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.