Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel development by activating 3 receptor tyrosine kinases (RTKs), VEGFR-1, -2, and -3, and by binding to coreceptors such as neuropilin-1 (NRP-1). We investigated how different VEGF-A isoforms, in particular VEGF-A₁₆₅a and VEGF-A₁₆₅b, control the balance between VEGFR-2 recycling, degradation, and signaling. Stimulation of cells with the NRP-1–binding VEGF-A₁₆₅a led to sequential NRP-1–mediated VEGFR-2 recycling through Rab5, Rab4, and Rab11 vesicles. Recycling was accompanied by dephosphorylation of VEGFR-2 between Rab4 and Rab11 vesicles and quantitatively and qualitatively altered signal output. In cells stimulated with VEGF-A₁₆₅b, an isoform unable to bind NRP-1, VEGFR-2 bypassed Rab11 vesicles and was routed to the degradative pathway specified by Rab7 vesicles. Deletion of the GIPC (synectin) binding motif of NRP-1 prevented transition of VEGFR-2 through Rab11 vesicles and attenuated signaling. Coreceptor engagement was specific for VEGFR-2 because EGFR recycled through Rab11 vesicles in the absence of known coreceptors. Our data establish a distinct role of NRP-1 in VEGFR-2 signaling and reveal a general mechanism for the function of coreceptors in modulating RTK signal output.

Mutations in the myotubularin (MTM)-related protein 2 ( MTMR2 ) gene are responsible for the severe autosomal recessive neuropathy Charcot–Marie–Tooth disease type 4B1. MTMR2 belongs to the MTM family of dual-specific phosphatases that use phosphatidylinositol (PI) 3,5-bisphosphate [PI(3,5)P ₂ ] and PI 3-phosphate [PI(3)P] as their substrate. Because these substrates are localized in the membrane bilayer, membrane targeting of Mtmr2 is an important regulatory mechanism. In hypoosmotically stressed COS cells with increased levels of PI(3,5)P ₂ , Mtmr2 is bound to the membrane of vacuoles formed under these conditions. Using several mutant forms of Mtmr2, we identified two domains that are necessary for membrane association: ( i ) A pleckstrin homology-GRAM domain; and ( ii ) a coiled-coil module. Protein-lipid overlay assays show that the pleckstrin homology-GRAM domain binds to PI(3,5)P ₂ and PI(5)P, a substrate and a product of the Mtmr2 enzyme, respectively. We also demonstrate that Mtmr2 forms a dimer and that the C-terminal coiled-coil is responsible for homodimerization, in addition to membrane association. Our data indicate that phosphoinositide–protein interactions, as well as protein–protein interactions, are necessary for the correct regulation of MTMR2.