Sphingosine-1-phosphate (S1P) plays an essential role in the immune system by promoting the egress of lymphocytes from lymphoid organs into blood and lymph via a direct interaction with one of its five cognate G protein–coupled receptors, S1PR1 (Baeyens and Schwab, 2020; Cartier and Hla, 2019; Cyster and Schwab, 2012; Pappu et al., 2007; Rosen et al., 2013; Spiegel and Milstien, 2003). After egressing from spleen, lymph nodes, or mucosal lymphoid tissues, T and B lymphocytes travel to other lymphoid organs in a cycle of continual pathogen surveillance. When an infection occurs, there is a temporary shutdown of lymphocyte egress from the responding lymphoid organ(s) and this enables increased accumulation of lymphocytes and enhances the immune response (Cyster and Schwab, 2012). Egress shutdown is mediated by type I interferon (IFN) inducing lymphocyte CD69 expression. CD69, a type II transmembrane C-type lectin protein, intrinsically inhibits the function of S1PR1 in T and B cells (Shiow et al., 2006). CD69 also regulates T cell egress from the thymus (Nakayama et al., 2002; Zachariah and Cyster, 2010). A disulfide-bond in the extracellular domain links CD69 as a homodimer (Ziegler et al., 1994). Biochemical studies demonstrated that CD69 may associate with S1PR1 through interactions between their transmembrane domains (TMs) to facilitate S1PR1 internalization and degradation (Bankovich et al., 2010). Unlike S1P, CD69 has been shown to bind S1PR1 but not the other S1PRs (Bankovich et al., 2010; Jenne et al., 2009; Shiow et al., 2006). However, the mechanism of CD69-induced S1PR1 internalization and thus functional inactivation has been unclear.

Importantly, several S1PR1 modulators (e.g. Fingolimod, also known as FTY720, Siponimod, Ozanimod, and Etrasimod), have been approved for treating the autoimmune diseases multiple sclerosis and ulcerative colitis (Brinkmann et al., 2010; Chun et al., 2021; Dal Buono et al., 2022; Kappos et al., 2010). These immunosuppressants are believed to act by inhibiting S1PR1 function and thereby preventing autoimmune effector lymphocytes exiting lymphoid organs, blocking the autoimmune attack. Either sphingosine or FTY720 is metabolically catalyzed by two intracellular sphingosine kinases into the phosphorylated form (S1P or FTY720-P) and then exported to the extracellular space via S1P transporters (Baeyens and Schwab, 2020; Spiegel et al., 2019). There, S1P binds to its receptors for initiation of the signal while FTY720-P activates the S1PR1 but causes a persistent internalization and degradation of S1PR1 to attenuate the signal (Brinkmann et al., 2010).

Recently, cryogenic electron microscopy (cryo-EM) structures of S1PR1 complexed with different small molecule ligands have been determined (Liu et al., 2022; Xu et al., 2022; Yu et al., 2022; Yuan et al., 2021). These findings reveal a mechanism of how S1PR1 engages its endogenous ligand S1P and its modulators to adopt the active conformation for recruiting the heterotrimeric G_i protein. The previously determined crystal structure of antagonist ML056-bound S1PR1 reveals its inactive state (Hanson et al., 2012). However, the molecular mechanism remains unknown of how CD69 binds to S1PR1 to trigger its internalization. Therefore, structural study on the S1PR1-CD69 complex will provide molecular insights into the CD69-mediated functional inhibition of S1PR1 and reveal how a class A GPCR can be regulated by a transmembrane protein modulator. In this manuscript, we determined the structure of CD69-bound S1PR1 coupled to G_αiβ1γ2 heterotrimer by cryo-EM at 3.15 Å resolution. Our findings reveal that TM of CD69 contacts TM4 of S1PR1 to activate the receptor allowing it to engage the α5 helix of G_αi in the absence of S1P ligand, thereby disrupting the receptor’s egress-promoting function.

Since serum contains an abundance of lipids including S1P, we expressed human S1PR1 or CD69 in HEK293 cells cultured in a medium with lipid-deficient serum. Then, we purified human S1PR1 protein alone to validate its activation in the presence of S1P using the GTPase-Glo assay (Figure 1A). We then tested the effect on S1PR1 of adding the CD69 homodimer in the absence of S1P. Remarkably, addition of CD69 alone caused a similar amount of G_i activation as addition of S1P indicating that CD69 functions as a protein agonist of S1PR1 (Figure 1A).

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To perform structural studies, we mixed lysates from HEK293 cells that independently expressed human CD69 and human S1PR1. The CD69-S1PR1 complex was then incubated with G_αiβ1γ2 heterotrimer and scFv16 (Maeda et al., 2018) at 1:1.2:1.4 molar ratio. After gel-filtration purification, the resulting complex was concentrated for cryo-EM analysis (Figure 1—figure supplement 1A). We obtained over 1 million particles from ~4000 cryo-EM images. The overall structure of the CD69-bound S1PR1 coupled to heterotrimeric G_i was determined at 3.15 Å resolution by 293,516 particles (Figure 1—figure supplement 1B–F; Table 1). The structure shows that one S1PR1 binds one CD69 homodimer and one G_i heterotrimer. It also revealed well-defined features for the canonical seven transmembrane helices (7-TMs) of S1PR1, the G_αi Ras-like domain, the G_β and G_γ subunits and scFv16 (Figure 1B, Figure 1—figure supplement 2A). The intracellular loop 3 (ICL3) and the C-terminus of S1PR1 and the intracellular and extracellular domains of CD69 were not found in the cryo-EM map indicating their flexibility in the complex. In contrast, the TMs of the CD69 homodimer were clearly defined in the map owing to their interactions with S1PR1 (Figure 1B, Figure 1—figure supplement 2A; the interacting TM helix is referred to as CD69-a). Because no lipid was supplemented into the protein during the expression and purification, there is no notable lipid ligand in the 7-TM bundle of S1PR1, which is different from the previous structural discoveries on S1PRs (Chen et al., 2022; Liu et al., 2022; Xu et al., 2022; Yu et al., 2022; Yuan et al., 2021; Zhao et al., 2022).

Structural comparison shows that the entire complex and the S1P bound S1PR1-G_i complex share a similar conformation with a root-mean-square deviation (RMSD) of 0.82 Å (Figure 1—figure supplement 3A). The receptors in both complexes can be aligned well; however, the F161^4.43 in TM4 presents a notable shift due to CD69 binding (Figure 1—figure supplement 3B). We docked the S1PR1 bound to the other TM of the CD69 homodimer which showed that the modeled receptor would sterically clash with the G_iα subunit (Figure 1—figure supplement 3C). This may explain why only one receptor binds one CD69 homodimer in the presence of the heterotrimeric G-protein.

Receptor activity-modifying protein 1 (RAMP1), a type I transmembrane domain protein, binds the calcitonin receptor-like receptor (CLR) class B GPCR to form the Calcitonin gene-related peptide (CGRP) receptor which is involved in the pathology of migraine (Russell et al., 2014). The structure of G_s-protein coupled CGRP receptor uncovers that TM of RAMP1 interacts with TMs 3–5 of CLR and the extracellular domains of RAMP1 and CLR have extensive interactions (Liang et al., 2018; Figure 1—figure supplement 4A). Both CLR and RAMP1 contribute to the engagement of their agonist CGRP. However, in our structure, CD69 acts as an agonist to activate S1PR1 through a direct binding to TM4 of S1PR1 in the absence of a canonical agonist (e.g. S1P or FTY720-P). The extracellular domain of CD69 is completely invisible in the complex and may not interact with the extracellular loops of S1PR1.

Another type of intramembrane interaction observed for GPCRs is the formation of either homodimers or heterodimers. The metabotropic glutamate receptor 2 (mGlu2), a Class-C GPCR, employs TM4 to maintain its inactive dimeric state or TM6 to assemble as a homodimer in the presence of its agonist (Du et al., 2021; Figure 1—figure supplement 4B). The structure of inactive mGlu2–mGlu7 heterodimer shows that TM5 plays a key role in the complex assembly (Du et al., 2021; Figure 1—figure supplement 4C). Moreover, TM1 of the class D GPCR Ste2 is responsible for engaging the TM1 of another Ste2 to form a homodimer (Velazhahan et al., 2021; Figure 1—figure supplement 4D). These findings elucidate that GPCRs could employ distinct TMs to recruit their transmembrane binding partners.

The TM of one protomer of CD69 homodimer interacts with the TM4 of S1PR1 (Figure 1C). The interface area between TMs is about 600 Ų. Structural analysis shows that residues V41, V45, V48, V49, T52, I56, I59, A60 of CD69 mediate its extensive interactions with the receptor (Figure 2A, Figure 1—figure supplement 2B). Residues L160^4.42, F161^4.43, I164^4.46, W168^4.50, V169^4.51, L172^4.54, I173^4.55, G176^4.58, I179^4.61 and M180^4.62 of S1PR1-TM4 contribute to the interaction with CD69 (Figure 2B, Figure 1—figure supplement 2B). However, the TM of another CD69 does not have any interactions with the receptor and heterotrimeric G_i protein (Figure 1C). Further structural comparison with the S1P-bound S1PR1-G_i complex indicates that the heterotrimeric G_i proteins in both complexes exhibit a similar state with a RMSD of 0.45 Å. Also, the intracellular regions of the heptahelical domain adopt a similar conformation to accommodate the G_i proteins. This finding implies that S1P and CD69 stimulate the receptor to engage the heterotrimeric G_i proteins in an analogous fashion.

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Figure 2—source data 1

Original uncropped western blots for data in Figure 2.

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Figure 2—source data 2

Uncropped western blots for data in Figure 2 with the relevant bands labeled.

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To validate our structural observations, we performed the co-immunoprecipitation (co-IP) assay using S1PR1 and CD69 variants. Compared to the wild-type S1PR1, two mutants (V169^4.51Y and M180^4.62Y) present reduced binding to CD69 (Figure 2C). The TGFα shedding assay showed that these two mutants retained normal activity in response to S1P (Figure 2D). We also tested two CD69 double mutations (V48F/V49F and I56F/I59F) for their association with S1PR1. The co-IP results show that the interaction between S1PR1 and either mutant is considerably attenuated, thus directly supporting the role of CD69-TM in the complex assembly (Figure 2C). Moreover, we have purified CD69(V48F/V49F) and CD69(I56F/I59F) individually and mixed with S1PR1 and G_αiβ1γ2 to conduct a GTPase-Glo assay (Figure 2—figure supplement 1). Consistent with the results of our co-IP assays, the activation of G_i proteins in the presence of either variant was decreased (Figure 2E). To further validate the physiological role of the CD69-S1PR1-G_i complex, we tested the two CD69 variants, for their influence on CD69-mediated S1PR1 internalization in WEHI231 B lymphoma cells. In accord with the biochemical data, CD69(V48F/V49F), and CD69(I56F/I59F) were both reduced in their ability to downregulate S1PR1 (Figure 2F).

The structures of the S1PR1 complex with its small molecule modulators (including S1P, FTY720-P, BAF312, and ML056) uncover that the TMs 3, 5, 6, and 7 contribute to accommodate the modulators in the orthosteric site (Hanson et al., 2012; Liu et al., 2022; Xu et al., 2022; Yu et al., 2022; Yuan et al., 2021). In contrast, S1PR1 employs its TM4 to associate with CD69 which functions as a protein agonist for triggering receptor activation. Structural comparison with the inactive state of ML056 bound S1PR1 reveals a unique mechanism of CD69-mediated S1PR1 activation (Figure 3A). The binding of CD69 induces a 4 Å shift at the intracellular end of TM4 causing the residues C167^4.49, I170^4.52, and L174^4.56 in TM4 to face TM3 (Figure 3B). C167^4.49 and I170^4.52 have hydrophobic contacts with the F133^3.41 in TM3, and L174^4.56 pushes the F210^5.47 in TM5 towards the edge of the receptor to form the hydrophobic interactions with W269^6.48 and F273^6.52 in TM6 (Figure 3C, Figure 1—figure supplement 2C). These interactions trigger the notable movement of TM5 and TM6 allowing the opening of intracellular regions to engage the heterotrimeric G_i proteins (Figure 3A and D). Residues A137^3.45, I170^4.52, L174^4.56, F210^5.47, W269^6.48, and F273^6.52 are conserved among S1PR1, S1PR2 and S1PR3, but not F133^3.41 and C167^4.49. Remarkably, further comparison shows that the key residues, which are crucial for the S1P binding and receptor activation, present similar conformations in the structures of S1P-bound S1PR1 and CD69-bound S1PR1, although S1P and CD69 have different structural natures and completely distinct binding sites in the receptor (Figure 3E).

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To date, five S1PRs have been identified. These receptors have different tissue distributions, and they also function via distinct kinds of G proteins (including G_i, G_q, and G_12/13) (Cartier and Hla, 2019). Previous work showed that CD69 specifically binds to S1PR1, and it does not associate with S1PR2, S1PR3 or S1PR5 (Bankovich et al., 2010; Jenne et al., 2009; Shiow et al., 2006). To dissect the binding specificity of CD69, we carried out the co-IP assays to show a very weak interaction between S1PR2 and CD69 (Figure 3—figure supplement 1A). Although the sequence homology among five S1PRs is high, residues L157^4.51 and L168^4.62 in S1PR2-TM4 are not conserved with those in S1PR1 and are determinants for specific recognition of CD69 (Figure 3—figure supplement 1B). We speculated that converting these two residues to those in S1PR1 may prompt the interaction between S1PR2 variant and CD69. Our co-IP result clearly shows that S1PR2(L157^4.51V/L168^4.62M) could interact with CD69 albeit the interactions are weaker than that between S1PR1 and CD69 (Figure 3—figure supplement 1A). This finding further demonstrates the essential role of S1PR1-TM4 in the CD69-mediated S1PR1 signaling.

All the known small molecule S1PR1 agonists or antagonists bind to the orthosteric site in the heptahelical domain (Hanson et al., 2012; Liu et al., 2022; Xu et al., 2022; Yu et al., 2022; Yuan et al., 2021). Interestingly, the CD69 binding site is akin to that of the allosteric agents which attach to receptors (Draper-Joyce et al., 2021; Mao et al., 2020; Yang et al., 2022), although the nature of these agents and CD69 is quite different. The diversity of the allosteric modulator binding sites in GPCRs has been revealed by numerous structures (Figure 3—figure supplement 2). When the orthosteric site is occupied, the positive allosteric modulator attaches to the receptor and then increases agonist affinity and/or efficacy. CD69 binds to the edge of S1PR1, but it acts as a protein agonist to directly activate the receptor in the absence of any agonists in the orthosteric site. Thus, our finding suggests CD69 is different from other S1PR1 agonists in that it functions via a direct binding to the edge of the receptor.

It remains unknown whether the antagonist of S1PR1 bound to the 7-TMs will affect the CD69-mediated regulation of S1PR1. We co-transfected S1PR1-GFP and CD69-mCherry into HEK293 cells in a lipid depleted medium. After 24 hr, the fluorescence images show that substantial receptors (~80%) have been internalized with CD69. However, when we added the Ex26, a potent S1PR1 antagonist (Cahalan et al., 2013), into the cells 6 hr after transfection, the images show that just ~50% receptors have been internalized (Figure 4A and B). This finding indicates that the CD69-mediated S1PR1 activation could be reversed when the 7-TMs pocket is preoccupied by an antagonist.

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It has been known that S1P, FTY720-P and CD69, could promote the internalization of S1PR1. However, the mechanisms of S1P- and FTY720-P-mediated internalization appear to be different. While both S1P and FTY720-P activate G_i-signaling, FTY720-P is considered as a β-arrestin-biased agonist, and FTY720-P-induced S1PR1 internalization is β-arrestin-dependent (Oo et al., 2007; Xu et al., 2022). The pathway of S1P-mediated internalization can be β-arrestin-dependent or independent (Galvani et al., 2015; Reeves et al., 2016). To test the mechanism of how CD69 induces the receptor internalization, we performed a fluorescence imaging assay to check the internalization of S1PR1 in the presence of either G_i inhibitor Pertussis toxin (PTX) or β-arrestin inhibitor Barbadin. The plasmids encoding S1PR1-GFP and CD69-mCherry were co-transfected into HEK293 cells in a lipid depleted medium. After 6 hr, we added PTX or Barbadin. On day 2, we calculated the fraction of internalized S1PR1 in each group by fluorescence imaging. The results show that Barbadin does not interfere with CD69-induced receptor internalization (Figure 4C and D), but PTX could prevent half of the receptors from internalization (Figure 4E and F). Our finding also supports that Barbadin was effective in reducing FTY70-P-induced S1PR1 internalization (Figure 4—figure supplement 1). Thus, CD69 agonism of S1PR1 induces G_i-dependent internalization of the complex.

Our studies provide a model for understanding how the lymphocyte activation marker CD69 controls lymphocyte egress and thus augments adaptive immunity. As an immediate early gene, CD69 is strongly transcriptionally induced in lymphocytes within an hour of exposure to type I IFN, toll-like receptor (TLR) ligands, or antigen receptor engagement (Grigorova et al., 2010; Shiow et al., 2006; Ziegler et al., 1994). Following induction, CD69 protein engages S1PR1 as an agonist, causing S1PR1 internalization and loss of the ability to sense S1P gradients. We speculate that even prior to internalization, CD69 disrupts S1PR1’s egress promoting function by acting as a high concentration agonist and thus making the receptor ‘blind’ to S1P distribution. Consistently, our functional analysis reveals that CD69 could not synergize with S1P to trigger S1PR1 activation (Figure 4—figure supplement 2).

Previous work has shown the critical importance of correctly distributed S1P and thus correctly localized S1PR1 activation for effective lymphocyte egress (Schwab et al., 2005). As well as promoting egress, S1PR1, transmits signals needed for maintaining T cell survival (Mendoza et al., 2017) and CD69 has been implicated in transmitting signals that influence T cell differentiation (Cibrián and Sánchez-Madrid, 2017; Kimura et al., 2017). Whether the CD69-S1PR1 complex contributes to these signals before undergoing degradation merits further study. GRK2 (Arnon et al., 2011; Oo et al., 2007; Watterson et al., 2002) and dynamin (Willinger et al., 2014) participate in S1PR1 internalization in response to S1P. In accord with these factors possibly having a role in CD69-mediated S1PR1 internalization, they have been shown to promote internalization of some receptors independently of β-arrestins (Moo et al., 2021). The selectivity of CD69 for S1PR1 is important for allowing activated CD69⁺ lymphocytes and natural killer cells to employ other S1PRs, such as S1PR2 and S1PR5, to carry out functions without interruption by CD69 (Jenne et al., 2009; Laidlaw et al., 2019; Moriyama et al., 2014). The lack of conservation of key residues that mediate the S1PR1-CD69 interaction in TM4 of S1PR2, S1PR5 and the other S1PRs provides an explanation for this selectivity (Figure 3—figure supplement 1B). In summary, we provide the first example of GPCR activation by interaction in cis with a transmembrane ligand and thereby explain the mechanism of lymphocyte egress shutdown. The structure also offers insights that may enable introduction of transcriptionally inducible GPCR switches into CAR-T cells and other engineered cell types.

The 3D cryo-EM density maps have been deposited in the Electron Microscopy Data Bank under the accession number EMD-29861. Atomic coordinates for the atomic model have been deposited in the Protein Data Bank under the accession number 8G94. All other data needed to evaluate the conclusions in the paper are present in the paper and/or the supplementary materials.

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Author details

1. Hongwen Chen

   Department of Molecular Genetics, The University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   Conceptualization, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1065-9808

2. Yu Qin

   Department of Molecular Genetics, The University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

3. Marissa Chou

   Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, United States

   Contribution

   Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

4. Jason G Cyster

   1. Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, United States
   2. Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Writing – original draft, Writing – review and editing

   For correspondence

   jason.cyster@ucsf.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4735-9745

5. Xiaochun Li

   1. Department of Molecular Genetics, The University of Texas Southwestern Medical Center, Dallas, United States
   2. Department of Biophysics, The University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Writing – original draft, Writing – review and editing

   For correspondence

   xiaochun.li@utsouthwestern.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0177-0803

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