%0 Journal Article %A Catherine B. Carbone %A Nadja kerm %A Ricardo A. Fernandes %A Enfu Hui %A Xiaolei Su %A K. Christopher Garcia %A Ronald D. Vale %T In Vitro Reconstitution of T Cell Receptor–Mediated Segregation of the CD45 Phosphatase %D 2017 %R 10.1101/097600 %J bioRxiv %P 097600 %X T cell signaling initiates upon binding of peptide-major histocompatibility complex (pMHC) on an antigen-presenting cell (APC) 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 co-receptor 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.SIGNIFICANCE STATEMENT The interface between a T cell and an antigen-presenting cell (APC) results in the formation of biochemically distinct plasma membrane domains that initiate signaling cascades. Here, using biochemical reconstitution and microscopy, we show that the binding energies of the TCRpMHC and PD-1-PD-L1 complexes are sufficient to create spatial organization at a model membrane-membrane interface. We show that spatial organization depends upon receptor-ligand binding affinity and the relative sizes of the extracellular domains. These biophysical parameters may be used to fine-tune signaling cascades in T cells. %U https://www.biorxiv.org/content/biorxiv/early/2017/06/07/097600.full.pdf