Abstract
The neural cell adhesion molecule NCAM binds glial cell line-derived 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 GFRalpha1. GFRalpha1 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 GFRalpha1 interact at the plasma membrane, but the molecular determinants involved have not been characterized nor is it clear whether their interaction is required for GFRalpha1 regulation of NCAM function. We have investigated the structure-function relationships underlying GFRalpha1 binding to NCAM in intact cells. The fourth Ig domain of NCAM was both necessary and sufficient for the interaction of NCAM with GFRalpha1. Moreover, although the N-terminal domain of GFRalpha1 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. GFRalpha1 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 GFRalpha1 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 GFRalpha1.
Highlights
Trophin high affinity binding has been shown to require both the p75NTR and Trk receptor subunits (4 – 6), but it is still unclear whether direct interaction between these two receptors underlies the generation of high affinity sites [7,8,9]
These results indicate that a preformed complex between GFR␣1 and NCAM is not required for high affinity glial cell line-derived neurotrophic factor (GDNF) binding to NCAM
We have identified the molecular determinants in NCAM and GFR␣1 that underlie their interaction at the plasma membrane of mammalian cells
Summary
Trophin high affinity binding has been shown to require both the p75NTR and Trk receptor subunits (4 – 6), but it is still unclear whether direct interaction between these two receptors underlies the generation of high affinity sites [7,8,9]. This allowed us to probe the role of NCAM/GFR␣1 interactions in NCAMmediated cell adhesion and high affinity GDNF binding.
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