scholarly journals Cryo-EM structure of the activated RET signaling complex reveals the importance of its cysteine-rich domain

2019 ◽  
Vol 5 (7) ◽  
pp. eaau4202 ◽  
Author(s):  
Janna M. Bigalke ◽  
Shintaro Aibara ◽  
Robert Roth ◽  
Göran Dahl ◽  
Euan Gordon ◽  
...  
Keyword(s):  
Calcium Binding ◽  
Neurological Diseases ◽  
Receptor Activation ◽  
Loss Of Function ◽  
Calcium Binding Site ◽  
Signaling Complex ◽  
Rich Domain ◽  
Extracellular Region ◽  
Domain 3 ◽  
Cysteine Rich Domain

Signaling through the receptor tyrosine kinase RET is essential during normal development. Both gain- and loss-of-function mutations are involved in a variety of diseases, yet the molecular details of receptor activation have remained elusive. We have reconstituted the complete extracellular region of the RET signaling complex together with Neurturin (NRTN) and GFRα2 and determined its structure at 5.7-Å resolution by cryo-EM. The proteins form an assembly through RET-GFRα2 and RET-NRTN interfaces. Two key interaction points required for RET extracellular domain binding were observed: (i) the calcium-binding site in RET that contacts GFRα2 domain 3 and (ii) the RET cysteine-rich domain interaction with NRTN. The structure highlights the importance of the RET cysteine-rich domain and allows proposition of a model to explain how complex formation leads to RET receptor dimerization and its activation. This provides a framework for targeting RET activity and for further exploration of mechanisms underlying neurological diseases.

Anaesthesia and neuromuscular disorders: what a neurologist needs to know

2020 ◽  
pp. practneurol-2020-002633
Author(s):  
Luuk R van den Bersselaar ◽  
Marc M J Snoeck ◽  
Madelief Gubbels ◽  
Sheila Riazi ◽  
Erik-Jan Kamsteeg ◽  
...  
Keyword(s):  
General Anaesthesia ◽  
Neuromuscular Disorders ◽  
Muscle Relaxants ◽  
Rich Domain ◽  
Specific Advice ◽  
Voltage Dependent ◽  
Life Threatening ◽  
Ryanodine Receptor 1 ◽  
Domain 3 ◽  
Cysteine Rich Domain

Neurologists are often asked for specific advice regarding patients with neuromuscular disease who require general anaesthesia. However, guidelines on specific neuromuscular disorders do not usually include specific guidelines or pragmatic advice regarding (regional and/or general) anaesthesia or procedural sedation. Furthermore, the medical literature on this subject is mostly limited to publications in anaesthesiology journals. We therefore summarise general recommendations and specific advice for anaesthesia in different neuromuscular disorders to provide a comprehensive and accessible overview of the knowledge on this topic essential for clinical neurologists. A preoperative multidisciplinary approach involving anaesthesiologists, cardiologists, chest physicians, surgeons and neurologists is crucial. Depolarising muscle relaxants (succinylcholine) should be avoided at all times. The dose of non-depolarising muscle relaxants must be reduced and their effect monitored. Patients with specific mutations in RYR1 (ryanodine receptor 1) and less frequently in CACNA1S (calcium channel, voltage-dependent, L type, alpha 1S subunit) and STAC3 (SH3 and cysteine rich domain 3) are at risk of developing a life-threatening malignant hyperthermia reaction.

scholarly journals Frizzled BRET sensors based on bioorthogonal labeling of unnatural amino acids reveal WNT-induced dynamics of the cysteine-rich domain.

2021 ◽  
Author(s):  
Maria Kowalski-Jahn ◽  
Hannes Schihada ◽  
Ainoleena Turku ◽  
Thomas Huber ◽  
Thomas P. Sakmar ◽  
...  
Keyword(s):  
Amino Acids ◽  
Unnatural Amino Acids ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Membrane Receptors ◽  
Rich Domain ◽  
Linker Domain ◽  
Cysteine Rich Domain

Frizzleds (FZD1-10) comprise a class of G protein-coupled receptors containing an extracellular cysteine-rich domain (CRD) that binds lipoglycoproteins of the Wingless/Int-1 family (WNTs). Despite the prominent role of the WNT/FZD system in health and disease, our understanding of how WNT binding to the FZD CRD is translated into receptor activation and transmembrane signaling remains limited. Current hypotheses dispute the roles for conformational dynamics and the involvement of the linker domain connecting the CRD with the seven-helical transmembrane core of FZD. To clarify the mechanism of WNT binding to FZD and to elucidate how WNT/FZD complexes achieve signaling pathway specificity, we devised conformational FZD-CRD biosensors based on bioluminescence-resonance-energy-transfer (BRET). Using FZD engineered with N-terminal nanoluciferase and fluorescently-labeled unnatural amino acids in the linker domain and extracellular loop 3, we show that WNT-3A and WNT-5A induce similar CRD conformational rearrangements despite promoting distinct downstream signaling pathways, and that CRD dynamics are not required for WNT/β-catenin signaling. Thus, the novel FZD-CRD biosensors we report provide insights into the stepwise binding, activation and signaling processes in FZDs. The sensor design is broadly applicable to explore fundamental events in signal transduction mediated by other membrane receptors.

scholarly journals Congenital myopathy results from misregulation of a muscle Ca2+ channel by mutant Stac3

2016 ◽  
Vol 114 (2) ◽  
pp. E228-E236 ◽  
Author(s):  
Jeremy W. Linsley ◽  
I-Uen Hsu ◽  
Linda Groom ◽  
Viktor Yarotskyy ◽  
Manuela Lavorato ◽  
...  
Keyword(s):  
Native American ◽  
Dynamic Imaging ◽  
Release Channel ◽  
Rich Domain ◽  
Dihydropyridine Receptors ◽  
Ec Coupling ◽  
Wide Range ◽  
Ryanodine Receptor 1 ◽  
Domain 3 ◽  
Cysteine Rich Domain

Skeletal muscle contractions are initiated by an increase in Ca2+ released during excitation–contraction (EC) coupling, and defects in EC coupling are associated with human myopathies. EC coupling requires communication between voltage-sensing dihydropyridine receptors (DHPRs) in transverse tubule membrane and Ca2+ release channel ryanodine receptor 1 (RyR1) in the sarcoplasmic reticulum (SR). Stac3 protein (SH3 and cysteine-rich domain 3) is an essential component of the EC coupling apparatus and a mutation in human STAC3 causes the debilitating Native American myopathy (NAM), but the nature of how Stac3 acts on the DHPR and/or RyR1 is unknown. Using electron microscopy, electrophysiology, and dynamic imaging of zebrafish muscle fibers, we find significantly reduced DHPR levels, functionality, and stability in stac3 mutants. Furthermore, stac3NAM myofibers exhibited increased caffeine-induced Ca2+ release across a wide range of concentrations in the absence of altered caffeine sensitivity as well as increased Ca2+ in internal stores, which is consistent with increased SR luminal Ca2+. These findings define critical roles for Stac3 in EC coupling and human disease.

Zinc co-ordination by the DHHC cysteine-rich domain of the palmitoyltransferase Swf1

2013 ◽  
Vol 454 (3) ◽  
pp. 427-435 ◽  
Author(s):  
Ayelén González Montoro ◽  
Rodrigo Quiroga ◽  
Javier Valdez Taubas
Keyword(s):  
Amino Acids ◽  
Tertiary Structure ◽  
Random Mutagenesis ◽  
Binding Pocket ◽  
Essential Amino Acids ◽  
Zinc Binding ◽  
Loss Of Function ◽  
Post Translational Modification ◽  
Rich Domain ◽  
Cysteine Rich Domain

S-acylation, commonly known as palmitoylation, is a widespread post-translational modification of proteins that consists of the thioesterification of one or more cysteine residues with fatty acids. This modification is catalysed by a family of PATs (palmitoyltransferases), characterized by the presence of a 50-residue long DHHC-CRD (Asp-His-His-Cys cysteine-rich domain). To gain knowledge on the structure–function relationships of these proteins, we carried out a random-mutagenesis assay designed to uncover essential amino acids in Swf1, the yeast PAT responsible for the palmitoylation of SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) proteins. We identified 21 novel loss-of-function mutations, which are mostly localized within the DHHC-CRD. Modelling of the tertiary structure of the Swf1 DHHC domain suggests that it could fold as a zinc-finger domain, co-ordinating two zinc atoms in a CCHC arrangement. All residues predicted to be involved in the co-ordination of zinc were found to be essential for Swf1 function in the screen. Moreover, these mutations result in unstable proteins, in agreement with a structural role for these zinc fingers. The conservation of amino acids predicted to form each zinc-binding pocket suggests a shared function, as the selective pressure to maintain them is lost upon mutation of one of them. A Swf1 orthologue that lacks one of the zinc-binding pockets is able to complement a yeast swf1∆ strain, possibly because a similar fold can be stabilized by hydrogen bonds instead of zinc co-ordination. Finally, we show directly that recombinant Swf1 DHHC-CRD is able to bind zinc. Sequence analyses of DHHC domains allowed us to present models of the zinc-binding properties for all PATs.

scholarly journals Notch-Jagged signaling complex defined by an interaction mosaic

2021 ◽  
Author(s):  
Matthieu R. Zeronian ◽  
Oleg Klykov ◽  
Júlia Portell i de Montserrat ◽  
Maria J. Konijnenberg ◽  
Anamika Gaur ◽  
...  
Keyword(s):  
Developmental Disorders ◽  
Regulatory Region ◽  
Activation Mechanism ◽  
Signaling Complex ◽  
Notch1 Signaling ◽  
Rich Domain ◽  
X Ray Scattering ◽  
Epidermal Growth ◽  
Notch1 Receptor ◽  
Cysteine Rich Domain

AbstractThe Notch signaling system links cellular fate to that of its neighbors, driving proliferation, apoptosis, and cell differentiation in metazoans, whereas dysfunction leads to debilitating developmental disorders and cancers. Other than a five-by-five domain complex, it is unclear how the 40 extracellular domains of the Notch1 receptor collectively engage the 19 domains of its canonical ligand Jagged1 to activate Notch1 signaling. Here, using cross-linking mass spectrometry (XL-MS), biophysical and structural techniques on the full extracellular complex and targeted sites,we identify five distinct regions, two on Notch1 and three on Jagged1, that form an interaction network.The Notch1 membrane-proximal regulatory region individually binds to the established Notch1 epidermal growth factor (EGF) 8-13 and Jagged1 C2-EGF3 activation sites, as well as to two additional Jagged1 regions, EGF 8-11 and cysteine-rich domain (CRD). XL-MS and quantitative interaction experiments show that the three Notch1 binding sites on Jagged1 also engage intramolecularly.These interactions, together with Notch1 and Jagged1 ectodomain dimensions and flexibility determined by small-angle X-ray scattering (SAXS), support the formation of backfolded architectures. Combined, the data suggest that critical Notch1 and Jagged1 regions are not distal, but engage directly to control Notch1 signaling, thereby redefining the Notch1-Jagged1 activation mechanism and indicating new routes for therapeutic applications.

scholarly journals The seven-span transmembrane receptor CD97 has a cellular ligand (CD55, DAF).

1996 ◽  
Vol 184 (3) ◽  
pp. 1185-1189 ◽  
Author(s):  
J Hamann ◽  
B Vogel ◽  
G M van Schijndel ◽  
R A van Lier
Keyword(s):  
Calcium Binding ◽  
Hormone Receptors ◽  
Regulatory Protein ◽  
Peptide Hormone ◽  
Calcium Binding Site ◽  
Cos Cells ◽  
Extracellular Region ◽  
Secretin Receptor ◽  
Short Consensus Repeat ◽  
Peptide Hormone Receptors

CD97 is an activation-induced antigen on leukocytes with a seven-span transmembrane (7-TM) region homologous to the secretin receptor superfamily. However, in contrast to this group of peptide hormone receptors, CD97 has an extended extracellular region with three EGF domains at the NH2 terminus, two of them with a calcium binding site. By demonstrating that lymphocytes and erythrocytes specifically adhere to CD97-transfected COS cells we here show that CD97 in parallel with its molecular evolution has acquired the ability to bind cellular ligands. A mAb selected on its capacity to block the adhesion between CD97 transfectants and red cells was found to be directed to the NH2-terminal short consensus repeat (SCR) of decay accelerating factor (DAF, CD55), a regulatory protein of the complement cascade. The specificity of the interaction of CD97 with CD55 was established by the observation that erythrocytes that lack CD55, obtained from patients with paroxysmal nocturnal hemoglobinuria (PNH) or the CD55, phenotype Inab, failed to adhere to CD97 transfectants. This is the first demonstration of a cellular ligand for a 7-TM receptor.

scholarly journals Mechanism and ultrasensitivity in Hedgehog signaling revealed by Patched1 disease mutations

2021 ◽  
Vol 118 (6) ◽  
pp. e2006800118
Author(s):  
Kostadin Petrov ◽  
Taciani de Almeida Magalhaes ◽  
Adrian Salic
Keyword(s):  
Mathematical Modeling ◽  
Catalytic Activity ◽  
Single Cell ◽  
Birth Defects ◽  
Hedgehog Signaling ◽  
Loss Of Function ◽  
Functional Assays ◽  
Rich Domain ◽  
Disease Mutations ◽  
Cysteine Rich Domain

Hedgehog signaling is fundamental in animal embryogenesis, and its dysregulation causes cancer and birth defects. The pathway is triggered when the Hedgehog ligand inhibits the Patched1 membrane receptor, relieving repression that Patched1 exerts on the GPCR-like protein Smoothened. While it is clear how loss-of-function Patched1 mutations cause hyperactive Hedgehog signaling and cancer, how other Patched1 mutations inhibit signaling remains unknown. Here, we develop quantitative single-cell functional assays for Patched1, which, together with mathematical modeling, indicate that Patched1 inhibits Smoothened enzymatically, operating in an ultrasensitive regime. Based on this analysis, we propose that Patched1 functions in cilia, catalyzing Smoothened deactivation by removing cholesterol bound to its extracellular, cysteine-rich domain. Patched1 mutants associated with holoprosencephaly dampen signaling by three mechanisms: reduced affinity for Hedgehog ligand, elevated catalytic activity, or elevated affinity for the Smoothened substrate. Our results clarify the enigmatic mechanism of Patched1 and explain how Patched1 mutations lead to birth defects.

Sign in / Sign up

Export Citation Format

Share Document