Netrin induces down-regulation of its receptor, Deleted in Colorectal Cancer, through the ubiquitin-proteasome pathway in the embryonic cortical neuron

Abstract

The proper regulation of temporal and spatial expression of the axon guidance cues and their receptors is critical for the normal wiring of nervous system during development. Netrins, a family of secreted guidance cues, are involved in the midline crossing of spinal commissural axons and in the guidance of cortical efferents. Axons normally lose the responsiveness to their attractants when they arrive at their targets, where the attractant is produced. However the molecular mechanism is still unknown. We investigated the molecular mechanism of down-regulation of netrin-1 signaling in the embryonic cortical neurons. Netrin-1 induced the ubiquitination and proteolytic cleavage of Deleted in Colorectal Cancer (DCC), a transmembrane receptor for netrin, in dissociated cortical neurons. A dramatic decrease of DCC level particularly on the cell surface was also observed after netrin-1 stimulation. Specific ubiquitin-proteasome inhibitors prevented the netrin-induced DCC cleavage and decrease of cell surface DCC. We suggest that the ligand-mediated down-regulation of DCC might participate in the loss of netrin-responsiveness in the developing nervous system.

Fig. 1

Effects of netrin on the Deleted in Colorectal Cancer (DCC) levels expressed in embryonic cortical neurons. Embryonic (E15) cortical neurons were cultured, treated with netrin-1 or control medium for the indicated time. The protein levels of DCC were analyzed either with immunofluorescent staining or western blot. (a) Representative immunofluorescent stainings stained with the anti-DCC antibody. Cultured cells were fixed and stained as described under ‘Experimental procedures’. Incubation with mouse IgG (MIgG) did not reveal any fluorescent structures. DCC immunostaining was mainly observed in the membrane part of cell bodies (white arrowhead) and many developing neurites. The obvious reduction of DCC staining was observed 2 h after netrin stimulation (Net-2 h), whereas conditioned medium from control HEK293 cells (HEK-2 h) produced no substantial effect on DCC immunostaining. Vacant arrowheads indicate the location of cell membranes. (b) Western blot showing the reduction of DCC levels 2 h after netrin stimulation. The protein levels of Fyn and actin remained unchanged. IB, immunoblot. (c) A quantitative result showing the time course of the change in DCC level following netrin stimulation. Data represent the means ± SE of three independent experiments.

Fig. 2

Netrin-induced ubiquitination of Deleted in Colorectal Cancer (DCC). Cultured embryonic neurons were treated with netrin for the indicated time, and cells were lysed. (a) A representative blot showing the ubiquitination of DCC. 10 mM N-ethylmaleimide was added to the lysis buffer to inhibit deubiquitination enzymes. DCC was immunoprecipitated (IP) with the anti-DCC antibody (AF-5) and immunostained with anti-ubiquitin antibody (Ubi). The ubiquitinated DCC was transiently shown as smear 20 min after netrin stimulation and disappeared after 2 h of netrin-1 stimulation (upper panel). The same blot was reprobed with an anti-DCC antibody (clone G97-449) (bottom panel). (b) A representative blot showing the laddering pattern of ubiquitinated DCC. A long exposure of the membrane showed a ladder of ubiquitinated DCC (multiple arrows). (c) A representative western blot showing the degradation of ubiquitinated DCC by proteasomes. The ubiquitinated DCC still remains after 2 h of netrin stimulation in the presence of MG132 (upper panel). The amounts of DCC in the immunoprecipitates were not decreased following 2 h of netrin stimulation in the presence of 10 μM MG132 (bottom panel).

Fig. 3

Netrin-induced limited proteolytic degradation of Deleted in Colorectal Cancer (DCC). Lysates from cortical neurons were immunoprecipitated with the anti-DCC antibody (AF-5) and immunostained with the same antibody. (a) A representative blot showing the proteolytic cleavage of DCC by netrin. Limited proteolytic fragments of DCC with various sizes appeared after netrin stimulation (upper panel). (b) A representative blot showing the effects of the proteasome inhibitors on DCC proteolysis. The pretreatment of MG132 and N-acetyl-Leu-Leu-NorLeu-Al (LnLL), but not leupeptin (Leup), almost completely inhibited netrin-induced DCC cleavage. (c) Representative immunofluorescent stainings showing the effect of proteasome inhibitors on the netrin-induced changes of DCC immunoreactivity. The netrin-induced decrease of DCC immunostaining was prevented by the pretreatment of MG132, but not by leupeptin.

Fig. 4

Netrin-induced decrease of cell surface Deleted in Colorectal Cancer (DCC). The cultured cells either untreated or treated with netrin-1 were cooled and labeled with the anti-DCC antibody (AF-5) on ice, then lysates were made after extensive washing. Antibody-labeled DCC was precipitated with protein A/G agarose beads and analyzed by immunoblot with the same antibody. (a) A representative blot showing the dramatic decrease of cell surface DCC by netrin in 30 min. (b) A quantitative result showing the time course of the change in DCC level following netrin stimulation. Data represent the means ± SE of three independent experiments. The decrease of DCC level by netrin stimulation in the cell surface was more profound than that in total DCC. (c) Effects of the proteasome inhibitors MG132 and N-acetyl-Leu-Leu-NorLeu-Al (LnLL) on the netrin-induced decrease of cell surface DCC. The netrin-induced decrease of cell surface DCC was in part blocked by the pretreatment of MG132 or LnLL.