scholarly journals Glycolipid-anchored proteins in neuroblastoma cells form detergent-resistant complexes without caveolin.

1995 ◽  
Vol 129 (3) ◽  
pp. 619-627 ◽  
Author(s):  
A Gorodinsky ◽  
D A Harris
Keyword(s):  
G Proteins ◽  
Tyrosine Kinases ◽  
Protein Complexes ◽  
Buoyant Density ◽  
Neuroblastoma Cells ◽  
Cell Types ◽  
Heterotrimeric G Proteins ◽  
Glycosyl Phosphatidylinositol ◽  
Src Family Tyrosine Kinases ◽  
Triton X

It has been known for a number of years that glycosyl-phosphatidylinositol (GPI)-anchored proteins, in contrast to many transmembrane proteins, are insoluble at 4 degrees C in nonionic detergents such as Triton X-100. Recently, it has been proposed that this behavior reflects the incorporation of GPI-linked proteins into large aggregates that are rich in sphingolipids and cholesterol, as well as in cytoplasmic signaling molecules such as heterotrimeric G proteins and src-family tyrosine kinases. It has been suggested that these lipid-protein complexes are derived from caveolae, non-clathrin-coated invaginations of the plasmalemma that are abundant in endothelial cells, smooth muscle, and lung. Caveolin, a proposed coat protein of caveolae, has been hypothesized to be essential for formation of the complexes. To further investigate the relationship between the detergent-resistant complexes and caveolae, we have characterized the behavior of GPI-anchored proteins in lysates of N2a neuroblastoma cells, which lack morphologically identifiable caveolae, and which do not express caveolin (Shyng, S.-L., J. E. Heuser, and D. A. Harris. 1994. J. Cell Biol. 125:1239-1250). We report here that the complexes prepared from N2a cells display the large size and low buoyant density characteristic of complexes isolated from sources that are rich in caveolae, and contain the same major constituents, including multiple GPI-anchored proteins, alpha and beta subunits of heterotrimeric G proteins, and the tyrosine kinases fyn and yes. Our results argue strongly that detergent-resistant complexes are not equivalent to caveolae in all cell types, and that in neuronal cells caveolin is not essential for the integrity of these complexes.

scholarly journals Comprehensive analysis of heterotrimeric G-protein complex diversity and their interactions with GPCRs in solution

2015 ◽  
Vol 112 (11) ◽  
pp. E1181-E1190 ◽  
Author(s):  
Matthias Hillenbrand ◽  
Christian Schori ◽  
Jendrik Schöppe ◽  
Andreas Plückthun
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Conformational Changes ◽  
Protein Complex ◽  
Protein Complexes ◽  
Heterotrimeric G Proteins ◽  
Agonist Binding ◽  
Transient Nature ◽  
The Stability ◽  
Neurotensin Receptor 1

Agonist binding to G-protein–coupled receptors (GPCRs) triggers signal transduction cascades involving heterotrimeric G proteins as key players. A major obstacle for drug design is the limited knowledge of conformational changes upon agonist binding, the details of interaction with the different G proteins, and the transmission to movements within the G protein. Although a variety of different GPCR/G protein complex structures would be needed, the transient nature of this complex and the intrinsic instability against dissociation make this endeavor very challenging. We have previously evolved GPCR mutants that display higher stability and retain their interaction with G proteins. We aimed at finding all G-protein combinations that preferentially interact with neurotensin receptor 1 (NTR1) and our stabilized mutants. We first systematically analyzed by coimmunoprecipitation the capability of 120 different G-protein combinations consisting of αi1or αsLand all possible βγ-dimers to form a heterotrimeric complex. This analysis revealed a surprisingly unrestricted ability of the G-protein subunits to form heterotrimeric complexes, including βγ-dimers previously thought to be nonexistent, except for combinations containing β5. A second screen on coupling preference of all G-protein heterotrimers to NTR1 wild type and a stabilized mutant indicated a preference for those Gαi1βγ combinations containing γ1and γ11. Heterotrimeric G proteins, including combinations believed to be nonexistent, were purified, and complexes with the GPCR were prepared. Our results shed new light on the combinatorial diversity of G proteins and their coupling to GPCRs and open new approaches to improve the stability of GPCR/G-protein complexes.

PDGF upregulates delayed rectifier via Src family kinases and sphingosine kinase in oligodendroglial progenitors

2003 ◽  
Vol 284 (1) ◽  
pp. C85-C93 ◽  
Author(s):  
Betty Soliven ◽  
Lan Ma ◽  
Hyun Bae ◽  
Bernard Attali ◽  
Alexander Sobko ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Growth Factor ◽  
Tyrosine Kinases ◽  
Cell Cycle Progression ◽  
Kinase Inhibitor ◽  
Sphingosine Kinase ◽  
Cell Types ◽  
Delayed Rectifier ◽  
Delayed Rectifier Current ◽  
Src Family Tyrosine Kinases

An increase in the expression of the delayed rectifier current ( I K) has been shown to correlate with mitogenesis in many cell types. However, pathways involved in the upregulation of I K by growth factors in oligodendroglial progenitors (OPs) have not been well-elucidated. In this study, we found that treatment with platelet-derived growth factor (PDGF) and basic fibroblast growth factor but not ciliary neurotrophic factor resulted in increased I K density and upregulation of Kv1.5 and Kv1.6 mRNA transcripts. The effect of PDGF on I K was blocked by mimosine, a cell cycle inhibitor, and by genistein, a tyrosine kinase inhibitor. Using inhibitors of PDGF-activated pathways, we found that PDGF-induced upregulation of Kv1.5 and I K density involves Src family tyrosine kinases, sphingosine kinase, and intracellular Ca2+ but not ERK1/2 or phosphatidylinositol 3-kinase pathways. Furthermore, agents that were effective inhibitors of PDGF-induced I Kupregulation also attenuated OP proliferation, supporting the concept that I K is an important link between PDGF-activated signaling cascades and cell cycle progression.

scholarly journals Two isoforms of murine hck, generated by utilization of alternative translational initiation codons, exhibit different patterns of subcellular localization.

1991 ◽  
Vol 11 (9) ◽  
pp. 4363-4370 ◽  
Author(s):  
P Lock ◽  
S Ralph ◽  
E Stanley ◽  
I Boulet ◽  
R Ramsay ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Molecular Basis ◽  
Mutational Analysis ◽  
Myeloid Cell ◽  
Cell Types ◽  
Subcellular Fractionation ◽  
Hemopoietic Cell ◽  
Translational Initiation ◽  
B Lymphoid ◽  
Triton X

Mammalian hck, a member of the src family of tyrosine kinases, is expressed predominantly in cells of the myeloid and B-lymphoid lineages. Using mutational analysis, we have investigated the molecular basis of two immunoreactive forms of murine hck of 56 and 59 kDa found in numerous hemopoietic cell types. Our results indicate that translation of murine p59hck initiates from a CTG codon located 21 codons 5' of an ATG that is utilized to generate p56hck. We provide evidence that two human hck isoforms are generated by the same mechanism. Subcellular fractionation studies reveal that while p59hck and p56hck are associated with membranes of various murine B-lymphoid and myeloid cell lines, p59hck alone is also located in the cytosol. In contrast to membrane-associated p59hck, which is metabolically labeled with [3H]myristic acid and exhibits amphiphilic properties in Triton X-114 detergent, cytosolic p59hck is hydrophilic, suggesting that it is not acylated. Possible mechanisms are proposed to account for these observations.

Use of DREADD Technology to Identify Novel Targets for Antidiabetic Drugs

2021 ◽  
Vol 61 (1) ◽  
pp. 421-440
Author(s):  
Lei Wang ◽  
Lu Zhu ◽  
Jaroslawna Meister ◽  
Derek B.J. Bone ◽  
Sai P. Pydi ◽  
...  
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Metabolic Diseases ◽  
Cell Types ◽  
Membrane Receptors ◽  
Designer Drug ◽  
Heterotrimeric G Proteins ◽  
Distinct Cell Type ◽  
The Individual

G protein–coupled receptors (GPCRs) form a superfamily of plasma membrane receptors that couple to four major families of heterotrimeric G proteins, Gs, Gi, Gq, and G12. GPCRs represent excellent targets for drug therapy. Since the individual GPCRs are expressed by many different cell types, the in vivo metabolic roles of a specific GPCR expressed by a distinct cell type are not well understood. The development of designer GPCRs known as DREADDs (designer receptors exclusively activated by a designer drug) that selectively couple to distinct classes of heterotrimeric G proteins has greatly facilitated studies in this area. This review focuses on the use of DREADD technology to explore the physiological and pathophysiological roles of distinct GPCR/G protein cascades in several metabolically important cell types. The novel insights gained from these studies should stimulate the development of GPCR-based treatments for major metabolic diseases such as type 2 diabetes and obesity.

scholarly journals Determination of the non-ionic detergent insolubility and phosphoprotein associations of glycosylphosphatidylinositol-anchored proteins expressed on T cells

1998 ◽  
Vol 334 (2) ◽  
pp. 325-333 ◽  
Author(s):  
Keith R. SOLOMON ◽  
Mark A. MALLORY ◽  
Robert W. FINBERG
Keyword(s):  
Signal Transduction ◽  
T Cells ◽  
Tyrosine Kinases ◽  
Activated T Cells ◽  
Lipid Microdomains ◽  
Signalling Molecules ◽  
Ionic Detergent ◽  
Src Family Tyrosine Kinases ◽  
Haemopoietic Cells ◽  
Triton X

Glycosylphosphatidylinositol (GPI)-anchored proteins are poorly solublized in non-ionic detergents such as Triton X-100 and Nonidet P40, but are easily solublized by detergents with high critical micelle concentrations such as octylglucoside. This solubility profile has been suggested to be due to the localization of GPI-anchored proteins to lipid microdomains rich in cholesterol and sphingolipids. Additionally, GPI-anchored proteins expressed on haemopoietic cells have been shown to associate with src-family tyrosine kinases and heterotrimeric G proteins. Despite these observations, the non-ionic detergent insolubility of GPI-anchored proteins on haemopoietic cells has not been quantified nor has a relationship between the non-ionic detergent insolubility of these proteins and their association with signal-transduction molecules been identified. Here we show that GPI-anchored proteins found on T-cell tumours and activated T cells, although significantly more insoluble then transmembrane proteins, are not uniform in their detergent insolubility. Whereas CD59 was between 4% and 13% soluble, CD48 was between 13% and 25% soluble, CD55 was between 20% and 30% soluble, and CD109 was between 34% and 75% soluble. The ability of these GPI-anchored proteins to associate with phosphoproteins was correlated with their detergent insolubility: the more detergent-insoluble that a GPI-anchored protein was, the greater the level of phosphoprotein associations. These experiments reveal a relationship between non-ionic detergent insolubility and association with signal-transduction molecules and suggest a cause-and-effect relationship between these two properties. In total, these experiments support the hypothesis that the association of GPI-anchored proteins with signalling molecules is due to their sorting to lipid microdomains.

scholarly journals Eph/Ephrin-Based Protein Complexes: The Importance of cis Interactions in Guiding Cellular Processes

2022 ◽  
Vol 8 ◽  
Author(s):  
Alessandra Cecchini ◽  
D. D. W. Cornelison
Keyword(s):  
Tyrosine Kinases ◽  
Cellular Response ◽  
Protein Complexes ◽  
Cell Types ◽  
Linear Process ◽  
Diverse Range ◽  
Ephb Receptor ◽  
Cellular Context ◽  
Associated Proteins ◽  
And Migration

Although intracellular signal transduction is generally represented as a linear process that transmits stimuli from the exterior of a cell to the interior via a transmembrane receptor, interactions with additional membrane-associated proteins are often critical to its success. These molecules play a pivotal role in mediating signaling via the formation of complexes in cis (within the same membrane) with primary effectors, particularly in the context of tumorigenesis. Such secondary effectors may act to promote successful signaling by mediating receptor-ligand binding, recruitment of molecular partners for the formation of multiprotein complexes, or differential signaling outcomes. One signaling family whose contact-mediated activity is frequently modulated by lateral interactions at the cell surface is Eph/ephrin (EphA and EphB receptor tyrosine kinases and their ligands ephrin-As and ephrin-Bs). Through heterotypic interactions in cis, these molecules can promote a diverse range of cellular activities, including some that are mutually exclusive (cell proliferation and cell differentiation, or adhesion and migration). Due to their broad expression in most tissues and their promiscuous binding within and across classes, the cellular response to Eph:ephrin interaction is highly variable between cell types and is dependent on the cellular context in which binding occurs. In this review, we will discuss interactions between molecules in cis at the cell membrane, with emphasis on their role in modulating Eph/ephrin signaling.

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.

Role of plasmalemmal caveolae in signal transduction

1998 ◽  
Vol 275 (5) ◽  
pp. L843-L851 ◽  
Author(s):  
Philip W. Shaul ◽  
Richard G. W. Anderson
Keyword(s):  
Signal Transduction ◽  
Buoyant Density ◽  
Cell Types ◽  
Mitogen Activated Protein Kinase ◽  
Multiple Components ◽  
Mitogen Activated Protein ◽  
Numerous Cell ◽  
Triton X ◽  
Oxide Synthase ◽  
G Protein Coupled

Caveolae are specialized plasmalemmal microdomains originally studied in numerous cell types for their involvement in the transcytosis of macromolecules. They are enriched in glycosphingolipids, cholesterol, sphingomyelin, and lipid-anchored membrane proteins, and they are characterized by a light buoyant density and resistance to solubilization by Triton X-100 at 4°C. Once the identification of the marker protein caveolin made it possible to purify this specialized membrane domain, it was discovered that caveolae also contain a variety of signal transduction molecules. This includes G protein-coupled receptors, G proteins and adenylyl cyclase, molecules involved in the regulation of intracellular calcium homeostasis, and their effectors including the endothelial isoform of nitric oxide synthase, multiple components of the tyrosine kinase-mitogen-activated protein kinase pathway, and numerous lipid signaling molecules. More recent work has indicated that caveolae further serve to compartmentalize, modulate, and integrate signaling events at the cell surface. This specialized plasmalemmal domain warrants direct consideration in future investigations of both normal and pathological signal transduction in pulmonary cell types.

scholarly journals Signals determining protein tyrosine kinase and glycosyl-phosphatidylinositol-anchored protein targeting to a glycolipid-enriched membrane fraction.

1994 ◽  
Vol 14 (8) ◽  
pp. 5384-5391 ◽  
Author(s):  
W Rodgers ◽  
B Crise ◽  
J K Rose
Keyword(s):  
Tyrosine Kinases ◽  
Membrane Fraction ◽  
Protein Tyrosine Kinase ◽  
Protein Targeting ◽  
Mdck Cells ◽  
Cell Types ◽  
Protein Tyrosine Kinases ◽  
Protein Tyrosine ◽  
Glycosyl Phosphatidylinositol ◽  
Common Signal

Glycosyl-phosphatidylinositol (GPI)-anchored membrane proteins and certain protein tyrosine kinases associate with a Triton X-100-insoluble, glycolipid-enriched membrane fraction in MDCK cells. Also, certain protein tyrosine kinases have been shown to associate with GPI-anchored proteins in other cell types. To characterize the interaction between GPI-anchored proteins and protein tyrosine kinases, GPI-anchored proteins were coexpressed with p56lck in HeLa cells. Both proteins were shown to target independently to the glycolipid-enriched membranes. Coimmunoprecipitation of GPI-anchored proteins and p56lck occurred only when both proteins were located in the glycolipid-enriched membranes, and gentle disruption of these membranes abolished the interaction. The GPI anchor was found to be the targeting signal for this membrane fraction in GPI-anchored proteins. Analysis of mutants indicated that p56lck was nearly quantitatively palmitoylated at Cys-5 but not palmitoylated at Cys-3. The nonpalmitoylated cysteine at position 3 was very important for association of p56lck with the membrane fraction, while palmitoylation at Cys-5 promoted only a low level of interaction. Because other src family protein tyrosine kinases that are associated with GPI-anchored proteins always contain a Cys-3, we propose that this residue, in addition to the N-terminal myristate, is part of a common signal targeting these proteins to a membrane domain that has been linked to transmembrane signaling.

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