Inactive VEGFR2(R1032Q) exerts pro‐oncogenic activity through heterodimerization with wild‐type receptor

The spike S of SARS-CoV-2 recognizes ACE2 on the host cell membrane to initiate entry. Soluble decoy receptors, in which the ACE2 ectodomain is engineered to block S with high affinity, potently neutralize infection and, because of close similarity with the natural receptor, hold out the promise of being broadly active against virus variants without opportunity for escape. Here, we directly test this hypothesis. We find that an engineered decoy receptor, sACE22.v2.4, tightly binds S of SARS-associated viruses from humans and bats, despite the ACE2-binding surface being a region of high diversity. Saturation mutagenesis of the receptor-binding domain followed by in vitro selection, with wild-type ACE2 and the engineered decoy competing for binding sites, failed to find S mutants that discriminate in favor of the wild-type receptor. We conclude that resistance to engineered decoys will be rare and that decoys may be active against future outbreaks of SARS-associated betacoronaviruses.

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Spatial intensity distribution analysis quantifies the extent and regulation of homodimerization of the secretin receptor

Biochemical Journal ◽

10.1042/bcj20170184 ◽

2017 ◽

Vol 474 (11) ◽

pp. 1879-1895 ◽

Cited By ~ 17

Author(s):

Richard J. Ward ◽

John D. Pediani ◽

Kaleeckal G. Harikumar ◽

Laurence J. Miller ◽

Graeme Milligan

Keyword(s):

G Protein ◽

Intensity Distribution ◽

Transmembrane Domain ◽

Distribution Analysis ◽

Wild Type ◽

Spatial Intensity Distribution ◽

Spatial Intensity ◽

Secretin Receptor ◽

Dimeric Form ◽

Type Receptor

Previous studies have indicated that the G-protein-coupled secretin receptor is present as a homodimer, organized through symmetrical contacts in transmembrane domain IV, and that receptor dimerization is critical for high-potency signalling by secretin. However, whether all of the receptor exists in the dimeric form or if this is regulated is unclear. We used measures of quantal brightness of the secretin receptor tagged with monomeric enhanced green fluorescent protein (mEGFP) and spatial intensity distribution analysis to assess this. Calibration using cells expressing plasma membrane-anchored forms of mEGFP initially allowed us to demonstrate that the epidermal growth factor receptor is predominantly monomeric in the absence of ligand and while wild-type receptor was rapidly converted into a dimeric form by ligand, a mutated form of this receptor remained monomeric. Equivalent studies showed that, at moderate expression levels, the secretin receptor exists as a mixture of monomeric and dimeric forms, with little evidence of higher-order complexity. However, sodium butyrate-induced up-regulation of the receptor resulted in a shift from monomeric towards oligomeric organization. In contrast, a form of the secretin receptor containing a pair of mutations on the lipid-facing side of transmembrane domain IV was almost entirely monomeric. Down-regulation of the secretin receptor-interacting G-protein Gαs did not alter receptor organization, indicating that dimerization is defined specifically by direct protein–protein interactions between copies of the receptor polypeptide, while short-term treatment with secretin had no effect on organization of the wild-type receptor but increased the dimeric proportion of the mutated receptor variant.

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Deletion of the kinase insert sequence of the platelet-derived growth factor beta-receptor affects receptor kinase activity and signal transduction

Molecular and Cellular Biology ◽

10.1128/mcb.10.2.801-809.1990 ◽

1990 ◽

Vol 10 (2) ◽

pp. 801-809

Author(s):

L Severinsson ◽

B Ek ◽

K Mellström ◽

L Claesson-Welsh ◽

C H Heldin

Keyword(s):

Growth Factor ◽

Protein Tyrosine Kinase ◽

Catalytic Efficiency ◽

Kinase Domain ◽

Platelet Derived Growth Factor ◽

Tyrosine Kinase Domain ◽

Wild Type ◽

Mutant Receptor ◽

Beta Receptor ◽

Type Receptor

A characteristic feature of the platelet-derived growth factor (PDGF) beta-receptor is the presence of an insert sequence in the protein tyrosine kinase domain. A receptor mutant which lacks the entire insert of 98 amino acids was expressed in CHO cells, and its functional characteristics were compared with those of the wild-type receptor. The mutant receptor bound PDGF-BB with high affinity and mediated internalization and degradation of the ligand with efficiency similar to that of the wild-type receptor but did not transduce a mitogenic signal. It was found to display a decreased autophosphorylation after ligand stimulation and had a decreased ability to phosphorylate exogenous substrates; phosphofructokinase was not phosphorylated at all, whereas a peptide substrate was phosphorylated, albeit at a lower rate compared with phosphorylation by the wild-type receptor. Furthermore, the mutant receptor did not mediate actin reorganization but mediated an increase in c-fos expression. The data indicate that the insert in the kinase domain of the PDGF beta-receptor is important for the substrate specificity or catalytic efficiency of the kinase; the deletion of the insert interferes with the transduction of some, but not all, of the signals that arise after activation of the receptor.

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Effects of Complementarity Determining Region Mutations on the Affinity of an α/β T Cell Receptor: Measuring the Energy Associated with CD4/CD8 Repertoire Skewing

Journal of Experimental Medicine ◽

10.1084/jem.189.3.461 ◽

1999 ◽

Vol 189 (3) ◽

pp. 461-470 ◽

Cited By ~ 22

Author(s):

Thomas C. Manning ◽

Evan A. Parke ◽

Luc Teyton ◽

David M. Kranz

Keyword(s):

T Cell ◽

T Cell Receptors ◽

Cell Receptor ◽

Minimal Energy ◽

Wild Type ◽

Mhc I ◽

Histocompatibility Complex ◽

Ligand Interactions ◽

Type Receptor ◽

Complementarity Determining Region

It has been proposed that the generally low affinities of T cell receptors (TCRs) for their peptide–major histocompatibility complex (pMHC) ligands (Kd ∼10−4 to 10−7 M) are the result of biological selection rather than an intrinsic affinity limitation imposed by the TCR framework. Using a soluble version of the 2C TCR, we have used complementarity determining region (CDR)-directed mutagenesis to investigate whether the affinity of this receptor for its allogeneic pMHC ligand can be improved upon. We report that several mutants at positions lying within CDR3α and CDR2β showed increased affinities for pMHC compared with the wild-type receptor. Additionally, we have investigated whether Vα mutations that have been implicated in the phenomenon of CD8+ repertoire skewing achieve this skewing by means of generalized increases in affinity for MHC-I molecules. Two mutants (S27F and S51P), which each promote skewing toward a CD8+ phenotype, exhibited significantly reduced affinity for pMHC-I, consistent with a quantitative-instructional model of CD4/CD8 lineage commitment. This model predicts that CD8 is downregulated on thymocytes that have TCR–ligand interactions above a minimal energy threshold. Together, the results (a) demonstrate that engineering higher affinity TCRs is feasible, and (b) provide TCR–pMHC energy values associated with CD4/CD8 repertoire skewing.

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Wild-Type Receptor

IUPAC Standards Online ◽

10.1515/iupac.85.0963 ◽

2016 ◽

Author(s):

Derek R. Buckle ◽

Paul W. Erhardt ◽

C. Robin Ganellin ◽

Toshi Kobayashi ◽

Thomas J. Perun ◽

...

Keyword(s):

Wild Type ◽

Type Receptor

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Selective Modulation of Wild Type Receptor Functions by Mutants of G-Protein-coupled Receptors

Journal of Biological Chemistry ◽

10.1074/jbc.274.18.12548 ◽

1999 ◽

Vol 274 (18) ◽

pp. 12548-12554 ◽

Cited By ~ 39

Author(s):

Christian Le Gouill ◽

Jean-Luc Parent ◽

Carolyn-Ann Caron ◽

Rémi Gaudreau ◽

Léonid Volkov ◽

...

Keyword(s):

G Protein ◽

G Protein Coupled Receptors ◽

Wild Type ◽

Selective Modulation ◽

G Protein Coupled ◽

Type Receptor

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Adenine nucleotides decrease the apparentKmof endogenous natriuretic peptide receptors for GTP

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00321.2007 ◽

2007 ◽

Vol 293 (6) ◽

pp. E1756-E1763 ◽

Cited By ~ 17

Author(s):

Laura K. Antos ◽

Lincoln R. Potter

Keyword(s):

Natriuretic Peptide ◽

Adenine Nucleotides ◽

Cyclase Activity ◽

Wild Type ◽

Peptide Receptors ◽

Natriuretic Peptide Receptors ◽

Independent Mechanism ◽

Initial Activation ◽

Type Receptor ◽

Guanylyl Cyclase Activity

Natriuretic peptide receptors A (NPR-A) and B (NPR-B) mediate most effects of natriuretic peptides by synthesizing cGMP. ATP increases the activity of these receptors by an unknown mechanism. We recently reported that a nonhydrolyzable form of ATP, adenylyl imidodiphosphate (AMPPNP), stabilizes but is not required for the activation of NPR-A and NPR-B in membranes from highly overexpressing cells. Here, we repeated these studies on receptors expressed in endogenous settings. Kinetic analysis indicated that both AMPPNP and ATP dramatically decrease the apparent Kmof both receptors for GTP but had little effect on the Vmax. The EC50for AMPPNP decreased as substrate concentration increased whereas the magnitude of the effect was greater at lower GTP concentrations. ATP increased the activity of a mutant receptor containing glutamates substituted for all known phosphorylation sites similarly to the wild-type receptor, consistent with a phosphorylation independent mechanism. Finally, the putative ATP binding sites were investigated. Mutation of the ATP modulatory domain region had no effect, but mutation of K535A dramatically diminished ANP-dependent cyclase activity in a manner that was unresponsive to ATP. Mutation of the highly conserved 630-KSS to AAA (all alanines) resulted in an expressed receptor that had no detectable guanylyl cyclase activity. We conclude that ATP is not required for the initial activation of NPRs but does increase activity over time by reducing the apparent Kmfor GTP.

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A constitutively active mutant of the α1B-adrenergic receptor can cause greater agonist-dependent down-regulation of the G-proteins Gqα and G11α than the wild-type receptor

Biochemical Journal ◽

10.1042/bj3200079 ◽

1996 ◽

Vol 320 (1) ◽

pp. 79-86 ◽

Cited By ~ 10

Author(s):

Tae Weon LEE ◽

Alan WISE ◽

Susanna COTECCHIA ◽

Graeme MILLIGAN

Keyword(s):

G Proteins ◽

Cell Lines ◽

Adrenergic Receptor ◽

Inositol Phosphate ◽

Wild Type ◽

Down Regulation ◽

Eta Receptor ◽

Type Receptor ◽

In Cells ◽

Constitutively Active

Rat 1 fibroblasts transfected to express either the wild-type hamster α1B-adrenergic receptor or a constitutively active mutant (CAM) form of this receptor resulting from the alteration of amino acid residues 288–294 to encode the equivalent region of the human β2-adrenergic receptor were examined. The basal level of inositol phosphate generation in cells expressing the CAMα1B-adrenergic receptor was greater than for the wild-type receptor. The addition of maximally effective concentrations of phenylephrine or noradrenaline resulted in substantially greater levels of inositol phosphate generation by the CAMα1B-adrenergic receptor, although this receptor was expressed at lower steady-state levels than the wild-type receptor. The potency of both phenylephrine and noradrenaline to stimulate inositol phosphate production was approx. 200-fold greater at the CAMα1B-adrenergic receptor than at the wild-type receptor. In contrast, endothelin 1, acting at the endogenously expressed endothelin ETA receptor, displayed similar potency and maximal effects in the two cell lines. The sustained presence of phenylephrine resulted in down-regulation of the α subunits of the phosphoinositidase C-linked, pertussis toxin-insensitive, G-proteins Gq and G11 in cells expressing either the wild-type or the CAMα1B-adrenergic receptor. The degree of down-regulation achieved was substantially greater in cells expressing the CAMα1B-adrenergic receptor at all concentrations of the agonist. However, in this assay phenylephrine displayed only a slightly greater potency at the CAMα1B-adrenergic receptor than at the wild-type receptor. There were no detectable differences in the basal rate of Gqα/G11α degradation between cells expressing the wild-type or the CAMα1B-adrenergic receptor. In both cell lines the addition of phenylephrine substantially increased the rate of degradation of these G-proteins, with a greater effect at the CAMα1B-adrenergic receptor. The enhanced capacity of agonist both to stimulate second-messenger production at the CAMα1B-adrenergic receptor and to regulate cellular levels of its associated G-proteins by stimulating their rate of degradation is indicative of an enhanced stoichiometry of coupling of this form of the receptor to Gq and G11.

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The ankyrin repeat-containing protein Akr1p is required for the endocytosis of yeast pheromone receptors.

Molecular Biology of the Cell ◽

10.1091/mbc.8.7.1317 ◽

1997 ◽

Vol 8 (7) ◽

pp. 1317-1327 ◽

Cited By ~ 33

Author(s):

S A Givan ◽

G F Sprague

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Surface ◽

Hybrid System ◽

Secretory Pathway ◽

Cytoplasmic Tail ◽

Ankyrin Repeat ◽

Wild Type ◽

Delta Cells ◽

Type Receptor ◽

Two Hybrid

The Saccharomyces cerevisiae a-factor receptor (Ste3p) requires its C-terminal cytoplasmic tail for endocytosis. Wild-type receptor is delivered to the cell surface via the secretory pathway but remains there only briefly before being internalized and delivered to the vacuole for degradation. Receptors lacking all or part of the cytoplasmic tail are not subject to this constitutive endocytosis. We used the cytoplasmic tail of Ste3p as bait in the two-hybrid system in an effort to identify other proteins involved in endocytosis. One protein identified was Akr1p, an ankyrin repeat-containing protein. We applied three criteria to demonstrate that Akr1p is involved in the constitutive endocytosis of Ste3p. First, when receptor synthesis is shut off, akr1 delta cells retain the ability to mate longer than do AKR1 cells. Second, Ste3p half-life is increased by greater than 5-fold in akr1 delta cells compared with AKR1 cells. Third, after a pulse of synthesis, newly synthesized receptor remains at the cell surface in akr1 delta mutants, whereas it is rapidly internalized in AKR1 cells. Specifically, in akr1 delta mutants, newly synthesized receptor is accessible to exogenous protease, and by indirect immunofluorescence, the receptor is located at the cell surface. akr1 delta cells are also defective for endocytosis of the alpha-factor receptor (Ste2p). Despite the block to constitutive endocytosis exhibited by akr1 delta cells, they are competent to carry out ligand-mediated endocytosis of Ste3p. In contrast, akr1 delta cells cannot carry out ligand-mediated endocytosis of Ste2p. We discuss the implications for Akr1p function in endocytosis and suggest a link to the regulation of ADP-ribosylation proteins (Arf proteins).

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Isolation and characterisation of the marmoset gonadotrophin releasing hormone receptor: Ser(140) of the DRS motif is substituted by Phe

Journal of Endocrinology ◽

10.1677/joe.0.1630447 ◽

1999 ◽

Vol 163 (3) ◽

pp. 447-456 ◽

Cited By ~ 22

Author(s):

B Byrne ◽

A McGregor ◽

PL Taylor ◽

R Sellar ◽

FE Rodger ◽

...

Keyword(s):

Inositol Phosphate ◽

Maximal Response ◽

Wild Type ◽

Releasing Hormone ◽

Agonist And Antagonist ◽

Gonadotrophin Releasing Hormone ◽

Antagonist Action ◽

G Protein Coupled ◽

Type Receptor ◽

D Value

In order to facilitate the understanding of gonadotrophin-releasing hormone (GnRH) agonist and antagonist action in the primate animal model, the marmoset GnRH receptor (GnRH-R) was cloned and characterised. It was shown to have 95% and 85% sequence identity with the human and rat GnRH-Rs, respectively, and, when transiently expressed in COS-7 cells, it exhibited high-affinity des-Gly(10), [d-Trp(6)]-GnRH binding, with a K(d) value similar to those of both the rat and human forms, but with a greatly reduced B(max) value. The ED(50) for production of GnRH-induced total inositol phosphate (IP) for the marmoset GnRH-R was also similar to those of the rat and the human, but the maximal response compared with the rat receptor was markedly reduced. In all mammalian forms of the GnRH-R cloned to date, the conserved DRY region of G-protein-coupled receptors is substituted with DRS. The most interesting feature of the marmoset GnRH-R was the substitution of this motif with DRF. In order to investigate the DRS to DRF substitution, a Ser(140)Phe rat GnRH-R mutant was generated. The mutant had a K(d) value similar to that of the wild-type rat receptor, although the B(max) value was slightly lower, indicating that expression of functional mutant receptor at the cell surface was reduced. The ED(50) value for IP production was also similar to that of the wild-type receptor, with a reduction in maximal response. The level of internalisation for the rat wild-type and mutant GnRH-R constructs was also assessed and the Ser(140)Phe mutant was shown to have an increased rate of receptor internalisation, suggesting a role for this residue in regulating internalisation. These results show that the marmoset GnRH-R exhibits a substitution in the DRS motif and that this substitution may play a part in desensitisation and internalisation events.

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