Nogo-A is a member of a family of potent neurite growth inhibitors. One of the active regions is the C-terminal Nogo-66 domain, which is shared by two other family members and which binds the cell surface receptor NgR. A central region called NogoΔ20 is specific to Nogo-A and exhibits inhibitor activity that is independent of NgR. Joset et al. found that when applied to a neuronal cell line (PC12 cells), cultured hippocampal cells, or cultured dorsal root ganglion (DRG) neurons, NogoΔ20 was internalized through a macropinocytotic mechanism that required the activity of Pincher, a member of the Eps15 homology domain–containing proteins (EHDs). Inhibition of clathrin-mediated endocytosis or caveolin-mediated endocytosis did not block NogoΔ20 internalization in PC12 cells. Overexpression of dominant-negative Pincher reduced growth cone collapse in hippocampal neurons treated with NogoΔ20, and cerebellar granule neurons overexpressing the dominant-negative Pincher exhibited longer neurites in the presence of NogoΔ20 than did untransfected cells exposed to NogoΔ20. NogoΔ20 is known to stimulate RhoA activity, and overexpression of dominant-negative Pincher reduced the NogoΔ20-stimulated activation of Rho family members in PC12 cells. NogoΔ20 was delivered to cell bodies of cultured DRG neurons through a process requiring microtubule-mediated retrograde transport, and this retrograde transport was blocked by overexpression of dominant-negative Pincher. Rho activity was increased in the DRG neurites shortly after NogoΔ20 application (30 minutes), and at later times (6 hours) increased Rho activity was evident in the cell bodies. In the neurites, active Rho colocalized with NogoΔ20-positive vesicles, leading the authors to propose that active Rho was delivered to cell bodies as part of NogoΔ20 signaling endosomes or NogoΔ20 signalosomes. In support of this hypothesis, endosomal fractions from PC12 cells exposed to NogoΔ20 exhibited higher amounts of active Rho than did fractions from untreated cells.