FIG. 7.

Plexin-B1 activation promotes the assembly of multimeric signaling complexes. (A) Plexin-B1 is tyrosine phosphorylated upon activation. Lysates of endothelial cells stably expressing myc-tagged Trk-A/Plexin-B1 chimeric receptors treated with 100 ng/ml NGF for the indicated periods of time were immunoprecipitated for the receptor (IP: myc) and immunoblotted for the presence of phosphorylated tyrosine residues (WB: P-Y). A phosphotyrosine band of the appropriate size begins to appear in the immunoprecipitated fraction at about 1 min (upper panel). Total receptor (WB: myc) was used as a loading control (lower panel). (B) 293T cells were cotransfected with myc-tagged Trk-A/Plexin-B1 and either DN-PYK2 (DN-PYK2), control DNA (C), or control DNA preceded by incubation with the Src inhibitor PP2 (PP2), followed by treatment with 100 ng/ml NGF. An immunoblot for myc was performed on phosphotyrosine-immunoprecipitated fractions (IP: P-Y and WB: myc). An increase in receptor phosphorylation is seen in response to NGF in control cells (lane 2) and cells pretreated with PP2 (lane 4) but not in cells expressing DN-PYK2 (lane 6). (C) PYK2, p85, and Src bind Plexin-B1 upon receptor activation. Lysates of endothelial cells stably expressing myc-tagged Trk-A/Plexin-B1 chimeric receptors treated with 100 ng/ml NGF for the indicated periods of time were immunoprecipitated for the receptor (IP: myc) and immunoblotted for the presence of PYK2, p85, and Src. Each of these proteins is seen associating with the immunoprecipitated receptor complex at 1 min following treatment. Total PYK2, p85, Src, and myc immunoblots were used as loading controls. As a negative control, a hemagglutinin immunoprecipitation was performed (IP: HA; right panels) which showed no proteins after 0 or 5 min treatment with NGF when probed for PYK2, p85, Src, and myc. Total PYK2, p85, Src, and myc were used as loading controls (WB lanes; lower panels).