scholarly journals Constitutive Activating and Inactivating Mutants of Eel Luteinizing Hormone Receptor

2020 ◽  
Author(s):  
Munkhzaya Byambaragchaa ◽  
Dong-An Kim ◽  
Dae-Jung Kim ◽  
Sun-Mee Hong ◽  
Myung-Hwa Kang ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Luteinizing Hormone ◽  
Cell Surface ◽  
Hormone Receptor ◽  
Luteinizing Hormone Receptor ◽  
Cell Surface Receptors ◽  
Wild Type ◽  
Surface Receptors ◽  
Agonist Stimulation ◽  
In Cells

We analyzed signal transduction of three constitutively activating mutants (M410T, L469R, and D590Y) and two inactivating mutants (D417N and Y558F) of the eel luteinizing hormone receptor (eel LHR), known to occur in human LHR. The objective of this study was to assess the functional effects of these mutations in signal transduction and cell surface loss of receptor. Mutant receptors were transiently expressed in Chinese hamster ovary (CHO-K1) cells. Eel LH-stimulated accumulation of cyclic adenosine monophosphate (cAMP) was measured by homogeneous time-resolved fluorescence (HTRF) assays. The loss of receptors from the cells surface was measured using an enzyme-linked immunosorbent assay (ELISA) in human embryonic kidney (HEK) 293 cells. The cAMP response in cells expressing the wild type eel LHR was increased in a dose-dependent manner using eel LH ligand stimulation. Compared with the wild type, cells expressing the activating mutants (M410T, L469R, and D590Y), exhibited a 4.0-, 19.1-, and 7.8-fold increase in basal cAMP response without agonist stimulation, respectively. Their maximal responses to agonist stimulation were approximately 65%, 52%, and 98%, respectively, of those of the wild type. The inactivating mutants (D417N and Y558F) did not completely impair signal transduction, and their maximal responses were only 33% and25 % of those of wild type. These data clearly showed that the eel LHR-L469R and D590Y, activating mutants enhanced the rate of the loss of cell surface receptors following treatment with eel LH. Thus, the loss of cell surface receptors in cells expressing mutant eel LHRs was consistent with the eel LH agonist-induced production of cAMP. Our results suggested that the activation of the eel LHR requires appropriate loss of LHR-ligand complexes from the cell surface.

scholarly journals Cyto-friendly polymerization at cell surfaces modulates cell fate by clustering cell-surface receptors

2020 ◽  
Vol 11 (16) ◽  
pp. 4221-4225 ◽  
Author(s):  
Jing Qi ◽  
Weishuo Li ◽  
Xiaoling Xu ◽  
Feiyang Jin ◽  
Di Liu ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Fate ◽  
Cell Surface Receptors ◽  
Cell Surfaces ◽  
Receptor Clustering ◽  
Surface Polymerization ◽  
Surface Receptors ◽  
Anti Cd20 ◽  
In Cells

Cell-surface polymerization of anti-CD20 aptamer modified macromer to induce CD20 receptor clustering, and effectively initiate the apoptotic signals in cells.

Intracellular signalling

2010 ◽  
pp. 169-176
Author(s):  
R. Andres Floto
Keyword(s):  
Signal Transduction ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Intracellular Signalling ◽  
Cell Surface Receptors ◽  
Surface Receptors ◽  
Transmission Of Information ◽  
Effective Transmission

This section outlines the general principles of intracellular signalling. Focusing on cell surface receptors, the requirements for effective transmission of information across the plasma membrane are outlined. The principal mechanisms utilized in mammalian signal transduction are described. For each, the pathological consequences of aberrant signalling and means by which pathways can be pharmacologically targeted are described in molecular terms....

scholarly journals Redirecting Retroviral Tropism by Insertion of Short, Nondisruptive Peptide Ligands into Envelope

2002 ◽  
Vol 76 (7) ◽  
pp. 3558-3563 ◽  
Author(s):  
Timothy J. Gollan ◽  
Michael R. Green
Keyword(s):  
Cell Surface ◽  
Leukemia Virus ◽  
Envelope Proteins ◽  
Peptide Ligands ◽  
Viral Envelope ◽  
Moloney Murine Leukemia Virus ◽  
Cell Surface Receptors ◽  
Wild Type ◽  
Surface Receptors

ABSTRACT A potentially powerful approach for in vivo gene delivery is to target retrovirus to specific cells through interactions between cell surface receptors and appropriately modified viral envelope proteins. Previously, relatively large (>100 residues) protein ligands to cell surface receptors have been inserted at or near the N terminus of retroviral envelope proteins. Although viral tropism could be altered, the chimeric envelope proteins lacked full activity, and coexpression of wild-type envelope was required for production of transducing virus. Here we analyze more than 40 derivatives of ecotropic Moloney murine leukemia virus (MLV) envelope, containing insertions of short RGD-containing peptides, which are ligands for integrin receptors. In many cases pseudotyped viruses containing only the chimeric envelope protein could transduce human cells. The precise location, size, and flanking sequences of the ligand affected transduction specificity and efficiency. We conclude that retroviral tropism can be rationally reengineered by insertion of short peptide ligands and without the need to coexpress wild-type envelope.

Rab11 regulates the recycling of the β2-adrenergic receptor through a direct interaction

2009 ◽  
Vol 418 (1) ◽  
pp. 163-172 ◽  
Author(s):  
Audrey Parent ◽  
Emilie Hamelin ◽  
Pascale Germain ◽  
Jean-Luc Parent
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Glutathione Transferase ◽  
Direct Interaction ◽  
Small Gtpase ◽  
Cell Surface Receptors ◽  
Wild Type ◽  
Surface Receptors ◽  
Early Endosomes ◽  
Intracellular Domains

The β2ARs (β2-adrenergic receptors) undergo ligand-induced internalization into early endosomes, but then are rapidly and efficiently recycled back to the plasma membrane, restoring the numbers of functional cell-surface receptors. Gathering evidence suggests that, during prolonged exposure to agonist, some β2ARs also utilize a slow recycling pathway through the perinuclear recycling endosomal compartment regulated by the small GTPase Rab11. In the present study, we demonstrate by co-immunoprecipitation studies that there is a β2AR–Rab11 association in HEK-293 cells (human embryonic kidney cells). We show using purified His6-tagged Rab11 protein and β2AR intracellular domains fused to GST (glutathione transferase) that Rab11 interacts directly with the C-terminal tail of β2AR, but not with the other intracellular domains of the receptor. Pull-down and immunoprecipitation assays revealed that the β2AR interacts preferentially with the GDP-bound form of Rab11. Arg333 and Lys348 in the C-terminal tail of the β2AR were identified as crucial determinants for Rab11 binding. A β2AR construct with these two residues mutated to alanine, β2AR RK/AA (R333A/K348A), was generated. Analysis of cell-surface receptors by ELISA revealed that the recycling of β2AR RK/AA was drastically reduced when compared with wild-type β2AR after agonist washout, following prolonged receptor stimulation. Confocal microscopy demonstrated that the β2AR RK/AA mutant failed to co-localize with Rab11 and recycle to the plasma membrane, in contrast with the wild-type receptor. To our knowledge, the present study is the first report of a direct interaction between the β2AR and a Rab GTPase, which is required for the accurate intracellular trafficking of the receptor.

scholarly journals Constitutive Activation and Inactivation of Mutations Inducing Cell Surface Loss of Receptor and Impairing of Signal Transduction of Agonist-Stimulated Eel Follicle-Stimulating Hormone Receptor

2020 ◽  
Vol 21 (19) ◽  
pp. 7075
Author(s):  
Munkhzaya Byambaragchaa ◽  
Jeong-Soo Kim ◽  
Hong-Kyu Park ◽  
Dae-Jung Kim ◽  
Sun-Mee Hong ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Cell Surface ◽  
Hormone Receptor ◽  
Follicle Stimulating Hormone ◽  
Chinese Hamster ◽  
Cell Surface Receptor ◽  
Enzyme Linked Immunosorbent Assay ◽  
Wild Type ◽  
Surface Receptor ◽  
Stimulating Hormone

In the present study, we investigated the signal transduction of mutants of the eel follicle-stimulating hormone receptor (eelFSHR). Specifically, we examined the constitutively activating mutant D540G in the third intracellular loop, and four inactivating mutants (A193V, N195I, R546C, and A548V). To directly assess functional effects, we conducted site-directed mutagenesis to generate mutant receptors. We measured cyclic adenosine monophosphate (cAMP) accumulation via homogeneous time-resolved fluorescence assays in Chinese hamster ovary (CHO-K1) cells and investigated cell surface receptor loss using an enzyme-linked immunosorbent assay in human embryonic kidney (HEK) 293 cells. The cells expressing eelFSHR-D540G exhibited a 23-fold increase in the basal cAMP response without agonist treatment. The cells expressing A193V, N195I, and A548V mutants had completely impaired signal transduction, whereas those expressing the R546C mutant exhibited little increase in cAMP responsiveness and a small increase in signal transduction. Cell surface receptor loss in the cells expressing inactivating mutants A193V, R546C, and A548V was clearly slower than in the cell expressing the wild-type eelFSHR. However, cell surface receptor loss in the cells expressing inactivating mutant N195I decreased in a similar manner to that of the cells expressing the wild-type eelFSHR or the activating mutant D540G, despite the completely impaired cAMP response. These results provide important information regarding the structure–function relationships of G protein-coupled receptors during signal transduction.

scholarly journals Cell Surface Receptors for Signal Transduction and Ligand Transport: A Design Principles Study

2007 ◽  
Vol 3 (6) ◽  
pp. e101 ◽  
Author(s):  
Harish Shankaran ◽  
Haluk Resat ◽  
H. Steven Wiley
Keyword(s):  
Signal Transduction ◽  
Cell Surface ◽  
Design Principles ◽  
Cell Surface Receptors ◽  
Surface Receptors ◽  
Ligand Transport
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