Direct identification of ligand-receptor interactions on living cells and tissues

Abstract

Many cellular responses are triggered by proteins, drugs or pathogens binding to cell-surface receptors, but it can be challenging to identify which receptors are bound by a given ligand. Here we describe TRICEPS, a chemoproteomic reagent with three moieties--one that binds ligands containing an amino group, a second that binds glycosylated receptors on living cells and a biotin tag for purifying the receptor peptides for identification by quantitative mass spectrometry. We validated this ligand-based, receptor-capture (LRC) technology using insulin, transferrin, apelin, epidermal growth factor, the therapeutic antibody trastuzumab and two DARPins targeting ErbB2. In some cases, we could also determine the approximate ligand-binding sites on the receptors. Using TRICEPS to label intact mature vaccinia viruses, we identified the cell surface proteins AXL, M6PR, DAG1, CSPG4 and CDH13 as binding factors on human cells. This technology enables the identification of receptors for many types of ligands under near-physiological conditions and without the need for genetic manipulations.

Lassa Virus Cell Entry Reveals New Aspects of Virus-Host Cell Interaction

“…Over the past years, TAM kinases have been implicated in viral entry by "apoptotic mimicry," which involves recognition of PS displayed in the viral lipid envelope by cellular PS receptors and is used by a broad range of enveloped viruses (37)(38)(39)(40)(41)(42). The coexpression of DG with TAM receptors in tissues infected by LASV suggests complex receptor use.…”

Laminin targeting of a peripheral nerve-highlighting peptide enables degenerated nerve visualization

“…No specific protein was selectively captured as determined by MS analysis, leading us to speculate that NP41 bound with low affinity to homogenates. Thus, to capture NP41-receptor interaction in situ, we developed a method for proximity labeling ligand-bound proteins using photooxidation and targeted BH labeling and also tested previously described glycoprotein capture using TRICEPS-based chemistry (8,9).…”

“…For target identification, NP41-FAM-HB or a D-enantiomer control was injected into mice, and nerves were excised before periodate application, then processed and analyzed as previously described (8). Quantification of individual peptides containing formerly glycosylated asparagine residues (N[115]-X-S/T motif) revealed laminin-γ1 and -α4 as enriched in the NP41-treated sample over the control (Fig.…”

“…A variety of methods to capture native ligand-receptor interactions have used chemical or photo-cross-linking followed by mass spectrometry (MS), some requiring that ligands retain binding activity after potentially disruptive chemical treatment (6)(7)(8)(9). In most cross-linking techniques, the ligand must reach an appropriately reactive site on the target for cross-linking to occur, while being conjugated to a potentially bulky purification tag, which can weaken specific binding.…”

“…Alternatively, ligand-directed receptor capture (LRC) using TRICEPS technology has been shown to enable carbohydratedirected tagging of receptors and subsequent identification by MS (8,9). Mild sodium periodate (NaIO 4 ) treatment oxidizes cisglycol groups in carbohydrates to aldehydes, and newly generated aldehydes cross-link to a BH-containing ligand.…”