scholarly journals Growth factor signaling to mTORC1 by amino acid–laden macropinosomes

2015 ◽  
Vol 211 (1) ◽  
pp. 159-172 ◽  
Author(s):  
Sei Yoshida ◽  
Regina Pacitto ◽  
Yao Yao ◽  
Ken Inoki ◽  
Joel A. Swanson
Keyword(s):  
Amino Acids ◽  
Growth Factors ◽  
Amino Acid ◽  
Growth Factor ◽  
Phorbol Esters ◽  
Growth Factor Receptor ◽  
Fluid Phase ◽  
Growth Factor Signaling ◽  
Murine Bone Marrow ◽  
Rapid Activation

The rapid activation of the mechanistic target of rapamycin complex-1 (mTORC1) by growth factors is increased by extracellular amino acids through yet-undefined mechanisms of amino acid transfer into endolysosomes. Because the endocytic process of macropinocytosis concentrates extracellular solutes into endolysosomes and is increased in cells stimulated by growth factors or tumor-promoting phorbol esters, we analyzed its role in amino acid–dependent activation of mTORC1. Here, we show that growth factor-dependent activation of mTORC1 by amino acids, but not glucose, requires macropinocytosis. In murine bone marrow–derived macrophages and murine embryonic fibroblasts stimulated with their cognate growth factors or with phorbol myristate acetate, activation of mTORC1 required an Akt-independent vesicular pathway of amino acid delivery into endolysosomes, mediated by the actin cytoskeleton. Macropinocytosis delivered small, fluorescent fluid-phase solutes into endolysosomes sufficiently fast to explain growth factor–mediated signaling by amino acids. Therefore, the amino acid–laden macropinosome is an essential and discrete unit of growth factor receptor signaling to mTORC1.

The structure and function of the epidermal growth factor receptor studied by using antisynthetic peptide antibodies

1985 ◽  
Vol 226 (1242) ◽  
pp. 127-134 ◽  
Keyword(s):  
Amino Acids ◽  
Epidermal Growth Factor Receptor ◽  
Amino Acid ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Amino Acid Sequence ◽  
Egf Receptor ◽  
Growth Factor Receptor ◽  
Binding Domain ◽  
Epidermal Growth

The human epidermal growth factor receptor has been purified and partial amino acid sequence obtained. A synthetic oligonucleotide was used to select complementary DNA clones from placental and A431 clone banks. The nucleotide sequence of a 5.8 kilobase transcript was determined and used to predict the total amino acid sequence of the receptor. We have predicted a model for the receptor which has an external ligand binding domain of 621 amino acids, a transmembrane region of 23 amino acids, and a cytoplasmic domain of 542 amino acids having protein tyrosine kinase activity. The kinase autophosphorylation sites have been mapped onto the primary amino acid sequence. Analysis of protein sequence databases have shown that the erb -B oncogene of avian erythroblastosis virus has acquired part of the avian EGF receptor gene. The hypothesis has been proposed that transformation by this virus is the result of expression of a truncated EGF receptor which lacks the majority of the EGF binding domain and delivers a continuous proliferation signal to transformed cells. We describe here the production of polyclonal and monoclonal antibodies to selected synthetic peptides from the EGF receptor and v-erb B sequences. Antisera to sequences encompassing the three major sites of autophosphorylation and the putative ATP binding site all recognize the native EGF receptor molecule. We have used these reagents to test our model of EGF receptor structure and v-erb B function.

scholarly journals Transforming growth factor alpha: mutation of aspartic acid 47 and leucine 48 results in different biological activities.

1988 ◽  
Vol 8 (3) ◽  
pp. 1247-1252 ◽  
Author(s):  
E Lazar ◽  
S Watanabe ◽  
S Dalton ◽  
M B Sporn
Keyword(s):  
Amino Acids ◽  
Biological Activity ◽  
Amino Acid ◽  
Growth Factor ◽  
Aspartic Acid ◽  
Transforming Growth Factor ◽  
Biologically Active ◽  
Single Amino Acid ◽  
Transforming Growth Factor Alpha ◽  
Factor Alpha

To study the relationship between the primary structure of transforming growth factor alpha (TGF-alpha) and some of its functional properties (competition with epidermal growth factor (EGF) for binding to the EGF receptor and induction of anchorage-independent growth), we introduced single amino acid mutations into the sequence for the fully processed, 50-amino-acid human TGF-alpha. The wild-type and mutant proteins were expressed in a vector by using a yeast alpha mating pheromone promoter. Mutations of two amino acids that are conserved in the family of the EGF-like peptides and are located in the carboxy-terminal part of TGF-alpha resulted in different biological effects. When aspartic acid 47 was mutated to alanine or asparagine, biological activity was retained; in contrast, substitutions of this residue with serine or glutamic acid generated mutants with reduced binding and colony-forming capacities. When leucine 48 was mutated to alanine, a complete loss of binding and colony-forming abilities resulted; mutation of leucine 48 to isoleucine or methionine resulted in very low activities. Our data suggest that these two adjacent conserved amino acids in positions 47 and 48 play different roles in defining the structure and/or biological activity of TGF-alpha and that the carboxy terminus of TGF-alpha is involved in interactions with cellular TGF-alpha receptors. The side chain of leucine 48 appears to be crucial either indirectly in determining the biologically active conformation of TGF-alpha or directly in the molecular recognition of TGF-alpha by its receptor.

scholarly journals Integrins can collaborate with growth factors for phosphorylation of receptor tyrosine kinases and MAP kinase activation: roles of integrin aggregation and occupancy of receptors.

1996 ◽  
Vol 135 (6) ◽  
pp. 1633-1642 ◽  
Author(s):  
S Miyamoto ◽  
H Teramoto ◽  
J S Gutkind ◽  
K M Yamada
Keyword(s):  
Signal Transduction ◽  
Growth Factors ◽  
Growth Factor ◽  
Map Kinase ◽  
Tyrosine Kinases ◽  
Map Kinases ◽  
Egf Receptor ◽  
Growth Factor Receptor ◽  
Kinase Activation ◽  
Transient Activation

Integrins mediate cell adhesion, migration, and a variety of signal transduction events. These integrin actions can overlap or even synergize with those of growth factors. We examined for mechanisms of collaboration or synergy between integrins and growth factors involving MAP kinases, which regulate many cellular functions. In cooperation with integrins, the growth factors EGF, PDGF-BB, and basic FGF each produced a marked, transient activation of the ERK (extracellular signal-regulated kinase) class of MAP kinase, but only if the integrins were both aggregated and occupied by ligand. Transmembrane accumulation of total tyrosine-phosphorylated proteins, as well as nonsynergistic MAP kinase activation, could be induced by simple integrin aggregation, whereas enhanced transient accumulation of the EGF-receptor substrate eps8 required integrin aggregation and occupancy, as well as EGF treatment. Each type of growth factor receptor was itself induced to aggregate transiently by integrin ligand-coated beads in a process requiring both aggregation and occupancy of integrin receptors, but not the presence of growth factor ligand. Synergism was also observed between integrins and growth factors for triggering tyrosine phosphorylation of EGF, PDGF, and FGF receptors. This collaborative response also required both integrin aggregation and occupancy. These studies identify mechanisms in the signal transduction response to integrins and growth factors that require various combinations of integrin aggregation and ligands for integrin or growth factor receptors, providing opportunities for collaboration between these major regulatory systems.

scholarly journals Gene expression of growth factor BMP15, GDF9, FGF2 and their receptors in bovine follicular cells

2018 ◽  
pp. 6778-6787 ◽  
Author(s):  
Pablo S Reineri ◽  
María S. Coria ◽  
María G. Barrionuevo ◽  
Olegario Hernández ◽  
Santiago Callejas ◽  
...  
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Growth Factor Receptor ◽  
Follicular Development ◽  
Fibroblast Growth ◽  
Rt Pcr ◽  
Follicular Cells

Introduction. Growth and follicular maturation involve transformations of various components of the follicle, such as the oocyte, granulosa and techa cells. Several growth factors, including differentiation growth factor 9 (GDF9), bone morphogenic protein 15 (BMP15) and basic fibroblast growth factor (FGF2) are important for follicular development and oocyte maturation, by its ability to increase the proliferation of granulosa, techa cells and the ovarian stroma. Objetive. Evaluate mRNA expression of GDF9, BMP15, FGF2 and their main receptors, transforming growth factor beta receptor 1 (TGFβ-R1), bone morphogenetic protein receptor, type IB (BMPR-IB) and fibroblast growth factor receptor 2 (FGFR2) in bovine follicular cells. Materials and methods. Total RNA was isolated from pooled samples of oocytes (OOs), cumulus cells (CCs) of cumulus oocyte complexes (COCs) and follicular cell pellets (PCs) of 70 ovaries obtained from 96 beef heifers, collected at a local abattoir. The expression pattern of growth factors and their receptors in follicular bovine cells was evaluated by reverse transcriptase polymerase chain reaction (RT-PCR). Results. The mRNA transcripts encoding GDF9, BMP15, FGF2, TGFβ-R1, BMPR-IB and FGFR2 genes were detected, by RT-PCR, in all studied cells. This is the first time that the expression of TGFβ-R1 and BMPR-IB receptors is reported in bovine oocytes. Conclusions. The presence of growth factors and receptor transcripts in the studied cells indicate that these factors could act as paracrine and autocrine regulators of folliculogenesis.

scholarly journals Multiple Transmembrane Amino Acid Requirements Suggest a Highly Specific Interaction between the Bovine Papillomavirus E5 Oncoprotein and the Platelet-Derived Growth Factor Beta Receptor

2002 ◽  
Vol 76 (16) ◽  
pp. 7976-7986 ◽  
Author(s):  
Valerie M. Nappi ◽  
Lisa M. Petti
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Growth Factor ◽  
Transmembrane Domain ◽  
Specific Interaction ◽  
Platelet Derived Growth Factor ◽  
Helical Conformation ◽  
Bovine Papillomavirus ◽  
E5 Protein ◽  
Amino Acid Requirements

ABSTRACT The bovine papillomavirus E5 protein activates the cellular platelet-derived growth factor β receptor (PDGFβR) tyrosine kinase in a ligand-independent manner. Evidence suggests that the small transmembrane E5 protein homodimerizes and physically interacts with the transmembrane domain of the PDGFβR, thereby inducing constitutive dimerization and activation of this receptor. Amino acids in the receptor previously found to be required for the PDGFβR-E5 interaction are a transmembrane Thr513 and a juxtamembrane Lys499. Here, we sought to determine if these are the only two receptor amino acids required for an interaction with the E5 protein. Substitution of large portions of the PDGFβR transmembrane domain indicated that additional amino acids in both the amino and carboxyl halves of the receptor transmembrane domain are required for a productive interaction with the E5 protein. Indeed, individual amino acid substitutions in the receptor transmembrane domain identified roles for the extracellular proximal transmembrane residues in the interaction. These data suggest that multiple amino acids within the transmembrane domain of the PDGFβR are required for a stable interaction with the E5 protein. These may be involved in direct protein-protein contacts or may support the proper transmembrane alpha-helical conformation for optimal positioning of the primary amino acid requirements.

Transforming Growth Factor α and Epidermal Growth Factor Receptor in Reactive and Malignant Mesothelial Proliferations

2004 ◽  
Vol 128 (1) ◽  
pp. 68-70
Author(s):  
Yun-Cai Cai ◽  
Victor Roggli ◽  
Eugene Mark ◽  
Philip T. Cagle ◽  
Armando E. Fraire
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factors ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Transforming Growth Factor ◽  
Malignant Tumors ◽  
Growth Factor Receptor ◽  
Transforming Growth Factor Α ◽  
Epidermal Growth ◽  
Factor Α

Abstract Background.—Growth factors such as transforming growth factor α (TGF-α) and epidermal growth factor receptor (EGFR) play an important role in cell proliferation. The immunohistochemical expression of these factors has been extensively studied in malignant tumors including mesothelioma. However, the comparative expression of these growth factors in mesothelioma and reactive mesothelial proliferations has been less well studied. Objective.—To evaluate the possible role of TGF-α and EGFR in the clinically important distinction between reactive mesothelial proliferations and malignant mesothelioma. Methods.—The expression of TGF-α and EGFR was studied in 39 cases of mesothelioma and 30 cases of reactive mesothelial proliferations by means of immunohistochemistry. Results.—Fourteen (70%) of 20 reactive mesothelial proliferations tested and 29 (76%) of 38 mesotheliomas tested expressed TGF-α. One (3%) of 30 reactive mesothelial proliferations and 17 (45%) of 39 mesotheliomas expressed EGFR. Conclusions.—These results suggest an up-regulation of EGFR in mesothelioma as compared with reactive mesothelial proliferations. This up-regulation further suggests a possible use of EGFR as an adjunct immunohistochemical test in the differential diagnosis of mesothelioma and reactive mesothelial proliferations.

Pip92: a short-lived, growth factor-inducible protein in BALB/c 3T3 and PC12 cells

1990 ◽  
Vol 10 (12) ◽  
pp. 6769-6774
Author(s):  
C H Charles ◽  
J S Simske ◽  
T P O'Brien ◽  
L F Lau
Keyword(s):  
Amino Acids ◽  
Growth Factors ◽  
Growth Factor ◽  
Neuronal Differentiation ◽  
Cellular Responses ◽  
Early Gene ◽  
Extracellular Signals ◽  
Pheochromocytoma Cell ◽  
Inducible Protein ◽  
Proline Rich Protein

pip92 is a cellular immediate-early gene inducible by serum growth factors in fibroblasts. It is also induced in the rat pheochromocytoma cell line PC12 by agents that cause proliferation, neuronal differentiation, and membrane depolarization. We show that the pip92-encoded polypeptide is a proline-rich protein of 221 amino acids, has an extremely short half-life, and is localized in the cytoplasm. We hypothesize that Pip92 plays a role in mediating the cellular responses to a variety of extracellular signals.

A tyrosine-phosphorylated carboxy-terminal peptide of the fibroblast growth factor receptor (Flg) is a binding site for the SH2 domain of phospholipase C-gamma 1

1991 ◽  
Vol 11 (10) ◽  
pp. 5068-5078
Author(s):  
M Mohammadi ◽  
A M Honegger ◽  
D Rotin ◽  
R Fischer ◽  
F Bellot ◽  
...  
Keyword(s):  
Amino Acid ◽  
Growth Factor ◽  
Binding Site ◽  
Fibroblast Growth Factor ◽  
Tryptic Peptide ◽  
Fibroblast Growth Factor Receptor ◽  
Growth Factor Receptor ◽  
Sh2 Domain ◽  
Fibroblast Growth ◽  
Wild Type

Phospholipase C-gamma (PLC-gamma) is a substrate of the fibroblast growth factor receptor (FGFR; encoded by the flg gene) and other receptors with tyrosine kinase activity. It has been demonstrated that the src homology region 2 (SH2 domain) of PLC-gamma and of other signalling molecules such as GTPase-activating protein and phosphatidylinositol 3-kinase-associated p85 direct their binding toward tyrosine-autophosphorylated regions of the epidermal growth factor or platelet-derived growth factor receptor. In this report, we describe the identification of Tyr-766 as an autophosphorylation site of flg-encoded FGFR by direct sequencing of a tyrosine-phosphorylated tryptic peptide isolated from the cytoplasmic domain of FGFR expressed in Escherichia coli. The same phosphopeptide was found in wild-type FGFR phosphorylated either in vitro or in living cells. Like other growth factor receptors, tyrosine-phosphorylated wild-type FGFR or its cytoplasmic domain becomes associated with intact PLC-gamma or with a fusion protein containing the SH2 domain of PLC-gamma. To delineate the site of association, we have examined the capacity of a 28-amino-acid tryptic peptide containing phosphorylated Tyr-766 to bind to various constructs containing SH2 and other domains of PLC-gamma. It is demonstrated that the tyrosine-phosphorylated peptide binds specifically to the SH2 domain but not to the SH3 domain or other regions of PLC-gamma. Hence, Tyr-766 and its flanking sequences represent a major binding site in FGFR for PLC-gamma. Alignment of the amino acid sequences surrounding Tyr-766 with corresponding regions of other FGFRs revealed conserved tyrosine residues in all known members of the FGFR family. We propose that homologous tyrosine-phosphorylated regions in other FGFRs also function as binding sites for PLC-gamma and therefore are involved in coupling to phosphatidylinositol breakdown.

scholarly journals Blockade of Platelet-Derived Growth Factor Signaling Inhibits Choroidal Neovascularization and Subretinal Fibrosis in Mice

2020 ◽  
Vol 9 (7) ◽  
pp. 2242
Author(s):  
Ye Liu ◽  
Kousuke Noda ◽  
Miyuki Murata ◽  
Di Wu ◽  
Atsuhiro Kanda ◽  
...  
Keyword(s):  
Growth Factor ◽  
Choroidal Neovascularization ◽  
Vision Loss ◽  
Growth Factor Receptor ◽  
Neutralizing Antibody ◽  
Age Related Macular Degeneration ◽  
Platelet Derived Growth Factor ◽  
Growth Factor Signaling ◽  
Age Related ◽  
Subretinal Fibrosis

Neovascular age related macular degeneration (nAMD) leads to severe vision loss worldwide and is characterized by the formation of choroidal neovascularization (CNV) and fibrosis. In the current study, we aimed to investigate the effect of blockade for platelet derived growth factor receptor-β (PDGFR-β) on the formation of choroidal neovascularization and fibrosis in the laser-induced CNV model in mice. Firstly, the presence of PDGFR-β in CNV lesions were confirmed. Intravitreal injection of PDGFR-β neutralizing antibody significantly reduced the size of CNV and subretinal fibrosis. Additionally, subretinal hyperreflective material (SHRM), a landmark feature on OCT as a risk factor for subretinal fibrosis formation in nAMD patients was also suppressed by PDGFR-β blockade. Furthermore, pericytes were abundantly recruited to the CNV lesions during CNV formation, however, blockade of PDGFR-β significantly reduced pericyte recruitment. In addition, PDGF-BB stimulation increased the migration of the rat retinal pericyte cell line, R-rPCT1, which was abrogated by the neutralization of PDGFR-β. These results indicate that blockade of PDGFR-β attenuates laser-induced CNV and fibrosis through the inhibition of pericyte migration.

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