motif mutants, EAY and ENY, or S1P3 as a control, co-transduced with CD69 and hNKRp1A in WEHI-231 cells. Cells are costained for the CD69 ectodomain and the Flag-S1P receptors and mutants as indicated. B, co-IP experiment for the S1P1 mutants or S1P3 as indicated. These data are representative of two experiments with similar results. C, flow cytometric analysis of desensitization mutants (S5A, Δ12) and S1P non-binding mutant (R120A), co-transduced with CD69 and hNKRp1A in WEHI-231 cells. Cells are co-stained for the CD69 ectodomain and the Flag-S1P receptors and mutants as indicated. Nonspecific bands are indicated with an asterisk.
Mentions: Previously we found that pertussis toxin treatment led to recovery of CD69 expression in S1P1 dominated cells, while not allowing recovery of S1P1 expression in CD69-dominated cells (4), suggesting different requirements for down-modulation of the complex depending on which partner is more abundantly expressed. By mutating the ERY motif of S1P1 (to EAY or ENY), we found that an intact G-protein-coupling motif (25) is needed for down-modulation of the complex, irrespective of whether CD69 or S1P1 is dominant (Fig. 9A). That is, rather than observing evidence for mutually exclusive surface expression of Flag-S1P1 and CD69 (as seen with wild-type S1P1 in Fig. 9A) the molecules were coexpressed on the cell surface (Fig. 9A, EAY and ENY). As expected, cells expressing NKRp1A as a control showed no evidence of Flag-S1P1 modulation (Fig. 9A, NKRp1A-stained plots). Despite the effect of the EAY and ENY mutations on modulation, these mutations did not disrupt the ability of CD69 and S1P1 to interact in the co-immunoprecipitation assay (Fig. 9B). Disruption of a desensitization motif in the C terminus of S1P1 by mutating 5 serines to alanines (S5A) (22) prevented S1P1 from mediating efficient down-modulation of CD69 without blocking CD69 down-modulation of S1P1 (Fig. 9C). That is, the cells expressing high amounts of mutant Flag-S1P1 were mostly co-expressing surface CD69 (Fig. 9C, S5A) in contrast to the suppression of CD69 expression on the cells expressing high amounts of wild-type Flag-S1P1 (Fig. 9C). This suggests a requirement for this serine-rich motif in S1P1-mediated modulation of CD69, but not in CD69-mediated modulation of S1P1. Deletion of the last 12 amino acids from the S1P1 C terminus, a region that contains further residues involved in ligand-mediated desensitization (23) did not affect down-modulation of the CD69-S1P1 complex (Fig. 9C, Δ12). Notably, mutation of S1P1 residue Arg-120 to Ala, a mutation that disrupts S1P binding (24), prevented efficient down-modulation of the CD69-S1P1 complex (Fig. 9C, R120A). This suggests that acquisition of a ligand-bound conformation might be necessary for efficient modulation of the CD69-S1P1 complex, both in cells dominated by CD69 and in cells dominated by S1P1.

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