scholarly journals Structural basis for activation of trimeric Gi proteins by multiple growth factor receptors via GIV/Girdin

2014 ◽  
Vol 25 (22) ◽  
pp. 3654-3671 ◽  
Author(s):  
Changsheng Lin ◽  
Jason Ear ◽  
Krishna Midde ◽  
Inmaculada Lopez-Sanchez ◽  
Nicolas Aznar ◽  
...  
Keyword(s):  
Growth Factor ◽  
G Proteins ◽  
Protein Interaction ◽  
Cross Talk ◽  
Tyrosine Kinases ◽  
Molecular Mechanisms ◽  
Growth Factor Receptors ◽  
Structural Basis ◽  
Nucleotide Exchange ◽  
Protein Protein Interaction

A long-standing issue in the field of signal transduction is to understand the cross-talk between receptor tyrosine kinases (RTKs) and heterotrimeric G proteins, two major and distinct signaling hubs that control eukaryotic cell behavior. Although stimulation of many RTKs leads to activation of trimeric G proteins, the molecular mechanisms behind this phenomenon remain elusive. We discovered a unifying mechanism that allows GIV/Girdin, a bona fide metastasis-related protein and a guanine-nucleotide exchange factor (GEF) for Gαi, to serve as a direct platform for multiple RTKs to activate Gαi proteins. Using a combination of homology modeling, protein–protein interaction, and kinase assays, we demonstrate that a stretch of ∼110 amino acids within GIV C-terminus displays structural plasticity that allows folding into a SH2-like domain in the presence of phosphotyrosine ligands. Using protein–protein interaction assays, we demonstrated that both SH2 and GEF domains of GIV are required for the formation of a ligand-activated ternary complex between GIV, Gαi, and growth factor receptors and for activation of Gαi after growth factor stimulation. Expression of a SH2-deficient GIV mutant (Arg 1745→Leu) that cannot bind RTKs impaired all previously demonstrated functions of GIV—Akt enhancement, actin remodeling, and cell migration. The mechanistic and structural insights gained here shed light on the long-standing questions surrounding RTK/G protein cross-talk, set a novel paradigm, and characterize a unique pharmacological target for uncoupling GIV-dependent signaling downstream of multiple oncogenic RTKs.

scholarly journals A predictive computational model reveals that GIV/girdin serves as a tunable valve for EGFR-stimulated cyclic AMP signals

2019 ◽  
Vol 30 (13) ◽  
pp. 1621-1633 ◽  
Author(s):  
Michael Getz ◽  
Lee Swanson ◽  
Debashish Sahoo ◽  
Pradipta Ghosh ◽  
Padmini Rangamani
Keyword(s):  
Growth Factor ◽  
G Protein ◽  
G Proteins ◽  
Cross Talk ◽  
Tyrosine Kinases ◽  
Control Valve ◽  
Camp Signaling ◽  
Nucleotide Exchange ◽  
Camp Concentration ◽  
Camp Levels

Cellular levels of the versatile second messenger cyclic (c)AMP are regulated by the antagonistic actions of the canonical G protein → adenylyl cyclase pathway that is initiated by G-protein–coupled receptors (GPCRs) and attenuated by phosphodiesterases (PDEs). Dysregulated cAMP signaling drives many diseases; for example, its low levels facilitate numerous sinister properties of cancer cells. Recently, an alternative paradigm for cAMP signaling has emerged in which growth factor–receptor tyrosine kinases (RTKs; e.g., EGFR) access and modulate G proteins via a cytosolic guanine-nucleotide exchange modulator (GEM), GIV/girdin; dysregulation of this pathway is frequently encountered in cancers. In this study, we present a network-based compartmental model for the paradigm of GEM-facilitated cross-talk between RTKs and G proteins and how that impacts cellular cAMP. Our model predicts that cross-talk between GIV, G αs, and G αi proteins dampens ligand-stimulated cAMP dynamics. This prediction was experimentally verified by measuring cAMP levels in cells under different conditions. We further predict that the direct proportionality of cAMP concentration as a function of receptor number and the inverse proportionality of cAMP concentration as a function of PDE concentration are both altered by GIV levels. Taking these results together, our model reveals that GIV acts as a tunable control valve that regulates cAMP flux after growth factor stimulation. For a given stimulus, when GIV levels are high, cAMP levels are low, and vice versa. In doing so, GIV modulates cAMP via mechanisms distinct from the two most often targeted classes of cAMP modulators, GPCRs and PDEs.

scholarly journals Receptor tyrosine kinases activate heterotrimeric G proteins via phosphorylation within the interdomain cleft of Gαi

2020 ◽  
Author(s):  
Nicholas A. Kalogriopoulos ◽  
Inmaculada Lopez-Sanchez ◽  
Changsheng Lin ◽  
Tony Ngo ◽  
Krishna Midde ◽  
...  
Keyword(s):  
Growth Factor ◽  
G Protein ◽  
G Proteins ◽  
Tyrosine Kinases ◽  
Molecular Mechanisms ◽  
Receptor Tyrosine Kinases ◽  
Second Messengers ◽  
Heterotrimeric G Proteins ◽  
Nucleotide Exchange ◽  
Key Steps

AbstractThe molecular mechanisms by which receptor tyrosine kinases (RTKs) and heterotrimeric G proteins, two major signaling hubs in eukaryotes, independently relay signals across the plasma membrane have been extensively characterized. How these hubs crosstalk has been a long-standing question, but answers remain elusive. Using linear-ion-trap mass spectrometry in combination with biochemical, cellular, and computational approaches, we unravel a mechanism of activation of heterotrimeric G proteins by RTKs and chart the key steps that mediate such activation. Upon growth factor stimulation, the guanine-nucleotide exchange modulator, GIV, dissociates Gαi•βγ trimers, scaffolds monomeric Gαi with RTKs, and facilitates the phosphorylation on two tyrosines located within the inter-domain cleft of Gαi. Phosphorylation triggers the activation of Gαi and inhibits second messengers (cAMP). Tumor-associated mutants reveal how constitutive activation of this pathway impacts cell’s decision to ‘go’ vs. ‘grow’. These insights define a tyrosine-based G protein signaling paradigm and reveal its importance in eukaryotes.Significance StatementGrowth factors and heterotrimeric G proteins are two of the most widely studied signaling pathways in eukaryotes; their crosstalk shapes some of the most fundamental cellular responses in both health and disease. Although mechanisms by which G protein pathways transactivate growth factor RTKs has been well-defined, how the reverse may happen is less understood. This study defines the key steps and cellular consequences of a fundamental mechanism of signal crosstalk that enables RTKs to transactivate heterotrimeric G protein, Gαi. Mutations found in tumors shed light on how derailing this mechanism impacts tumor cell behavior. Thus, findings not only show how cells integrate extracellular signals via pathway crosstalk, but also demonstrate the relevance of this pathway in cancers.

scholarly journals Receptor tyrosine kinases activate heterotrimeric G proteins via phosphorylation within the interdomain cleft of Gαi

2020 ◽  
Vol 117 (46) ◽  
pp. 28763-28774
Author(s):  
Nicholas A. Kalogriopoulos ◽  
Inmaculada Lopez-Sanchez ◽  
Changsheng Lin ◽  
Tony Ngo ◽  
Krishna K. Midde ◽  
...  
Keyword(s):  
G Proteins ◽  
Tyrosine Kinases ◽  
Molecular Mechanisms ◽  
Receptor Tyrosine Kinases ◽  
Ion Trap ◽  
Second Messengers ◽  
Protein Signaling ◽  
Heterotrimeric G Proteins ◽  
Nucleotide Exchange ◽  
Key Steps

The molecular mechanisms by which receptor tyrosine kinases (RTKs) and heterotrimeric G proteins, two major signaling hubs in eukaryotes, independently relay signals across the plasma membrane have been extensively characterized. How these hubs cross-talk has been a long-standing question, but answers remain elusive. Using linear ion-trap mass spectrometry in combination with biochemical, cellular, and computational approaches, we unravel a mechanism of activation of heterotrimeric G proteins by RTKs and chart the key steps that mediate such activation. Upon growth factor stimulation, the guanine-nucleotide exchange modulator dissociates Gαi•βγ trimers, scaffolds monomeric Gαi with RTKs, and facilitates the phosphorylation on two tyrosines located within the interdomain cleft of Gαi. Phosphorylation triggers the activation of Gαi and inhibits second messengers (cAMP). Tumor-associated mutants reveal how constitutive activation of this pathway impacts cell’s decision to “go” vs. “grow.” These insights define a tyrosine-based G protein signaling paradigm and reveal its importance in eukaryotes.

scholarly journals Targeting fibroblast growth factor receptors to combat aggressive ependymoma

2021 ◽  
Author(s):  
Daniela Lötsch ◽  
Dominik Kirchhofer ◽  
Bernhard Englinger ◽  
Li Jiang ◽  
Konstantin Okonechnikov ◽  
...  
Keyword(s):  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Molecular Mechanisms ◽  
Radial Glia ◽  
Growth Factor Receptors ◽  
Dominant Negative ◽  
Mrna Levels ◽  
Fibroblast Growth ◽  
Cell Models ◽  
Fibroblast Growth Factor Receptors

AbstractEpendymomas (EPN) are central nervous system tumors comprising both aggressive and more benign molecular subtypes. However, therapy of the high-risk subtypes posterior fossa group A (PF-A) and supratentorial RELA-fusion positive (ST-RELA) is limited to gross total resection and radiotherapy, as effective systemic treatment concepts are still lacking. We have recently described fibroblast growth factor receptors 1 and 3 (FGFR1/FGFR3) as oncogenic drivers of EPN. However, the underlying molecular mechanisms and their potential as therapeutic targets have not yet been investigated in detail. Making use of transcriptomic data across 467 EPN tissues, we found that FGFR1 and FGFR3 were both widely expressed across all molecular groups. FGFR3 mRNA levels were enriched in ST-RELA showing the highest expression among EPN as well as other brain tumors. We further identified high expression levels of fibroblast growth factor 1 and 2 (FGF1, FGF2) across all EPN subtypes while FGF9 was elevated in ST-EPN. Interrogation of our EPN single-cell RNA-sequencing data revealed that FGFR3 was further enriched in cycling and progenitor-like cell populations. Corroboratively, we found FGFR3 to be predominantly expressed in radial glia cells in both mouse embryonal and human brain datasets. Moreover, we detected alternative splicing of the FGFR1/3-IIIc variant, which is known to enhance ligand affinity and FGFR signaling. Dominant-negative interruption of FGFR1/3 activation in PF-A and ST-RELA cell models demonstrated inhibition of key oncogenic pathways leading to reduced cell growth and stem cell characteristics. To explore the feasibility of therapeutically targeting FGFR, we tested a panel of FGFR inhibitors in 12 patient-derived EPN cell models revealing sensitivity in the low-micromolar to nano-molar range. Finally, we gain the first clinical evidence for the activity of the FGFR inhibitor nintedanib in the treatment of a patient with recurrent ST-RELA. Together, these preclinical and clinical data suggest FGFR inhibition as a novel and feasible approach to combat aggressive EPN.

scholarly journals Vav Family Proteins Couple to Diverse Cell Surface Receptors

2000 ◽  
Vol 20 (17) ◽  
pp. 6364-6373 ◽  
Author(s):  
Sheri L. Moores ◽  
Laura M. Selfors ◽  
Jessica Fredericks ◽  
Timo Breit ◽  
Keiko Fujikawa ◽  
...  
Keyword(s):  
Growth Factor ◽  
Cell Surface ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Receptor Activation ◽  
Cell Surface Receptors ◽  
Nucleotide Exchange ◽  
Surface Receptors ◽  
Downstream Signaling

ABSTRACT Vav proteins are guanine nucleotide exchange factors for Rho family GTPases which activate pathways leading to actin cytoskeletal rearrangements and transcriptional alterations. Vav proteins contain several protein binding domains which can link cell surface receptors to downstream signaling proteins. Vav1 is expressed exclusively in hematopoietic cells and tyrosine phosphorylated in response to activation of multiple cell surface receptors. However, it is not known whether the recently identified isoforms Vav2 and Vav3, which are broadly expressed, can couple with similar classes of receptors, nor is it known whether all Vav isoforms possess identical functional activities. We expressed Vav1, Vav2, and Vav3 at equivalent levels to directly compare the responses of the Vav proteins to receptor activation. Although each Vav isoform was tyrosine phosphorylated upon activation of representative receptor tyrosine kinases, integrin, and lymphocyte antigen receptors, we found unique aspects of Vav protein coupling in each receptor pathway. Each Vav protein coprecipitated with activated epidermal growth factor and platelet-derived growth factor (PDGF) receptors, and multiple phosphorylated tyrosine residues on the PDGF receptor were able to mediate Vav2 tyrosine phosphorylation. Integrin-induced tyrosine phosphorylation of Vav proteins was not detected in nonhematopoietic cells unless the protein tyrosine kinase Syk was also expressed, suggesting that integrin activation of Vav proteins may be restricted to cell types that express particular tyrosine kinases. In addition, we found that Vav1, but not Vav2 or Vav3, can efficiently cooperate with T-cell receptor signaling to enhance NFAT-dependent transcription, while Vav1 and Vav3, but not Vav2, can enhance NFκB-dependent transcription. Thus, although each Vav isoform can respond to similar cell surface receptors, there are isoform-specific differences in their activation of downstream signaling pathways.

SH2 domains of the p85 alpha subunit of phosphatidylinositol 3-kinase regulate binding to growth factor receptors

1992 ◽  
Vol 12 (3) ◽  
pp. 991-997
Author(s):  
C J McGlade ◽  
C Ellis ◽  
M Reedijk ◽  
D Anderson ◽  
G Mbamalu ◽  
...  
Keyword(s):  
Growth Factor ◽  
Tyrosine Kinases ◽  
Growth Factor Receptors ◽  
Alpha Subunit ◽  
Receptor Protein ◽  
Regulatory Subunit ◽  
Pdgf Receptor ◽  
Colony Stimulating Factor ◽  
Sh2 Domains ◽  
Stimulating Factor

The binding of cytoplasmic signaling proteins such as phospholipase C-gamma 1 and Ras GTPase-activating protein to autophosphorylated growth factor receptors is directed by their noncatalytic Src homology region 2 (SH2) domains. The p85 alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase, which associates with several receptor protein-tyrosine kinases, also contains two SH2 domains. Both p85 alpha SH2 domains, when expressed individually as fusion proteins in bacteria, bound stably to the activated beta receptor for platelet-derived growth factor (PDGF). Complex formation required PDGF stimulation and was dependent on receptor tyrosine kinase activity. The bacterial p85 alpha SH2 domains recognized activated beta PDGF receptor which had been immobilized on a filter, indicating that SH2 domains contact autophosphorylated receptors directly. Several receptor tyrosine kinases within the PDGF receptor subfamily, including the colony-stimulating factor 1 receptor and the Steel factor receptor (Kit), also associate with PI 3-kinase in vivo. Bacterially expressed SH2 domains derived from the p85 alpha subunit of PI 3-kinase bound in vitro to the activated colony-stimulating factor 1 receptor and to Kit. We infer that the SH2 domains of p85 alpha bind to high-affinity sites on these receptors, whose creation is dependent on receptor autophosphorylation. The SH2 domains of p85 are therefore primarily responsible for the binding of PI 3-kinase to activated growth factor receptors.

Sign in / Sign up

Export Citation Format

Share Document