Disruption of the GDNF Binding Site in NCAM Dissociates Ligand Binding and Homophilic Cell Adhesion

Abstract

Most plasma membrane proteins are capable of sensing multiple cell-cell and cell-ligand interactions, but the extent to which this functional versatility is founded on their modular design is less clear. We have identified the third immunoglobulin domain of the Neural Cell Adhesion Molecule (NCAM) as the necessary and sufficient determinant for its interaction with Glial Cell Line-derived Neurotrophic Factor (GDNF). Four charged contacts were identified by molecular modeling as the main contributors to binding energy. Their mutation abolished GDNF binding to NCAM but left intact the ability of NCAM to mediate cell adhesion, indicating that the two functions are genetically separable. The GDNF-NCAM interface allows complex formation with the GDNF family receptor ␣1, shedding light on the molecular architecture of a multicomponent GDNF receptor.Members of the glial cell line-derived neurotrophic factor (GDNF) 3 family regulate cell survival, differentiation, and migration in the peripheral and central nervous systems as well as in a few peripheral organs. The four members of this ligand family, i.e. GDNF, Neurturin, Artemin, and Persephin, share ϳ40% of their amino acid sequence. GDNF and Artemin are being developed as therapeutic agents against Parkinson disease and peripheral neuropathies, respectively (1-3). Signaling by GDNF family ligands is mediated by alternative multicomponent receptor complexes containing a ligand binding, glycosylphosphatidylinositol-anchored subunit termed GDNF family receptor ␣ (GFR␣) (4 -9), together with either the RET receptor tyrosine kinase (10, 11) or the neural cell adhesion molecule (NCAM) (12) as signaling subunits. Four related GFR␣ proteins, termed GFR␣1 to 4, with different ligand specificity have been identified (13). In collaboration with RET, GFR␣1 mediates the effects of GDNF on neuronal differentiation and migration in the developing enteric nervous system (14, 15) and ureter morphogenesis during kidney development (16,17). On the other hand, in the presence of NCAM, GDNF and GFR␣1 stimulate neurite outgrowth in vitro (12) and synaptogenesis in vitro and in vivo (18) in hippocampal neurons, stimulate migration of neuronal precursors in the rostral migratory stream (12,19), and regulate Schwann cell migration and function (12,20). Despite the importance of GDNF signaling for normal development and its possible therapeutic applications, the molecular architecture of these receptor complexes is not yet understood.Crystal structures have been described for GDNF (21), a fragment of the ligand binding domain of GFR␣1 (22), and the complex between Artemin and the ligand binding domain of its cognate GFR␣3 receptor (23). The latter validated previous mutagenesis studies performed on GDNF ligands and GFR␣ receptors (24 -26) and demonstrated that the ligand binding domain of GFR␣ receptors is formed by a single compact module that interacts with the poles of the elongated dimer of GDNF family ligands. In addition, a model of the extracellular region of RET based on four consecu...

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

“…Notably, soluble Ig3 was able to inhibit GDNFinduced neurite outgrowth, supporting the possibility that Ig3 is sufficient for GDNF binding. Consistent with these findings, Sjöstrand et al (2007) showed that Ig3 is necessary and sufficient for GDNF binding. Together, these results indicate that the binding site for GDNF is contained within NCAM Ig3.…”

“…Using surface plasmon resonance (SPR) analysis, we found, in accordance with Sjöstrand et al (2007), that NCAM Ig3 is necessary for GDNF binding. We identified a motif in the primary sequence of GDNF that is involved in binding to NCAM and demonstrate that a peptide, termed Gliafin, encompassing this motif, mimics GDNF with regard to binding, signaling, and effects on neurite outgrowth.…”

“…The discrepancy is most likely attributable to the fact that we used NCAM fragments (recombinantly produced Ig modules), whereas Paratcha et al (2003) tested GDNF binding to full-length NCAM expressed on the cell surface of transfected COS cells. Although GDNF has been shown to bind the Ig3 module (Sjöstrand et al, 2007), other NCAM modules in a full-length molecule may modulate the affinity. Additionally, the test systems applied in the two studies have important differences.…”

Assembly and activation of neurotrophic factor receptor complexes

“…NCAM has also been shown to directly interact with GFRa1, even in the absence of GDNF, through its fourth Ig domain (Sjös-trand and Ibáñez, 2008). Molecular modeling studies have suggested a 2:2:2 stoichiometry for the GDNF/ GFRa1/NCAM complex (Sjöstrand et al, 2007). The observation that dimeric factors interact with receptor dimers raises two important but sometimes overlooked questions: (i) are receptor homodimers induced upon ligand binding or already preformed prior to ligand engagement?, and (ii) is homodimerization required for receptor activation and downstream signaling?…”

“…Unlike Ret, however, NCAM is able to bind GDNF on its own, although signal transduction appears to require the higher affinity afforded in the presence of the GFRa1 subunit (Paratcha et al, 2003). NCAM biding to GDNF requires a distinct determinant in the third Ig domain of the NCAM molecule, and NCAM point mutants have been generated that interfere with GDNF binding without affecting cell adhesion (Sjöstrand et al, 2007;Nielsen, 2009). The NCAM complexes involved in cell-cell adhesion are believed to be also dimeric, in which a cis-dimer is formed through Ig domains I and II bending over each other, and this structure would then interact in trans with another dimer forming a rather compact cluster.…”