Ubiquitination and Endocytosis of Cell Adhesion Molecule DM-GRASP Regulate Its Cell Surface Presence and Affect Its Role for Axon Navigation

Abstract

DM-GRASP, cell adhesion molecule of the immunoglobulin superfamily, has been shown to promote growth and navigation of axons. We here demonstrate that clustering of DM-GRASP in the plasma membrane induces its rapid internalization via dynamin-and clathrin-dependent endocytosis, which is controlled by phosphatidylinositol 3-kinase and mitogen-activated protein kinase ERK. The clustering of DM-GRASP activates ERK; the intensity and duration of ERK activation by DM-GRASP do not depend on rapid clathrin-mediated internalization of DM-GRASP. Moreover, the preference of retinal ganglion cell axons for DM-GRASP-coated micro-lanes requires clathrin-mediated endocytosis for the appropriate axonal turning reactions at substrate borders. Because the intracellular domain of DM-GRASP does not contain motifs for direct interactions with the endocytosis machinery, we performed a yeast two-hybrid screen to identify intracellular proteins mediating the uptake of DM-GRASP and isolated ubiquitin. Immunoprecipitation of DM-GRASP coexpressed with ubiquitin revealed that one or two ubiquitin(s) are attached to the intracellular domain of cell surface-resident DM-GRASP. Furthermore, elevated ubiquitination levels result in a decrease of cell surface-resident DM-GRASP as well as in the amount of total DM-GRASP. The endocytosis rate is not affected, but the delivery to multivesicular bodies is increased, indicating that DM-GRASP ubiquitination enhances its sorting into the degradation pathway. Together, our data show that ubiquitination and endocytosis of DM-GRASP in concert regulate its cell surface concentration, which is crucial for its function in axon navigation.

Translation of the cell adhesion molecule ALCAM in axonal growth cones – regulation and functional importance

“…The amount of ALCAM in the growth cone plasma membrane depends not only on synthesis but also on the degradation of ALCAM, and is crucial for proper growth cone functioning (Thelen et al, 2008); ALCAM is mainly present in the axonal plasma membrane and is also enriched at the growth cone in vitro (supplementary material Fig. S1).…”

“…In addition, targeting, sorting and filtering at the axon initial segment are impediments for a (swift) appearance of membrane proteins in the axon (Song et al, 2009). The endocytosis of ALCAM in the growth cone results in its degradation and not in recycling (Thelen et al, 2008), also indicating that there is a need for a local synthesis of this CAM. In vivo, ALCAM endocytosis could be triggered by contact of RGC growth cones with ALCAM-presenting RGC axons.…”

“…Thus the onset, level and growth cone subregion of ALCAM synthesis might be regulated by environmental cues that stimulate these two kinases. It is noteworthy in this context that ERK inhibition reduces not only ALCAM's the growth cone translation of ALCAM but also its endocytosis and degradation (Thelen et al, 2008); therefore, the reduced axonal elongation and navigation, observed upon ERK inhibition, is rather due to a reduced re-sensitization of ALCAM (see below) and not to changed ALCAM levels in the cell membrane. (6) The rapid reappearance of ALCAM in the growth cone plasma membrane (10 minutes after triggering of endocytosis) is prevented by inhibition of its translation.…”

“…When ALCAM is functionally neutralized by antibody inhibition or knocked out, RGC axon fasciculation and entry into the optic nerve is reduced (Avci et al, 2004;Weiner et al, 2004). Inhibition of ALCAM interactions (Avci et al, 2004) or of its internalization (Thelen et al, 2008) abolishes the axonal preference for ALCAMcontaining lanes, demonstrating the impact of physiological levels of ALCAM in the plasma membrane. Highly defined nanospaced ALCAM, offered as cell culture substrate, revealed the importance of a critical density of this CAM for axon growth (Thelen et al, 2007;Jaehrling et al, 2009).…”

“…with itself (Tanaka et al, 1991;van Kempen et al, 2001), and heterophilically with the IgSF-CAM CAML1 (also known as NgCAM) (DeBernardo and Chang, 1996;Buhusi et al, 2009). ALCAM is an integral membrane protein, which is ubiquitinated, rapidly endocytosed [in a manner dependent on clathrin or extracellular-signal-regulated kinase (ERK)] and delivered to the degradation pathway; this does not cause, however, changes in the level of ALCAM in the growth cone plasma membrane (Thelen et al, 2008).…”