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Proc Natl Acad Sci U S A. 2017 Oct 31;114(44):E9338-E9345. doi: 10.1073/pnas.1710358114. Epub 2017 Oct 17.

In vitro reconstitution of T cell receptor-mediated segregation of the CD45 phosphatase.

Carbone CB1,2,3, Kern N1,2, Fernandes RA4,5,6, Hui E1,2,3, Su X1,2,3, Garcia KC4,5,6, Vale RD7,2,3.

Author information

1
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158.
2
Howard Hughes Medical Institute, University of California, San Francisco, CA 94158.
3
Howard Hughes Medical Institute Summer Institute, Marine Biological Laboratory, Woods Hole, MA 02543.
4
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305.
5
Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305.
6
Howard Hughes Medical Institute, Stanford University Medical School, Stanford, CA 94305.
7
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158; ron.vale@ucsf.edu.

Abstract

T cell signaling initiates upon the binding of peptide-loaded MHC (pMHC) on an antigen-presenting cell to the T cell receptor (TCR) on a T cell. TCR phosphorylation in response to pMHC binding is accompanied by segregation of the transmembrane phosphatase CD45 away from TCR-pMHC complexes. The kinetic segregation hypothesis proposes that CD45 exclusion shifts the local kinase-phosphatase balance to favor TCR phosphorylation. Spatial partitioning may arise from the size difference between the large CD45 extracellular domain and the smaller TCR-pMHC complex, although parsing potential contributions of extracellular protein size, actin activity, and lipid domains is difficult in living cells. Here, we reconstitute segregation of CD45 from bound receptor-ligand pairs using purified proteins on model membranes. Using a model receptor-ligand pair (FRB-FKBP), we first test physical and computational predictions for protein organization at membrane interfaces. We then show that the TCR-pMHC interaction causes partial exclusion of CD45. Comparing two developmentally regulated isoforms of CD45, the larger RABC variant is excluded more rapidly and efficiently (∼50%) than the smaller R0 isoform (∼20%), suggesting that CD45 isotypes could regulate signaling thresholds in different T cell subtypes. Similar to the sensitivity of T cell signaling, TCR-pMHC interactions with Kds of ≤15 µM were needed to exclude CD45. We further show that the coreceptor PD-1 with its ligand PD-L1, immunotherapy targets that inhibit T cell signaling, also exclude CD45. These results demonstrate that the binding energies of physiological receptor-ligand pairs on the T cell are sufficient to create spatial organization at membrane-membrane interfaces.

KEYWORDS:

CD45; PD-1; TCR; kinetic segregation; signaling

PMID:
29042512
PMCID:
PMC5676914
DOI:
10.1073/pnas.1710358114
[Indexed for MEDLINE]
Free PMC Article

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