RET receptor tyrosine kinase plays vital developmental and neuroprotective roles in metazoans. GDNF family ligands (GFLs) when bound to cognate GFRα co-receptors recognize and activate RET ...stimulating its cytoplasmic kinase function. The principles for RET ligand-co-receptor recognition are incompletely understood. Here, we report a crystal structure of the cadherin-like module (CLD1-4) from zebrafish RET revealing interdomain flexibility between CLD2 and CLD3. Comparison with a cryo-electron microscopy structure of a ligand-engaged zebrafish RETECD-GDNF-GFRα1a complex indicates conformational changes within a clade-specific CLD3 loop adjacent to the co-receptor. Our observations indicate that RET is a molecular clamp with a flexible calcium-dependent arm that adapts to different GFRα co-receptors, while its rigid arm recognizes a GFL dimer to align both membrane-proximal cysteine-rich domains. We also visualize linear arrays of RETECD-GDNF-GFRα1a suggesting that a conserved contact stabilizes higher-order species. Our study reveals that ligand-co-receptor recognition by RET involves both receptor plasticity and strict spacing of receptor dimers by GFL ligands.
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•X-ray structure of zebrafish RETCLD1-4 module reveals conformational flexibility•Conformational differences between RETCLD1-4 and a liganded RETECD cryo-EM structure•Spatial separation of RETECD CRD dimer C termini imposed by each ligand dimer•Differences in GDNF and GDF15 co-receptor engagement of RET and multimerization evidence
Adams et al. use X-ray crystallography and cryo-electron microscopy to probe conformational changes in RET due to binding of GDNF ligand-GFRα1 co-receptor. The study shows a two-site clamping mechanism with flexible adaptations near the RET calcium sites but near identical spacings of the RET cysteine-rich domains established by GDNF/GDF15 ligand dimers.
Protein glycosylation events that happen early in the secretory pathway are often dysregulated during tumorigenesis. These events can be probed, in principle, by monosaccharides with bioorthogonal ...tags that would ideally be specific for distinct glycan subtypes. However, metabolic interconversion into other monosaccharides drastically reduces such specificity in the living cell. Here, we use a structure-based design process to develop the monosaccharide probe NE-(S)-azidopropionylgalactosamine (GalNAzMe) that is specific for cancer-relevant Ser/Thr(O)–linked N-acetylgalactosamine (GalNAc) glycosylation. By virtue of a branched N-acylamide side chain, GalNAzMe is not interconverted by epimerization to the corresponding N-acetylglucosamine analog by the epimerase N-acetylgalactosamine–4-epimerase (GALE) like conventional GalNAc–based probes. GalNAzMe enters O-GalNAc glycosylation but does not enter other major cell surface glycan types including Asn(N)-linked glycans. We transfect cells with the engineered pyrophosphorylase mut-AGX1 to biosynthesize the nucleotidesugar donor uridine diphosphate (UDP)-GalNAzMe from a sugar-1-phosphate precursor. Tagged with a bioorthogonal azide group, GalNAzMe serves as an O-glycan–specific reporter in superresolution microscopy, chemical glycoproteomics, a genome-wide CRISPR-knockout (CRISPR-KO) screen, and imaging of intestinal organoids. Additional ectopic expression of an engineered glycosyltransferase, “bump-and-hole” (BH)–GalNAc-T2, boosts labeling in a programmable fashion by increasing incorporation of GalNAzMe into the cell surface glycoproteome. Alleviating the need for GALE-KO cells in metabolic labeling experiments, GalNAzMe is a precision tool that allows a detailed view into the biology of a major type of cancer-relevant protein glycosylation.
Metabolic oligosaccharide engineering (MOE) has fundamentally contributed to our understanding of protein glycosylation. Efficient MOE reagents are activated into nucleotide-sugars by cellular ...biosynthetic machineries, introduced into glycoproteins and traceable by bioorthogonal chemistry. Despite their widespread use, the metabolic fate of many MOE reagents is only beginning to be mapped. While metabolic interconnectivity can affect probe specificity, poor uptake by biosynthetic salvage pathways may impact probe sensitivity and trigger side reactions. Here, we use metabolic engineering to turn the weak alkyne-tagged MOE reagents Ac4GalNAlk and Ac4GlcNAlk into efficient chemical tools to probe protein glycosylation. We find that bypassing a metabolic bottleneck with an engineered version of the pyrophosphorylase AGX1 boosts nucleotide-sugar biosynthesis and increases bioorthogonal cell surface labeling by up to two orders of magnitude. A comparison with known azide-tagged MOE reagents reveals major differences in glycoprotein labeling, substantially expanding the toolbox of chemical glycobiology.
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 (RETECD), GDNF, and GFRα1 ternary complex, defining the basis for ligand recognition. RETECD 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 RETECD 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.
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•Extracellular architecture of RETECD is revealed by SAXS•EM structure for a RETECD-GDNF-GFRα complex reveals a composite ligand-binding site•A GFRα1-binding hotspot contacts the invariant RETCLD2-3 calcium sites•RETCRD couples ligand recognition and receptor homodimerization, exploited by MEN2A
RET is crucial for vertebrate development and causes three different human diseases. The basis for RET recognition of its bipartite GFL-GFRα ligands has been unclear. Goodman et al. describe a flower-shaped structure for a mammalian RET ectodomain-GDNF-GFRα1 complex. The structure reveals a composite binding site in RET that is driven by GFRα1 contacts and able to accommodate multiple GFL ligands. GFL-GFRα binding promotes a homotypic interaction between membrane-proximal regions of RET, a feature hijacked by oncogenic crosslinking mutations found in MEN2A patients.
The elaboration of polarity is central to organismal development and to the maintenance of functional epithelia. Among the controls determining polarity are the PAR proteins, PAR6, aPKCι and PAR3, ...regulating both known and unknown effectors. Here, we identify FARP2 as a 'RIPR' motif-dependent partner and substrate of aPKCι that is required for efficient polarisation and junction formation. Binding is conferred by a FERM/FA domain-kinase domain interaction and detachment promoted by aPKCι-dependent phosphorylation. FARP2 is shown to promote GTP loading of Cdc42, which is consistent with it being involved in upstream regulation of the polarising PAR6-aPKCι complex. However, we show that aPKCι acts to promote the localised activity of FARP2 through phosphorylation. We conclude that this aPKCι-FARP2 complex formation acts as a positive feedback control to drive polarisation through aPKCι and other Cdc42 effectors.This article has an associated First Person interview with the first author of the paper.
Metabolic oligosaccharide engineering (MOE) has fundamentally contributed to our understanding of protein glycosylation. Efficient MOE reagents are activated into nucleotide-sugars by cellular ...biosynthetic machineries, introduced into glycoproteins and traceable by bioorthogonal chemistry. Despite their widespread use, the metabolic fate of many MOE reagents is only beginning to be mapped. While metabolic interconnectivity can affect probe specificity, poor uptake by biosynthetic salvage pathways may impact probe sensitivity and trigger side reactions. Here, we use metabolic engineering to turn the weak alkyne-tagged MOE reagents Ac
GalNAlk and Ac
GlcNAlk into efficient chemical tools to probe protein glycosylation. We find that bypassing a metabolic bottleneck with an engineered version of the pyrophosphorylase AGX1 boosts nucleotide-sugar biosynthesis and increases bioorthogonal cell surface labeling by up to two orders of magnitude. A comparison with known azide-tagged MOE reagents reveals major differences in glycoprotein labeling, substantially expanding the toolbox of chemical glycobiology.
The synthesis, structure–activity relationships (SAR) and structural data of a series of indolin-2-one inhibitors of RET tyrosine kinase are described. These compounds were designed to explore the ...available space around the indolinone scaffold within RET active site. Several substitutions at different positions were tested and biochemical data were used to draw a molecular model of steric and electrostatic interactions, which can be applied to design more potent and selective RET inhibitors. The crystal structures of RET kinase domain in complex with three inhibitors were solved. All three compounds bound in the ATP pocket and formed two hydrogen bonds with the kinase hinge region. Crystallographic analysis confirmed predictions from molecular modelling and helped refine SAR results. These data provide important information for the development of indolinone inhibitors for the treatment of RET-driven cancers.
Trafficking of the galanin R2 receptor (GALR2) fused with enhanced GFP (EGFP) was studied by using confocal fluorescence microscopy. The fusion protein was predominantly localized on the plasma ...membrane with some intracellular fluorescent structures (vesicles), mainly in the perinuclear region. Incubation with galanin resulted in a concentration-dependent increase in intracellular Ca2+ concentration levels, suggesting that the GALR2-EGFP conjugate is functional. After blocking endocytosis with methyl-β-cyclodextrin GALR2-EGFP expression was increased on the surface and decreased in the cytoplasm. Blocking endocytic recycling with monensin caused an increase of intracellular GALR2-EGFP accumulation and a decrease of fluorescence on the plasma membrane. GALR2-EGFP on the plasma membrane was internalized within 5-10 min after treatment with galanin or AR-M1896, a selective GALR2 agonist, with a dramatic reduction in plasma membrane localization and appearance in intracellular vesicles. Neither M35 nor M40, two galanin analogues with putative antagonistic action, prevented GALR2 agonist-induced internalization of GALR2-EGFP, suggesting that they are not antagonists at this receptor under the present circumstances. Galanin stimulation at low temperature caused GALR2-EGFP aggregation and clustering on the surface but no translocation to cytoplasm. After coincubation with galanin the GALR2-EGFP was colocalized with internalized Texas red-transferrin, a marker of the clathrin endocytic pathway. Hyperosmotic sucrose inhibited internalization of GALR2-EGFP. Taken together these findings indicate that GALR2 undergoes constitutive endocytosis and recycling and that both ligand-independent and ligand-dependent internalization use the clathrin-dependent endocytic recycling pathway.
Atypical protein kinase Cι (PKCι) has roles in cell growth, cellular polarity, and migration, and its abundance is frequently increased in cancer. We identified a protein interaction surface ...containing a dibasic motif (RIPR) that bound a distinct subset of PKCι substrates including lethal giant larvae 2 (LLGL2) and myosin X, but not other substrates such as Par3. Further characterization demonstrated that Arg471 in this motif was important for binding to LLGL2, whereas Arg474 was critical for interaction with myosin X, indicating that multiple complexes could be formed through this motif. A somatic mutation of the dibasic motif (R471C) was the most frequent mutation of PKCι in human cancer, and the intact dibasic motif was required for normal polarized epithelial morphogenesis in three-dimensional cysts. Thus, the R471C substitution is a change-of-function mutation acting at this substrate-specific recruitment site to selectively disrupt the polarizing activity of PKCι.
The Arp2/3 complex regulates many cellular processes by stimulating formation of branched actin filament networks. Because three of its seven subunits exist as two different isoforms, mammals produce ...a family of Arp2/3 complexes with different properties that may be suited to different physiological contexts. To shed light on how isoform diversification affects Arp2/3 function, we determined a 4.2 Å resolution cryo-EM structure of the most active human Arp2/3 complex containing ARPC1B and ARPC5L, and compared it with the structure of the least active ARPC1A-ARPC5-containing complex. The architecture of each isoform-specific Arp2/3 complex is the same. Strikingly, however, the N-terminal half of ARPC5L is partially disordered compared to ARPC5, suggesting that this region of ARPC5/ARPC5L is an important determinant of complex activity. Confirming this idea, the nucleation activity of Arp2/3 complexes containing hybrid ARPC5/ARPC5L subunits is higher when the ARPC5L N-terminus is present, thereby providing insight into activity differences between the different Arp2/3 complexes.
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