The ErbB4 receptor isoforms JM-a and JM-b differ within their extracellular juxtamembrane (eJM) domains. Here we used ErbB4 isoforms as a model to address whether structural variation in the eJM domain of receptor tyrosine kinases (RTK) affects downstream signaling. Analysis of ErbB4 interactome and super-resolution cell imaging indicated association of the JM-a- and JM-b-like receptors with specific signaling complexes at different cell surface compartments. A JM-a-specific sequence motif was discovered, and its presence in the eJM domains of several other human RTKs predicted selective STAT activation indicating a conserved RTK signaling mechanism. The RTKs with the JM-a-like eJM motif activated STAT5a, and those that were JM-b-like (lacking the JM-a-like motif) activated STAT5b and STAT3. TYK2 was found to be necessary for JM-b-stimulated STAT5b activation. The activation of STAT5a by the JM-a-like receptors, in turn, involved specific interaction with oligosaccharides N-linked to cell surface glycoproteins such as β1 integrin. These findings provide evidence for a mechanism linking a ubiquitous extracellular motif in RTKs with selective intracellular STAT signaling.

To control whether the interaction between ErbB4 and β1 integrin interaction was selective for the JM-a isoform, anti-ErbB4 immunoprecipitates from MDA-MB-468 cells overexpressing either ErbB4 JM-a or JM-b were analyzed by a β1-integrin-targeted mass spectrometry. The targeted PRM samples were analyzed on an Easy-nLC 1200 liquid chromatography system coupled to an Orbitrap Lumos Fusion instrument (Thermo Fisher Scientific) equipped with a nanoelectrospray source. Peptides were loaded on an in-house packed 100 μm × 2 cm precolumn packed with ReproSil-Pur 5 μm 200 Å C18-AQ beads using 0.1% formic acid in water (buffer A) and separated by reverse phase chromatography on a 75 μm × 15 cm analytical column packed with ReproSil-Pur 5 μm 200 Å C18-AQ beads. All separations were performed using a 40 minute gradient at a flow rate of 300 nl/minute. The gradient ranged from 6% buffer B (80% acetonitrile in 0.1% formic acid) to 36% buffer B in 30 minutes. Buffer B concentration was ramped up to 100% in 5 minutes, and 100% buffer B was run 5 minutes for washout. A scheduled PRM method was used to simultaneously target all selected β1 integrin peptides. The β1 integrin peptides were selected using an online PRM method designer Picky with default parameters, except miscleaved peptides were allowed, and the maximal number of features monitored in parallel was set to 31. The PRM method consisted of a targeted MS/MS spectra acquisition with a resolution of 30,000, automatic gain control target of 5e4, isolation window of m/z 1.6, maximum injection time set at 54 ms, and normalized collision energy = 27.

MDA-MB-468 cells overexpressing either ErbB4 JM-a or JM-b were starved overnight and crosslinked with 1 mM DTBP. Cells were lysed with buffer containing 0.1% Triton X-100, 1 mM EDTA, 5 mM NaF, 10 mM Tris-HCl, pH 7.4, and a protease inhibitor cocktail (ThermoFisher Scientific) and anti-ErbB4 immonoprecipitation was performed. Proteins were eluted with 6 M guanidine hydrochloride, 5 mM tris(2-carboxyethyl)phosphine, 10 mM chloroacetamide and 100 mM Tris, pH 8.5 in 95 °C for 10 minutes. Guanidine hydrochloride was diluted to 2 M and eluted samples were digested with 1:50 (w/v) trypsin (Thermo Scientific) overnight at 37 °C. Following digestion, the reactions were quenched with 10% trifluoroacetic acid at a final concentration of approximately 0.5% (pH ~2), desalted on tC18 SepPak 96-well plate (Waters), and dried by vacuum centrifugation.