The CD28 Transmembrane Domain Contains an Essential Dimerization Motif

Area of Science:

• Immunology
• Molecular Biology
• Cell Biology

Background:

• CD28 is vital for T cell responses and regulatory T cell function.
• CD28 functions as a disulfide-linked homodimer, but the mechanisms of ligand binding and signaling are unclear.
• Inside-out signaling may alter CD28 conformation for bivalent binding.

Purpose of the Study:

• To investigate the role of the CD28 transmembrane domain in dimer formation and signaling.
• To identify conserved motifs within the CD28 transmembrane domain involved in dimerization.

Main Methods:

• Examined the CD28 transmembrane domain for dimerization motifs.
• Utilized bacterial and mammalian expression systems to assess dimerization.
• Employed Förster Resonance Energy Transfer (FRET) by flow cytometry.
• Introduced mutations using CRISPR gene editing.

Main Results:

• Identified a conserved YxxxxT motif in the CD28 transmembrane domain, similar to CTLA-4 and CD3ζ.
• Demonstrated that the CD28 transmembrane domain drives dimerization in bacterial and mammalian cells.
• Showed that mutations in the YxxxxT motif impair dimerization.
• Found that mutating this motif leads to reduced CD28 cell surface expression.

Conclusions:

• The YxxxxT motif within the CD28 transmembrane domain is critical for CD28 dimer assembly and/or stable surface expression.
• This motif likely plays a key role in regulating CD28 function in immune responses.