scholarly journals Domain deletion in the extracellular portion of the EGF-receptor reduces ligand binding and impairs cell surface expression.

1990 ◽  
Vol 1 (2) ◽  
pp. 173-188 ◽  
Author(s):  
I Lax ◽  
F Bellot ◽  
A M Honegger ◽  
A Schmidt ◽  
A Ullrich ◽  
...  
Keyword(s):  
Cell Surface ◽  
Ligand Binding ◽  
Protein Tyrosine Kinase ◽  
Egf Receptor ◽  
Surface Display ◽  
Growth Factor Receptor ◽  
Cell Surface Expression ◽  
Receptor Interaction ◽  
Amino Terminal ◽  
Mutant Receptors

Cultured NIH-3T3 cells were transfected with cDNA constructs encoding human epidermal growth factor-receptor (EGF-R)* and two deletion mutants in the extracellular portion of the receptor molecule. One mutant is devoid of 124 amino-terminal amino acids, and the other lacks 76 residues. Mutant receptors were not delivered to the cell surface unless the transfected cells contained also endogenous EGF-Rs, suggesting that receptor interaction complements the mutation and allows surface display of mutant receptors. Immunoprecipitation experiments revealed an association between mutant and endogenous EGF-Rs when both proteins were expressed in the same cell. Hence, receptor-oligomers may exist in the plane of the membrane even in the absence of ligand binding, and oligomerization may play a role in normal trafficking of EGF-Rs to the cell surface. Mutant receptors retained partial ligand binding activity as 125I-labeled EGF was covalently cross-linked to both mutant receptors, and EGF stimulated, albeit weakly, their protein tyrosine kinase activity. Both mutant EGF-Rs bind EGF with a 10-fold lower affinity than that of the solubilized wild type EGF-R. These results provide further evidence that the region flanked by the two cysteine-rich domains plays a crucial role in defining ligand-binding specificity of EGF-R.

scholarly journals EHD1 and RUSC2 Control Basal Epidermal Growth Factor Receptor Cell Surface Expression and Recycling

2020 ◽  
Vol 40 (7) ◽  
Author(s):  
Eric C. Tom ◽  
Insha Mushtaq ◽  
Bhopal C. Mohapatra ◽  
Haitao Luan ◽  
Aaqib M. Bhat ◽  
...  
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cell Surface ◽  
Surface Display ◽  
Growth Factor Receptor ◽  
Cell Surface Display ◽  
Cell Surface Expression ◽  
Golgi Compartment ◽  
Epidermal Growth

ABSTRACT Epidermal growth factor receptor (EGFR) is a prototype receptor tyrosine kinase and an oncoprotein in many solid tumors. Cell surface display of EGFR is essential for cellular responses to its ligands. While postactivation endocytic trafficking of EGFR has been well elucidated, little is known about mechanisms of basal/preactivation surface display of EGFR. Here, we identify a novel role of the endocytic regulator EHD1 and a potential EHD1 partner, RUSC2, in cell surface display of EGFR. EHD1 and RUSC2 colocalize with EGFR in vesicular/tubular structures and at the Golgi compartment. Inducible EHD1 knockdown reduced the cell surface EGFR expression with accumulation at the Golgi compartment, a phenotype rescued by exogenous EHD1. RUSC2 knockdown phenocopied the EHD1 depletion effects. EHD1 or RUSC2 depletion impaired the EGF-induced cell proliferation, demonstrating that the novel, EHD1- and RUSC2-dependent transport of unstimulated EGFR from the Golgi compartment to the cell surface that we describe is functionally important, with implications for physiologic and oncogenic roles of EGFR and targeted cancer therapies.

scholarly journals Analysis of the Role of N-Linked Glycosylation in Cell Surface Expression, Function, and Binding Properties of SARS-CoV-2 Receptor ACE2

2021 ◽  
Author(s):  
Raymond Rowland ◽  
Alberto Brandariz-Nuñez
Keyword(s):  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Receptor Interaction ◽  
Minimal Effect ◽  
Binding Properties ◽  
Virus Receptor

Understanding the role of glycosylation in the virus-receptor interaction is important for developing approaches that disrupt infection. In this study, we showed that deglycosylation of both ACE2 and S had a minimal effect on the spike-ACE2 interaction.

scholarly journals Allosteric activation of preformed EGF receptor dimers by binding of a single ligand

2021 ◽  
Author(s):  
Endang Purba ◽  
Ei-ichiro Saita ◽  
Reetesh Akhouri ◽  
Lars-Göran Öfverstedt ◽  
Gunnar Wilken ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Single Molecule ◽  
Molecular Mechanisms ◽  
Egf Receptor ◽  
Electron Tomography ◽  
Growth Factor Receptor ◽  
Full Length ◽  
Cancer Therapies ◽  
Receptor Dimer

Abstract Aberrant activation of the epidermal growth factor receptor (EGFR) by mutations has been implicated in a variety of human cancers. Elucidation of the structure of the full-length receptor is essential to understand the molecular mechanisms underlying its activation. Unlike previously anticipated, here, we report that purified full-length EGFR adopts a homodimeric form in vitro before and after activation. Cryo-electron tomography analysis of the purified receptor also showed that the extracellular domains of the receptor dimer, which are conformationally flexible before activation, are stabilised by ligand binding. Consistently, optical single-molecule observation also demonstrated that binding of only one ligand activates the receptor dimer on the cell surface. Based on these results, we propose an allosteric model for the activation of EGFR dimers by ligand binding. Our results demonstrate how oncogenic mutations spontaneously activate the receptor and shed light on the development of novel cancer therapies.

scholarly journals Misfolding Ectodomain Mutations of the Lutropin Receptor Increase Efficacy of Hormone Stimulation

2016 ◽  
Vol 30 (1) ◽  
pp. 62-76 ◽  
Author(s):  
E. Charmandari ◽  
R. Guan ◽  
M. Zhang ◽  
L. G. Silveira ◽  
Q. R. Fan ◽  
...  
Keyword(s):  
Cell Surface ◽  
Leydig Cells ◽  
Cellular Response ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Lutropin Receptor ◽  
Mutant Receptors ◽  
Type Receptor ◽  
Leydig Cell Hypoplasia ◽  
Inactivating Mutations

Abstract We demonstrate 2 novel mutations of the LHCGR, each homozygous, in a 46,XY patient with severe Leydig cell hypoplasia. One is a mutation in the signal peptide (p.Gln18_Leu19ins9; referred to here as SP) that results in an alteration of the coding sequence of the N terminus of the mature mutant receptor. The other mutation (p.G71R) is also within the ectodomain. Similar to many other inactivating mutations, the cell surface expression of recombinant human LHR(SP,G71R) is greatly reduced due to intracellular retention. However, we made the unusual discovery that the intrinsic efficacy for agonist-stimulated cAMP in the reduced numbers of receptors on the cell surface was greatly increased relative to the same low number of cell surface wild-type receptor. Remarkably, this appears to be a general attribute of misfolding mutations in the ectodomains, but not serpentine domains, of the gonadotropin receptors. These findings suggest that there must be a common, shared mechanism by which disparate mutations in the ectodomain that cause misfolding and therefore reduced cell surface expression concomitantly confer increased agonist efficacy to those receptor mutants on the cell surface. Our data further suggest that, due to their increased agonist efficacy, extremely small changes in cell surface expression of misfolded ectodomain mutants cause larger than expected alterations in the cellular response to agonist. Therefore, for inactivating LHCGR mutations causing ectodomain misfolding, the numbers of cell surface mutant receptors on fetal Leydig cells of 46,XY individuals exert a more exquisite effect on the relative severity of the clinical phenotypes than already appreciated.

scholarly journals Loss-of-Function Mutations in the Human Luteinizing Hormone Receptor Predominantly Cause Intracellular Retention

Endocrinology ◽  
2016 ◽  
Vol 157 (11) ◽  
pp. 4364-4377 ◽  
Author(s):  
Claire Louise Newton ◽  
Ross Calley Anderson ◽  
Arieh Anthony Katz ◽  
Robert Peter Millar
Keyword(s):  
Luteinizing Hormone ◽  
Cell Surface ◽  
Ligand Binding ◽  
Hormone Receptors ◽  
Receptor Activation ◽  
Luteinizing Hormone Receptor ◽  
Cell Surface Expression ◽  
Receptor Function ◽  
Loss Of Function ◽  
Pharmacological Chaperone

Mutations in G protein–coupled receptors (GPCRs) have been identified for many endocrine hormone signaling deficiencies. Inactivating mutations can impair ligand binding, receptor activation/coupling to signaling pathways, or can cause receptor misfolding and consequent impaired expression at the cell membrane. Here we examine the cell surface expression, ligand binding, and signaling of a range of mutant human luteinizing hormone receptors (LHRs) identified as causing reproductive dysfunction in human patients. The data obtained reveal how mutations in GPCRs can have diverse and severely deleterious effects on receptor function. Furthermore, it was found that impaired functionality of the majority of the mutant LHRs was due to reduced expression at the cell surface (14/20) while only two mutations caused impaired binding affinity and two impaired in signaling. An additional two mutations were found to cause no impairment of receptor function. These data demonstrate that the majority of LHR mutations lead to intracellular retention and highlight the potential for novel pharmacological chaperone therapeutics that can “rescue” expression/function of retained mutant GPCRs.

scholarly journals Characterization, cell-surface expression and ligand-binding properties of different truncated N-terminal extracellular domains of the ionotropic glutamate receptor subunit GluR1

1996 ◽  
Vol 315 (1) ◽  
pp. 217-225 ◽  
Author(s):  
R. A. Jeffrey McILHINNEY ◽  
Elek MOLNÁR
Keyword(s):  
Cell Surface ◽  
Ligand Binding ◽  
Glutamate Receptor ◽  
Cell Membranes ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Receptor Subunit ◽  
Binding Studies ◽  
Terminal Domain ◽  
Glutamate Receptor Subunit

To identify the location of the first transmembrane segment of the GluR1 glutamate receptor subunit artificial stop codons have been introduced into the N-terminal domain at amino acid positions 442, 510 and 563, namely just before and spanning the proposed first two transmembrane regions. The resultant truncated N-terminal fragments of GluR1, termed NT1, NT2 and NT3 respectively were expressed in Cos-7 cells and their cellular distribution and cell-surface expression analysed using an N-terminal antibody to GluR1. All the fragments were fully glycosylated and were found to be associated with cell membranes but none was secreted. Differential extraction of the cell membranes indicated that both NT1 and NT2 behave as peripheral membrane proteins. In contrast NT3, like the full subunit, has integral membrane protein properties. Furthermore only NT3 is expressed at the cell surface as determined by immunofluorescence and cell-surface biotinylation. Protease protection assays indicated that only NT3 had a cytoplasmic tail. Binding studies using the selective ligand [3H]α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate ([3H]AMPA) demonstrated that NT3 does not bind ligand. Together these results indicate that the first transmembrane domain of the GluR1 subunit lies between residues 509 and 562, that the N-terminal domain alone cannot form a functional ligand-binding site and that this domain can be targeted to the cell surface provided that it has a transmembrane-spanning region.

scholarly journals Individual cells traffic the Vasopressin 2 Receptor to their cell surface with different success

2021 ◽  
Author(s):  
Eline J Koers ◽  
Bradley A Morgan ◽  
Iain B Styles ◽  
Dmitry J Veprintsev
Keyword(s):  
Cell Surface ◽  
Receptor Cell ◽  
Surface Expression ◽  
G Protein Coupled Receptors ◽  
Cell Surface Expression ◽  
Subcellular Localisation ◽  
Equal Distribution ◽  
Mutant Receptors ◽  
G Protein Coupled ◽  
Correct Expression

G protein coupled receptors (GPCRs) translate the actions of hormones and neurotransmitters into intracellular signalling events. Mutations in GPCRs can prevent their correct expression and trafficking to the cell surface and cause disease. Single cell subcellular localisation measurements reveal that while some cells appear to traffic the majority of the vasopressin 2 receptor (V2R) molecules to the cell surface, others retain a greater number of receptors in the ER or have approximately equal distribution. Mutations in the V2R affect the proportion of cells able to send this GPCR to their cell surface but surprisingly they do not prevent all cells from correctly trafficking the mutant receptors. These findings reveal the potential for rescue of mutant receptor cell surface expression by pharmacological manipulation of the GPCR folding and trafficking machinery.

scholarly journals YLMY Tyrosine Residue within the Cytoplasmic Tail of Newcastle Disease Virus Fusion Protein Regulates Its Surface Expression to Modulate Viral Budding and Pathogenicity

2021 ◽  
Vol 9 (3) ◽  
Author(s):  
Yawen Bu ◽  
Qingyuan Teng ◽  
Delan Feng ◽  
Lu Sun ◽  
Jia Xue ◽  
...  
Keyword(s):  
Cell Surface ◽  
Fusion Protein ◽  
Surface Expression ◽  
Protein Processing ◽  
Cell Surface Expression ◽  
Tyrosine Residue ◽  
F Protein ◽  
Tyrosine Residues ◽  
Amino Terminal ◽  
Viral Budding

The amino-terminal cytoplasmic domains of paramyxovirus fusion glycoproteins include trafficking signals that influence protein processing and cell surface expression. This study clarified that tyrosine residues at different positions in the YLMY motif in the cytoplasmic region of the F protein regulate F protein transportation, thereby affecting viral replication and pathogenicity.

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