To further explore the interaction of LRRTM proteins with glypica

To further explore the interaction of LRRTM proteins with glypicans Anticancer Compound Library and neurexins, we performed Fc pulldown assays in 293T cells. LRRTM4-Fc pulled down HA-GPC4 from cell lysate, whereas Fc, Nrx1β(-S4)-Fc, or Nrx1β(+S4)-Fc did not interact with HA-GPC4 (Figure S1F). In reciprocal experiments, GPC4-Fc pulled down myc-LRRTM4 from cell

lysate but did not bind to FLRT3-myc (Figure S1G), confirming that GPC4 and LRRTM4 can bind each other. Under these conditions, LRRTM2 displayed a weak interaction with GPC4 (Figures S1F and S1G), which we did not detect in cell surface binding assays (Figures 1E, 1F, 1I, and 1J). This suggests that LRRTM2 may have a low affinity for glypican, which would agree with the minor presence of glypican in the LRRTM2-Fc pulldown (two spectral counts; Figure 1D). Together, our results indicate that LRRTM4 has two binding partners: neurexin and glypican. Whereas neurexins interact with both LRRTM2 and LRRTM4, glypican is a preferential binding partner

of LRRTM4. To determine whether LRRTM4 and GPC4 can interact directly, we performed cell-free binding assays in which we mixed recombinant His-tagged LRRTM4 ectodomain with purified Fc proteins. Fc proteins were precipitated with protein A/G agarose beads and bound proteins were analyzed by western blot. His-LRRTM4 coprecipitated with GPC4-Fc and Nrx1β(-S4)-Fc, but not with learn more Fc or LPHN3-Fc (Figure 2A), confirming a direct interaction between the LRRTM4 ectodomain and GPC4. We next analyzed whether LRRTM4 can simultaneously bind to its two binding partners, neurexin and glypican. We purified recombinant HA-GPC4 from HEK293T-conditioned media by affinity chromatography

using HA antibodies (Figure S4A) and mixed HA-GPC4 with Nrx1β(-S4)-Fc and His-LRRTM4 or His-FLRT3. We then precipitated neurexin with protein A/G agarose to test whether pulldown of LRRTM4 bound to neurexin would also bring down glypican. Nrx1β(-S4)-Fc precipitated His-LRRTM4, but not His-FLRT3. HA-GPC4 did not come down with neurexin-bound second LRRTM4 (Figure 2B). In the reciprocal experiment, HA-GPC4 was precipitated with HA antibody-coupled beads to test whether pulldown of LRRTM4 bound to glypican can bring down neurexin. We found that HA-GPC4 precipitated His-LRRTM4, but not His-FLRT3. Nrx1β(-S4)-Fc did not coprecipitate with glypican-bound LRRTM4 (Figure 2C). In separate experiments, we further established that Nrx1β(-S4)-Fc or Nrx1β(+S4)-Fc does not bind to glypican (Figures S1F, S2A, and S2B). These data suggest that LRRTM4 forms separate complexes with neurexin and glypican and argue against the existence of a tripartite complex. We next investigated the aspects of glypican processing that are important for LRRTM4 binding. Glypicans consist of a core protein with a cysteine-rich globular domain and a stalk-like domain containing three HS GAG attachment sites.

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