RET Recognition of GDNF-GFRα1 Ligand by a Composite Binding Site Promotes Membrane-Proximal Self-Association

Abstract

The RET receptor tyrosine kinase is essential to vertebrate development and implicated in multiple human diseases. RET binds a cell surface bipartite ligand comprising a GDNF family ligand and a GFRα coreceptor, resulting in RET transmembrane signaling. We present a hybrid structural model, derived from electron microscopy (EM) and low-angle X-ray scattering (SAXS) data, of the RET extracellular domain (RET(ECD)), GDNF, and GFRα1 ternary complex, defining the basis for ligand recognition. RET(ECD) envelopes the dimeric ligand complex through a composite binding site comprising four discrete contact sites. The GFRα1-mediated contacts are crucial, particularly close to the invariant RET calcium-binding site, whereas few direct contacts are made by GDNF, explaining how distinct ligand/coreceptor pairs are accommodated. The RET(ECD) cysteine-rich domain (CRD) contacts both ligand components and makes homotypic membrane-proximal interactions occluding three different antibody epitopes. Coupling of these CRD-mediated interactions suggests models for ligand-induced RET activation and ligand-independent oncogenic deregulation.

Structure and function of RET in multiple endocrine neoplasia type 2

“…To stimulate RET tyrosine kinase activity from the extracellular environment, these GFLs first need to assemble into a complex with their glycosylphosphatidylinositol (GPI)-anchored co-receptors, the GDNF receptor-α family (GFRα1-4), after which the GFL-GFRα complex recruits RET to form cognate and non-cognate heterodimers. The RET-GFL-GFRα complex (the RET 'ternary' complex) has a 2:2:2 stoichiometry, meaning a dimer of GDNF binds two co-receptor molecules that at the same time recruit two RET receptor molecules and exhibits positive cooperativity (Schlee et al 2006, Goodman et al 2014. Recently, the architecture of RET extracellular domain (ECD) was revealed by small angle X-ray scattering (SAXS) and electron microscopy (EM).…”

Exon Skipping in the RET Gene Encodes Novel Isoforms That Differentially Regulate RET Protein Signal Transduction

“…Although it was originally thought that CLD1-3 of RET was involved in direct binding from results obtained through mutagenesis studies, a direct interaction between CLD1-3 and the GDNF-GFR␣1 complex has not been observed (53,54). Instead, it has been proposed that CLD1-3 contributes to the stability of the tertiary structure of the GDNF-GFR␣1-RET complex by forming a secondary dimerization site to trap the GDNF-GFR␣1 binary complex (14). These studies predict that RET ⌬E3 , which forms a fused CLD1-CLD2 region, likely gives rise to a less stable tertiary GDNF-GFR␣1-RET complex compared with full-length RET.…”

“…Molecular Modeling-This analysis was performed as described previously (14). Models of cadherin-like domains 1-4 were taken from PDB code 4UX8, defined by a combination of EM and small angle x-ray scattering analyses.…”

“…This region by itself was shown to be insufficient to bind ligand but retains at least one important binding epitope (i.e. necessary for ligand binding but not sufficient) (14). Many receptor tyrosine kinases have extracellular domains that have autoinhibitory functions in the absence of ligand.…”

“…Domain of RET-To understand the impact deletions in the extracellular domain would have on RET, we analyzed the deletions in terms of a CLD1-4 model derived from electron microscopy and small angle x-ray scattering analyses (14). The main consequence of deletion of exon 3 is the removal of half of CLD1 (residues 113-150) and half of CLD2 (residues 154 -208).…”