scholarly journals Cryo–electron microscopy structure and analysis of the P-Rex1–Gβγ signaling scaffold

2019 ◽  
Vol 5 (10) ◽  
pp. eaax8855 ◽  
Author(s):  
Jennifer N. Cash ◽  
Sarah Urata ◽  
Sheng Li ◽  
Sandeep K. Ravala ◽  
Larisa V. Avramova ◽  
...  
Keyword(s):  
G Protein ◽  
Neutrophil Migration ◽  
Signaling Proteins ◽  
Pdz Domains ◽  
Exchange Mass Spectrometry ◽  
Cryo Electron Microscopy ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
Carboxyl Terminal ◽  
G Protein Coupled

PIP3-dependent Rac exchanger 1 (P-Rex1) is activated downstream of G protein–coupled receptors to promote neutrophil migration and metastasis. The structure of more than half of the enzyme and its regulatory G protein binding site are unknown. Our 3.2 Å cryo-EM structure of the P-Rex1–Gβγ complex reveals that the carboxyl-terminal half of P-Rex1 adopts a complex fold most similar to those of Legionella phosphoinositide phosphatases. Although catalytically inert, the domain coalesces with a DEP domain and two PDZ domains to form an extensive docking site for Gβγ. Hydrogen-deuterium exchange mass spectrometry suggests that Gβγ binding induces allosteric changes in P-Rex1, but functional assays indicate that membrane localization is also required for full activation. Thus, a multidomain assembly is key to the regulation of P-Rex1 by Gβγ and the formation of a membrane-localized scaffold optimized for recruitment of other signaling proteins such as PKA and PTEN.

Scaffolding mechanism of arrestin-2 in the cRaf/MEK1/ERK signaling cascade

2021 ◽  
Vol 118 (37) ◽  
pp. e2026491118
Author(s):  
Changxiu Qu ◽  
Ji Young Park ◽  
Min Woo Yun ◽  
Qing-tao He ◽  
Fan Yang ◽  
...  
Keyword(s):  
Mapk Signaling ◽  
Signaling Proteins ◽  
Structural Mechanism ◽  
Mapk Kinase ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
G Protein Coupled ◽  
Binding Interfaces ◽  
Homologous Desensitization

Arrestins were initially identified for their role in homologous desensitization and internalization of G protein–coupled receptors. Receptor-bound arrestins also initiate signaling by interacting with other signaling proteins. Arrestins scaffold MAPK signaling cascades, MAPK kinase kinase (MAP3K), MAPK kinase (MAP2K), and MAPK. In particular, arrestins facilitate ERK1/2 activation by scaffolding ERK1/2 (MAPK), MEK1 (MAP2K), and Raf (MAPK3). However, the structural mechanism underlying this scaffolding remains unknown. Here, we investigated the mechanism of arrestin-2 scaffolding of cRaf, MEK1, and ERK2 using hydrogen/deuterium exchange–mass spectrometry, tryptophan-induced bimane fluorescence quenching, and NMR. We found that basal and active arrestin-2 interacted with cRaf, while only active arrestin-2 interacted with MEK1 and ERK2. The ATP binding status of MEK1 or ERK2 affected arrestin-2 binding; ATP-bound MEK1 interacted with arrestin-2, whereas only empty ERK2 bound arrestin-2. Analysis of the binding interfaces suggested that the relative positions of cRaf, MEK1, and ERK2 on arrestin-2 likely facilitate sequential phosphorylation in the signal transduction cascade.

scholarly journals Activation of phospholipase C β by Gβγ and Gαq involves C-terminal rearrangement to release auto-inhibition

2019 ◽  
Author(s):  
Isaac Fisher ◽  
Meredith Jenkins ◽  
Greg Tall ◽  
John E Burke ◽  
Alan V. Smrcka
Keyword(s):  
Phospholipase C ◽  
Inositol Phosphates ◽  
G Protein Coupled Receptors ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
Hdx Ms ◽  
G Protein Coupled ◽  
Inhibitory Interactions

AbstractPhospholipase C (PLC) enzymes hydrolyse phosphoinositide lipids to inositol phosphates and diacylglycerol. Direct activation of PLCβ by Gαq and/or Gβγ subunits mediates signalling by Gq and some Gi coupled G protein-coupled receptors (GPCRs), respectively. PLCβ isoforms contain a unique C-terminal extension, consisting of proximal and distal C-terminal domains (CTD) separated by a flexible linker. The structure of PLCβ3 bound to Gαq is known, however, for both Gαq and Gβγ, the mechanism for PLCβ activation on membranes is unknown. We examined PLCβ2 dynamics on membranes using hydrogen deuterium exchange mass spectrometry (HDX-MS). Gβγ caused a robust increase in dynamics of the distal C-terminal domain (CTD). Gαq showed decreased deuterium incorporation at the Gαq binding site on PLCβ. In vitro Gβγ-dependent activation of PLC is inhibited by the distal CTD. The results suggest that disruption of auto-inhibitory interactions with the CTD, respectively, leads to increased PLCβ hydrolase activity.

Different conformational dynamics of PDZ1 and PDZ2 in full-length EBP50 analyzed by hydrogen/deuterium exchange mass spectrometry

2015 ◽  
Vol 93 (4) ◽  
pp. 290-297 ◽  
Author(s):  
Ji Young Park ◽  
Nguyen Minh Duc ◽  
Dong Kyun Kim ◽  
Su Youn Lee ◽  
Sheng Li ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Conformational Dynamics ◽  
Deuterium Exchange ◽  
Full Length ◽  
Hydrogen Deuterium Exchange ◽  
Pdz Domains ◽  
Ligand Selectivity ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry

Ezrin-radixin-moesin-binding protein 50 (EBP50) is a scaffolding protein expressed in polarized epithelial cells in various organs, including the liver, kidney, and small intestine, in which it regulates the trafficking and targeting cellular proteins. EBP50 contains two postsynaptic density-95/disk-large/ZO-1 homology (PDZ) domains (e.g., PDZ1 and PDZ2) and an ezrin/radixin/moesin-binding (EB) domain. PDZ domains are one of the major scaffolding domains regulating protein–protein interactions with critical biological roles in cell polarity, migration, proliferation, recognition, and cell–cell interaction. PDZ1 and PDZ2 in EBP50 have different ligand selectivity, although several high-resolution structural studies of isolated PDZ1 and PDZ2 showed similar structures. We studied the conformations of full-length EBP50 and isolated PDZ1 and PDZ2 using hydrogen/deuterium exchange mass spectrometry (HDX-MS). The deuterium uptake profiles of isolated PDZ1 and PDZ2 were similar to those of full-length EBP50. Interestingly, PDZ1 was more dynamic than PDZ2, and these PDZ domains underwent different conformational changes upon ligand binding. These results might explain the differences in ligand-selectivity between PDZ1 and PDZ2.

scholarly journals The G Protein-Coupled Receptor Kinases (GRKs) in Chemokine Receptor-Mediated Immune Cell Migration: From Molecular Cues to Physiopathology

Cells ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 75
Author(s):  
Marta Laganà ◽  
Géraldine Schlecht-Louf ◽  
Françoise Bachelerie
Keyword(s):  
G Protein ◽  
Chemokine Receptor ◽  
Immune Cell ◽  
Neutrophil Migration ◽  
G Protein Coupled Receptor ◽  
Receptor Kinases ◽  
Immune Cell Migration ◽  
And Migration ◽  
G Protein Coupled ◽  
Gpcr Desensitization

Although G protein-coupled receptor kinases (GRKs) have long been known to regulate G protein-coupled receptor (GPCR) desensitization, their more recently characterized functions as scaffolds and signalling adapters underscore that this small family of proteins governs a larger array of physiological functions than originally suspected. This review explores how GRKs contribute to the complex signalling networks involved in the migration of immune cells along chemokine gradients sensed by cell surface GPCRs. We outline emerging evidence indicating that the coordinated docking of several GRKs on an active chemokine receptor determines a specific receptor phosphorylation barcode that will translate into distinct signalling and migration outcomes. The guidance cues for neutrophil migration are emphasized based on several alterations affecting GRKs or GPCRs reported to be involved in pathological conditions.

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