A generic cell surface ligand system for studying cell-cell recognition.

Dose-response experiments are a mainstay of receptor biology studies and can reveal valuable insights into receptor function. Such studies of receptors that bind cell surface ligands are currently limited by the difficulty in manipulating the surface density of ligands at a cell-cell interface. Here, we describe a generic cell surface ligand system that allows precise manipulation of cell surface ligand densities over several orders of magnitude. These densities are robustly quantifiable, a major advance over previous studies. We validate the system for a range of immunoreceptors, including the T-cell receptor (TCR), and show that this generic ligand stimulates via the TCR at a similar surface density as its native ligand. We also extend our work to the activation of chimeric antigen receptors. This novel system allows the effect of varying the surface density, valency, dimensions, and affinity of the ligand to be investigated. It can be readily broadened to other receptor-cell surface ligand interactions and will facilitate investigation into the activation of, and signal integration between, cell surface receptors.

TCR-pMHC kinetics under force in a cell-free system show no intrinsic catch bond, but a minimal encounter duration before binding.

The T cell receptor (TCR)-peptide-MHC (pMHC) interaction is the only antigen-specific interaction during T lymphocyte activation. Recent work suggests that formation of catch bonds is characteristic of activating TCR-pMHC interactions. However, whether this binding behavior is an intrinsic feature of the molecular bond, or a consequence of more complex multimolecular or cellular responses, remains unclear. We used a laminar flow chamber to measure, first, 2D TCR-pMHC dissociation kinetics of peptides of various activating potency in a cell-free system in the force range (6 to 15 pN) previously associated with catch-slip transitions and, second, 2D TCR-pMHC association kinetics, for which the method is well suited. We did not observe catch bonds in dissociation, and the off-rate measured in the 6- to 15-pN range correlated well with activation potency, suggesting that formation of catch bonds is not an intrinsic feature of the TCR-pMHC interaction. The association kinetics were better explained by a model with a minimal encounter duration rather than a standard on-rate constant, suggesting that membrane fluidity and dynamics may strongly influence bond formation.

Mutant prolactin receptor and familial hyperprolactinemia.

Hyperprolactinemia that is not associated with gestation or the puerperium is usually due to tumors in the anterior pituitary gland and occurs occasionally in hereditary multiple endocrine neoplasia syndromes. Here, we report data from three sisters with hyperprolactinemia, two of whom presented with oligomenorrhea and one with infertility. These symptoms were not associated with pituitary tumors or multiple endocrine neoplasia but were due to a heterozygous mutation in the prolactin receptor gene, PRLR, resulting in an amino acid change from histidine to arginine at codon 188 (His188Arg). This substitution disrupted the high-affinity ligand-binding interface of the prolactin receptor, resulting in a loss of downstream signaling by Janus kinase 2 (JAK2) and signal transducer and activator of transcription 5 (STAT5). Thus, the familial hyperprolactinemia appears to be due to a germline, loss-of-function mutation in PRLR, resulting in prolactin insensitivity.

The large ectodomains of CD45 and CD148 regulate their segregation from and inhibition of ligated T-cell receptor.

T-cell receptor (TCR) triggering results in a cascade of intracellular tyrosine phosphorylation events that ultimately leads to T-cell activation. It is dependent on changes in the relative activities of membrane-associated tyrosine kinases and phosphatases near the engaged TCR. CD45 and CD148 are transmembrane tyrosine phosphatases with large ectodomains that have activatory and inhibitory effects on TCR triggering. This study investigates whether and how the ectodomains of CD45 and CD148 modulate their inhibitory effect on TCR signaling. Expression in T cells of forms of these phosphatases with truncated ectodomains inhibited TCR triggering. In contrast, when these phosphatases were expressed with large ectodomains, they had no inhibitory effect. Imaging studies revealed that truncation of the ectodomains enhanced colocalization of these phosphatases with ligated TCR at the immunological synapse. Our results suggest that the large ectodomains of CD45 and CD148 modulate their inhibitory effect by enabling their passive, size-based segregation from ligated TCR, supporting the kinetic-segregation model of TCR triggering.

Biophysical assay for tethered signaling reactions reveals tether-controlled activity for the phosphatase SHP-1.

Tethered enzymatic reactions are ubiquitous in signaling networks but are poorly understood. A previously unreported mathematical analysis is established for tethered signaling reactions in surface plasmon resonance (SPR). Applying the method to the phosphatase SHP-1 interacting with a phosphorylated tether corresponding to an immune receptor cytoplasmic tail provides five biophysical/biochemical constants from a single SPR experiment: two binding rates, two catalytic rates, and a reach parameter. Tether binding increases the activity of SHP-1 by 900-fold through a binding-induced allosteric activation (20-fold) and a more significant increase in local substrate concentration (45-fold). The reach parameter indicates that this local substrate concentration is exquisitely sensitive to receptor clustering. We further show that truncation of the tether leads not only to a lower reach but also to lower binding and catalysis. This work establishes a new framework for studying tethered signaling processes and highlights the tether as a control parameter in clustered receptor signaling.