Identification of NCAM‐binding peptides promoting neurite outgrowth via a heterotrimeric G‐protein‐coupled pathway

Abstract

A combinatorial library of undecapeptides was produced and utilized for the isolation of peptide binding to the fibronectin type 3 modules (F3I-F3II) of the neural cell adhesion molecule (NCAM). The isolated peptides were sequenced and produced as dendrimers. Two of the peptides (denoted EN-FIN2 and ENFIN11) were confirmed to bind to F3I-F3II of NCAM by surface plasmon resonance. The peptides induced neurite outgrowth in primary cerebellar neurons and PC12E2 cells, but had no apparent neuroprotective properties. NCAM is known to activate different intracellular pathways, including signaling through the fibroblast growth factor receptor, the Src-related non-receptor tyrosine kinase Fyn, and heterotrimeric G-proteins. Interestingly, neurite outgrowth stimulated by ENFIN2 and ENFIN11 was independent of signaling through fibroblast growth factor receptor and Fyn, but could be inhibited with pertussis toxin, an inhibitor of certain heterotrimeric G-proteins. Neurite outgrowth induced by trans-homophilic NCAM was unaffected by the peptides, whereas knockdown of NCAM completely abrogated ENFIN2-and ENFIN11-induced neuritogenesis. These observations suggest that ENFIN2 and ENFIN11 induce neurite outgrowth in an NCAM-dependent manner through G-protein-coupled signal transduction pathways. Thus, ENFIN2 and ENFIN11 may be valuable for exploring this particular type of NCAMmediated signaling.

Role of Glial Cell Line-Derived Neurotrophic Factor (GDNF)–Neural Cell Adhesion Molecule (NCAM) Interactions in Induction of Neurite Outgrowth and Identification of a Binding Site for NCAM in the Heel Region of GDNF

“…1 D). Neurons were transfected with either a plasmid encoding shRNA for NCAM or control shRNA (Hansen et al, 2007). To confirm the knockdown of NCAM expression, the transfected neurons were stained for NCAM.…”

“…A vector encoding short hairpin RNA (shRNA) for NCAM (Hansen et al, 2007) was used to knock down the expression of NCAM. A vector encoding shRNA not targeting any known expressed rat sequence (Hansen et al, 2007) was used as a control.…”

“…A vector encoding short hairpin RNA (shRNA) for NCAM (Hansen et al, 2007) was used to knock down the expression of NCAM. A vector encoding shRNA not targeting any known expressed rat sequence (Hansen et al, 2007) was used as a control. For uncoupling of signaling through NCAM-180 and NCAM-140, respectively, expression vectors encoding the cytoplasmic domain of either NCAM-180 or NCAM-140 (Kolkova et al, 2000b) were used, and a matching empty vector served as a control.…”

Unique Intracellular Trafficking Processes Associated With Neural Cell Adhesion Molecule and Its Intracellular Signaling

“…Both IF and biochemical analyses indicated that FGFR in cells stimulated by NCAM is channeled to a recycling pathway back to the cells surface. After 60 min of treatment, FGFR in FGF-treated cells was found in lysosomes, whereas those by NCAM were in the recycling endosome compartment and exhibited with its FNIII domain induced neurite outgrowth through a pathway that involves heterotrimeric G proteins, and that is independent of FGFR and Fyn (Hansen et al 2007). The above illustrates the complex diversity in NCAMassociated signaling.…”

Analysis of Non-canonical Fibroblast Growth Factor Receptor 1 (FGFR1) Interaction Reveals Regulatory and Activating Domains of Neurofascin

“…L1 converges with NCAM signaling upstream of the MAPK pathway at the level of Raf (18,21,28,29). NCAM may induce alternative signaling pathways, including protein kinase A-dependent signaling or G-proteins (18,30). NCAM signaling to the nucleus may include activation of CREB and c-Fos or NF-B (29,31,32).…”