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The cytoplasmic domain of the integrin lymphocyte function-associated antigen 1 beta subunit: sites required for
M L Hibbs1, S Jakes, S A Stacker
1Center for Blood Research, Harvard Medical School, Boston, Massachusetts 02115.
Insights
Specific regions of lymphocyte function-associated antigen 1 (LFA-1) cytoplasmic domain are crucial for binding to intercellular adhesion molecule 1 (ICAM-1). Serine phosphorylation is not solely responsible for this binding, suggesting complex regulation of integrin adhesiveness.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Leukocyte integrins, such as lymphocyte function-associated antigen 1 (LFA-1), mediate cell adhesion.
- LFA-1 binding to intercellular adhesion molecule 1 (ICAM-1) is critical for immune cell interactions.
- The cytoplasmic domain of LFA-1 plays a regulatory role in integrin activation.
Purpose of the Study:
- To identify the specific regions within the LFA-1 cytoplasmic domain required for ICAM-1 binding.
- To investigate the role of serine phosphorylation at Ser 756 in LFA-1/ICAM-1 interactions.
- To determine if serine phosphorylation is essential for phorbol ester-stimulated LFA-1 binding.
Main Methods:
- Site-directed mutagenesis of the LFA-1 cytoplasmic domain.
- Phosphoamino acid analysis to identify phosphorylation sites.
- Assays to measure LFA-1 binding to ICAM-1 in response to phorbol ester stimulation.
Main Results:
- The NH2-terminal 28 amino acids of the LFA-1 cytoplasmic domain are dispensable for ICAM-1 binding.
- A 5-amino acid segment (including Thr 758-760 and Phe 766) in the COOH-terminal third is essential for binding.
- Ser 756 is a major phosphorylation site, but its phosphorylation can be dissociated from phorbol ester-stimulated binding, indicating it's not the sole determinant.
- The identified regulatory sites are conserved across multiple integrin beta subunits (β1, β3, β7).
Conclusions:
- Specific residues within the LFA-1 cytoplasmic domain are critical for regulating ICAM-1 binding.
- Serine phosphorylation at Ser 756 is not the sole driver of LFA-1 activation and binding.
- These findings reveal conserved mechanisms for integrin adhesiveness regulation, with potential implications for broader integrin function.
Abstract:
We have defined the regions of the cytoplasmic domain of the leukocyte integrin lymphocyte function-associated antigen 1 (LFA-1) that are required for active binding of its extracellular domain to intercellular adhesion molecule 1 (ICAM-1). The NH2-terminal 28 amino acids in the cytoplasmic domain are dispensable, but a segment of 5 amino acids including three contiguous threonines (758-760) and Phe 766 in the COOH-terminal third of the cytoplasmic domain are required for binding to ICAM-1. Mutation and phosphoamino acid analysis show that Ser 756 is the major residue phosphorylated in response to phorbol ester. Furthermore, multiple mutations demonstrate that serine phosphorylation can be dissociated from phorbol ester-stimulated binding of LFA-1 to ICAM-1. The sites we have defined are previously unremarked, are well conserved in the beta 1, beta 3, and beta 7 integrin subunits, and may be of broad importance in regulating adhesiveness of integrins.