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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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Requirement for CD69 interaction with S1P1 to inhibit lymphocyte chemotaxis and egress from lymph nodes. A, transwell migration assay testing CD69 and relevant CD69/NKRp1A chimeric molecules for their ability to inhibit S1P1-dependent S1P migration. Migration to SDF-1α, a CXCR4 ligand, is shown as a negative control. B, schematic of cell transfer experiment. Donor cells were activated for 24 h with anti-CD3/CD28 antibodies before transduction of the indicated recombinant retroviruses. These three CMTMR and congenically marked cell populations were then mixed and injected into recipient mice. Half of these mice were analyzed 24 h post-transfer and half where treated at 24 h with α4 and αL integrin-neutralizing antibodies to block LN entry and LN cells were analyzed 18 h later. C, flow cytometric analysis showing transferred CD4 cells, distinguishing 6N6-Δ31-transduced cells (CMTMR−) from CD69-transduced cells (CMTMR+) in the same LN preparation. Transduced cells with high GFP reporter expression were gated and the percent of these cells among total LN cells is shown. D, ratio of transduced cells remaining in LNs following 18 h of α4 plus αL antibody treatment compared with the starting number of cells. This result is representative of two independent experiments.
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Figure 11: Requirement for CD69 interaction with S1P1 to inhibit lymphocyte chemotaxis and egress from lymph nodes. A, transwell migration assay testing CD69 and relevant CD69/NKRp1A chimeric molecules for their ability to inhibit S1P1-dependent S1P migration. Migration to SDF-1α, a CXCR4 ligand, is shown as a negative control. B, schematic of cell transfer experiment. Donor cells were activated for 24 h with anti-CD3/CD28 antibodies before transduction of the indicated recombinant retroviruses. These three CMTMR and congenically marked cell populations were then mixed and injected into recipient mice. Half of these mice were analyzed 24 h post-transfer and half where treated at 24 h with α4 and αL integrin-neutralizing antibodies to block LN entry and LN cells were analyzed 18 h later. C, flow cytometric analysis showing transferred CD4 cells, distinguishing 6N6-Δ31-transduced cells (CMTMR−) from CD69-transduced cells (CMTMR+) in the same LN preparation. Transduced cells with high GFP reporter expression were gated and the percent of these cells among total LN cells is shown. D, ratio of transduced cells remaining in LNs following 18 h of α4 plus αL antibody treatment compared with the starting number of cells. This result is representative of two independent experiments.

Mentions: Previous work established that CD69 is necessary for inhibition of lymphocyte egress following IFNα/β exposure and correlated this requirement with the ability of CD69 to inhibit S1P1 function (4). To further test whether CD69 inhibition of egress depends on its ability to interact with and inhibit S1P1, we tested the activity of wild type and an S1P1 non-binding mutant of CD69 (6N6-Δ31, Fig. 2B) at inhibiting T cell migration to S1P and egress from lymph nodes (Fig. 11). Consistent with the inability to interact with S1P1, 6N6-Δ31 had no inhibitory activity on S1P1-mediated cell migration to S1P (Fig. 11A). The reciprocal construct, N6N-Δ31 that has some ability to interact with S1P1 (Fig. 2B) showed partial inhibition of migration (Fig. 11A). Interestingly, construct 6N6-stalk that contains the CD69 ectodomain and scores positive in the interaction assay (Fig. 2B), failed to inhibit migration (Fig. 11A). Wild-type CD69 strongly inhibited migration to S1P as expected, while not affecting the response to the chemokine SDF1α (CXCL12) (Fig. 11A). Together, these findings highlight the need for the CD69 transmembrane and HEGSI motif for the migration inhibitory effect but also suggest that additional interaction surfaces cooperate to achieve the full migration block.


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)

Requirement for CD69 interaction with S1P1 to inhibit lymphocyte chemotaxis and egress from lymph nodes. A, transwell migration assay testing CD69 and relevant CD69/NKRp1A chimeric molecules for their ability to inhibit S1P1-dependent S1P migration. Migration to SDF-1α, a CXCR4 ligand, is shown as a negative control. B, schematic of cell transfer experiment. Donor cells were activated for 24 h with anti-CD3/CD28 antibodies before transduction of the indicated recombinant retroviruses. These three CMTMR and congenically marked cell populations were then mixed and injected into recipient mice. Half of these mice were analyzed 24 h post-transfer and half where treated at 24 h with α4 and αL integrin-neutralizing antibodies to block LN entry and LN cells were analyzed 18 h later. C, flow cytometric analysis showing transferred CD4 cells, distinguishing 6N6-Δ31-transduced cells (CMTMR−) from CD69-transduced cells (CMTMR+) in the same LN preparation. Transduced cells with high GFP reporter expression were gated and the percent of these cells among total LN cells is shown. D, ratio of transduced cells remaining in LNs following 18 h of α4 plus αL antibody treatment compared with the starting number of cells. This result is representative of two independent experiments.
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Figure 11: Requirement for CD69 interaction with S1P1 to inhibit lymphocyte chemotaxis and egress from lymph nodes. A, transwell migration assay testing CD69 and relevant CD69/NKRp1A chimeric molecules for their ability to inhibit S1P1-dependent S1P migration. Migration to SDF-1α, a CXCR4 ligand, is shown as a negative control. B, schematic of cell transfer experiment. Donor cells were activated for 24 h with anti-CD3/CD28 antibodies before transduction of the indicated recombinant retroviruses. These three CMTMR and congenically marked cell populations were then mixed and injected into recipient mice. Half of these mice were analyzed 24 h post-transfer and half where treated at 24 h with α4 and αL integrin-neutralizing antibodies to block LN entry and LN cells were analyzed 18 h later. C, flow cytometric analysis showing transferred CD4 cells, distinguishing 6N6-Δ31-transduced cells (CMTMR−) from CD69-transduced cells (CMTMR+) in the same LN preparation. Transduced cells with high GFP reporter expression were gated and the percent of these cells among total LN cells is shown. D, ratio of transduced cells remaining in LNs following 18 h of α4 plus αL antibody treatment compared with the starting number of cells. This result is representative of two independent experiments.
Mentions: Previous work established that CD69 is necessary for inhibition of lymphocyte egress following IFNα/β exposure and correlated this requirement with the ability of CD69 to inhibit S1P1 function (4). To further test whether CD69 inhibition of egress depends on its ability to interact with and inhibit S1P1, we tested the activity of wild type and an S1P1 non-binding mutant of CD69 (6N6-Δ31, Fig. 2B) at inhibiting T cell migration to S1P and egress from lymph nodes (Fig. 11). Consistent with the inability to interact with S1P1, 6N6-Δ31 had no inhibitory activity on S1P1-mediated cell migration to S1P (Fig. 11A). The reciprocal construct, N6N-Δ31 that has some ability to interact with S1P1 (Fig. 2B) showed partial inhibition of migration (Fig. 11A). Interestingly, construct 6N6-stalk that contains the CD69 ectodomain and scores positive in the interaction assay (Fig. 2B), failed to inhibit migration (Fig. 11A). Wild-type CD69 strongly inhibited migration to S1P as expected, while not affecting the response to the chemokine SDF1α (CXCL12) (Fig. 11A). Together, these findings highlight the need for the CD69 transmembrane and HEGSI motif for the migration inhibitory effect but also suggest that additional interaction surfaces cooperate to achieve the full migration block.

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