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Related Experiment Video

Updated: Aug 9, 2025

A Pipeline to Investigate the Structures and Signaling Pathways of Sphingosine 1-Phosphate Receptors
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Transmembrane protein CD69 acts as an S1PR1 agonist.

Hongwen Chen1, Yu Qin1, Marissa Chou2

  • 1Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

Biorxiv : the Preprint Server for Biology
|February 24, 2023
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Summary

Cluster of Differentiation 69 (CD69) acts as a protein agonist for Sphingosine-1-phosphate receptor 1 (S1PR1), promoting its internalization and inhibiting lymphocyte egress for immune regulation.

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Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries
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Area of Science:

  • Structural Biology
  • Immunology
  • Biochemistry

Background:

  • Sphingosine-1-phosphate receptor 1 (S1PR1) activation by S1P is crucial for lymphocyte egress, immune surveillance, and T cell activity.
  • Cluster of Differentiation 69 (CD69) negatively regulates lymphocyte egress by interacting with S1PR1, but the mechanism was unclear.
  • It was unknown if a transmembrane protein could act as a G protein-coupled receptor (GPCR) agonist.

Approach:

  • Determined the cryo-electron microscopy (cryo-EM) structure of CD69-bound S1PR1 complexed with the heterotrimeric Gi protein.
  • Analyzed the interaction interface between CD69 and S1PR1, including specific transmembrane helix contacts.
  • Performed functional analyses using site-directed mutagenesis to assess the impact of interface mutations on receptor interactions and internalization.

Key Points:

  • The CD69 homodimer's transmembrane helix directly contacts S1PR1, allosterically activating it.
  • This interaction induces S1PR1 transmembrane helix movements, leading to Gi protein engagement.
  • Mutations at the CD69-S1PR1 interface disrupt binding and reduce receptor internalization.

Conclusions:

  • CD69 functions as a *cis*-acting protein agonist of S1PR1, a novel mechanism for GPCR activation.
  • CD69 binding promotes Gi-dependent S1PR1 internalization and impairs S1P gradient sensing.
  • This interaction ultimately inhibits lymphocyte egress, impacting immune cell trafficking and function.