Molecular modeling of the extracellular domain of the RET receptor tyrosine kinase reveals multiple cadherin-like domains and a calcium-binding site

Using bioinformatic tools, mutagenesis, and binding studies, we have invest igated the structural organization of the extracellular region of the RET r eceptor tyrosine kinase, a functional receptor for glial cell line-derived neurotrophic factor (GDNF). Multiple sequence alignments of seven vertebrat e sequences and one invertebrate RET sequence delineated four distinct N-te rminal domains, each of about 110 residues, containing many of the consensu s motifs of the cadherin fold. Based on these alignments and the crystal st ructures of epithelial and neural cadherins, we have generated molecular mo dels of each of the four cadherin-like domains in the extracellular region of human RET. The modeled structures represent realistic models from both e nergetic and geometrical points of view and are consistent with previous ob servations gathered from biochemical analyses of the effects of Hirschsprun g's disease mutations affecting the folding and stability of the RET molecu le, as well as our own site-directed mutagenesis studies of RET cadherin-li ke domain 1. We have also investigated the role of Ca2+ in ligand binding b y RET and found that Ca2+ ions are required for RET binding to GDNF but not for GDNF binding to the GFR alpha1 co-receptor. In agreement with these re sults, RET, but not GFR alpha1, was found to bind Ca2+ directly. Our result s indicate that the overall architecture of the extracellular region of RET is more closely related to cadherins than previously thought. The models o f the cadherin-like domains of human RET represent valuable tools with whic h to guide future site-directed mutagenesis studies aimed at identifying re sidues involved in ligand binding and receptor activation.