Neural Cell Adhesion Molecule (N-CAM) Homophilic Binding Mediated by the Two N-terminal Ig Domains Is Influenced by Intramolecular Domain-Domain Interactions

Neural cell adhesion molecule (NCAM) is a cell surface adhesion glycoprotein that plays an important role in the development and stability of nervous tissue. The homophilic binding mechanism of NCAM is still a subject of debate on account of findings that appear to support different mechanisms. This paper describes single molecule force measurements with both full-length NCAM and NCAM mutants that lack different immunoglobulin (Ig) domains. By systematically applying an external, time-dependent force to the bond, we obtained parameters that describe the energy landscape of NCAM-NCAM bonds. Histograms of the rupture forces between the full-length NCAM extracellular domains revealed two binding events, one rupturing at higher forces than the other. These bond rupture data show that the two bonds have the same dissociation rates. Despite the energetic and kinetic similarities, the bond strengths differ significantly, and are mechanically distinct. Measurements with NCAM domain deletion mutants mapped the weaker bond to the Ig1-2 segment, and the stronger bond to the Ig3 domain. Finally, the quantitative agreement between the fragment adhesion and the strengths of both NCAM bonds shows that the domain deletions considered in this study do not alter the intrinsic strengths of either of the two bonds.

Section: Discussionsupporting

confidence: 90%

Section: Discussionsupporting

confidence: 81%

See 1 more Smart Citation

Single Molecule Adhesion Measurements Reveal Two Homophilic Neural Cell Adhesion Molecule Bonds with Mechanically Distinct Properties

Wieland

Gewirth

Leckband

2005

“…Most models of NCAM homophilic interaction agree on the requirement of domain 3 for efficient NCAM-mediated cell adhesion (28,(33)(34)(35). We therefore tested the effects of mutations that disrupt GDNF binding on the ability of NCAM to mediate homophilic cell-cell interactions.…”

Section: Resultsmentioning

confidence: 99%

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

Sjöstrand

Carlsson

Paratcha

et al. 2007

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...

“…The fact that this domain predominantly interacted with the fourth Ig domain of NCAM would also indicate an important role for the latter in NCAM-mediated cell adhesion. Different molecular models have been proposed to explain NCAM-mediated cell adhesion (21)(22)(23)(24), some of which exclude a direct role of the fourth NCAM Ig domain. In this regard, it should also be noted that the N-terminal domain of GFR␣1 could conceivably be affecting NCAM-mediated cell adhesion by sterically interfering with trans-homophilic NCAM interactions mediated by other domains in the NCAM molecule.…”

Section: Discussionmentioning

confidence: 99%

Insights into GFRα1 Regulation of Neural Cell Adhesion Molecule (NCAM) Function from Structure-Function Analysis of the NCAM/GFRα1 Receptor Complex

Sjöstrand

Ibáñez

2008

The neural cell adhesion molecule NCAM binds glial cell linederived neurotrophic factor (GDNF) through specific determinants located in its third immunoglobulin (Ig) domain. However, high affinity GDNF binding and downstream signaling depend upon NCAM co-expression with the GDNF co-receptor GFR␣1. GFR␣1 promotes high affinity GDNF binding to NCAM and down-regulates NCAM-mediated homophilic cell adhesion, but the mechanisms underlying these effects are unknown. NCAM and GFR␣1 interact at the plasma membrane, but the molecular determinants involved have not been characterized nor is it clear whether their interaction is required for GFR␣1 regulation of NCAM function. We have investigated the structure-function relationships underlying GFR␣1 binding to NCAM in intact cells. The fourth Ig domain of NCAM was both necessary and sufficient for the interaction of NCAM with GFR␣1. Moreover, although the N-terminal domain of GFR␣1 had previously been shown to be dispensable for GDNF binding, we found that it was both necessary and sufficient for the efficient interaction of this receptor with NCAM. GFR␣1 lacking its N-terminal domain was still able to potentiate GDNF binding to NCAM and assemble into a tripartite receptor complex but showed a reduced capacity to attenuate NCAM-mediated cell adhesion. On its own, the GFR␣1 N-terminal domain was sufficient to decrease NCAM-mediated cell adhesion. These results indicate that direct receptor-receptor interactions are not required for high affinity GDNF binding to NCAM but play an important role in the regulation of NCAM-mediated cell adhesion by GFR␣1.