scholarly journals Functional rescue of a constitutively desensitized β2AR through receptor dimerization

1998 ◽  
Vol 330 (1) ◽  
pp. 287-293 ◽  
Author(s):  
E. Terence HEBERT ◽  
P. Thomas LOISEL ◽  
Lynda ADAM ◽  
Nathalie ETHIER ◽  
ST. Stephane ONGE ◽  
...  
Keyword(s):  
Adrenergic Receptors ◽  
Prolonged Exposure ◽  
Wild Type ◽  
Receptor Dimerization ◽  
Metabolic Labelling ◽  
Functional Consequences ◽  
Receptor Mutant ◽  
Physical Interactions ◽  
Type Receptor ◽  
Positive Effect

We have recently demonstrated that wild-type β2-adrenergic receptors (β2AR) form homodimers and that disruption of receptor dimerization inhibits signalling via Gs [Hebert, Moffett, Morello, Loisel, Bichet, Barret and Bouvier (1996) J. Biol. Chem. 271, 16384-16392]. Here taking advantage of the altered functional properties of a non-palmitoylated, constitutively desensitized mutant β2AR (C341Gβ2AR), we sought to study whether physical interactions between mutant and wild-type β2AR expressed in Sf9 cells could occur and have functional consequences. Using metabolic labelling with [3H]palmitate and co-immunoprecipitation we demonstrated the existence of heterodimerization between wild-type and C341Gβ2AR. Furthermore, we show that, in co-expression experiments, wild-type receptors have a dominant positive effect resulting in the functional complementation of C341Gβ2AR. Indeed, when expressed alone, the mutant C341G receptor displays altered functional characteristics in that (1) the response of the receptor to agonist is reduced as compared to the wild-type receptor and (2) the desensitization of the receptor in response to prolonged exposure to agonist is minimal. In contrast, when C341G and the wild-type β2AR were expressed together, both the response to agonist and subsequent desensitization (at a constant level of total receptor) were equivalent to the wild-type β2AR expressed alone. This dominant positive effect was also seen when C341G was co-expressed with a second receptor mutant in which the two protein kinase A phosphorylation sites (S261, 262, 345, 346A β2AR) were mutated. Taken together these data suggest that intermolecular interactions between receptors may have both functional and structural implications for G-protein-mediated signalling.

Evidence to support a spectrum of active states for the glucagon receptor

2004 ◽  
Vol 32 (6) ◽  
pp. 1037-1039 ◽  
Author(s):  
N. Strudwick ◽  
N. Bhogal ◽  
N.A. Evans ◽  
F.E. Blaney ◽  
J.B.C. Findlay
Keyword(s):  
Partial Agonist ◽  
Transmembrane Helix ◽  
Complex Model ◽  
Wild Type ◽  
Glucagon Receptor ◽  
Activating Mutations ◽  
Ternary Complex Model ◽  
Receptor Mutant ◽  
G Protein Coupled ◽  
Type Receptor

The ternary complex model suggests that G-protein-coupled receptors resonate between inactive (R) and active (R*) forms. Physiologically, R sites ordinarily predominate with a few R* sites giving rise to basal activity. Agonists recognize, stabilize and increase the R* population, thus altering intracellular activity. There is evidence to suggest the possibility of a spectrum of conformations between R and R*. Our aim is to study the consequences of putative GR (glucagon receptor)-activating mutations using glucagon and partial agonist des-His1-[Glu9]glucagon amide (glucagon-NH2). Alanine substitution in TM (transmembrane) helix 2 of Arg173 or of His177 detrimentally affected glucagon and glucagon-NH2 response maxima. TM2 receptor mutant, Phe181-Ala, displayed reduced maximum cAMP accumulation in response to glucagon-NH2. Thr353-Cys (TM6) and Glu406-Ala (TM7) receptors demonstrated constitutive activity and enhanced EC50 values for glucagon-NH2; Arg346-Ala (TM6) and Asn404-Ala (TM7) receptors were activated by sub-fmol glucagon concentrations, yet were not constitutively active and demonstrated wild-type receptor-like EC50 values for glucagon-NH2. Unlike Arg346-Ala receptors, Thr353-Cys, Asn404-Ala and Glu406-Ala receptors demonstrated improved EC50 values for glucagon, whereas their maximal responses to and their affinity for glucagon were comparable with the wild-type receptor. In contrast, despite slightly reduced glucagon-NH2 affinity, Arg346-Ala, Thr353-Cys, Asn404-Ala and Glu406-Ala receptors displayed glucagon-NH2 response maxima that exceeded those seen for wild-type receptors. Interestingly, we observed biphasic glucagon-mediated signalling responses. Our results are consistent with the concept of different agonists promoting the formation of distinct active states from partially active R*low to fully active R*high forms.

scholarly journals Adverse Adipose Phenotype and Hyperinsulinemia in Gravid Mice Deficient in Placental Growth Factor

Endocrinology ◽  
2008 ◽  
Vol 149 (5) ◽  
pp. 2176-2183 ◽  
Author(s):  
Bianca Hemmeryckx ◽  
Rita van Bree ◽  
Berthe Van Hoef ◽  
Lisbeth Vercruysse ◽  
H. Roger Lijnen ◽  
...  
Keyword(s):  
Growth Factor ◽  
Adrenergic Receptors ◽  
Growth Factor Receptor ◽  
Placental Growth Factor ◽  
Peroxisome Proliferator ◽  
Vascular Endothelial ◽  
Wild Type ◽  
Peroxisome Proliferator Activated Receptor ◽  
Adipocyte Hypertrophy ◽  
Endothelial Growth Factor

Pregnancy-induced metabolic changes are regulated by signals from an expanded adipose organ. Placental growth factor (PlGF), acting through vascular endothelial growth factor receptor-1, may be among those signals. There is a steep rise in circulating PlGF during normal pregnancy, which is repressed in gravidas who develop preeclampsia. PlGF-deficiency in mice impairs adipose vascularization and development. Here we studied young-adult PlGF-deficient (PlGF−/−) and wild-type mice on a high-fat diet in the nongravid state and at embryonic day (E) 13.5 or E18.5 of gestation. Litter size and weight were normal, but E18.5 placentas were smaller in PlGF−/− pregnancies. PlGF−/− mice showed altered intraadipose dynamics, with the following: 1) less blood vessels and fewer brown, uncoupling protein (UCP)-1-positive, adipocytes in white sc and perigonadal fat compartments and 2) white adipocyte hypertrophy. The mRNA expression of β3-adrenergic receptors, peroxisome proliferator-activated receptor-γ coactivator-1α, and UCP-1 was decreased accordingly. Moreover, PlGF−/− mice showed hyperinsulinemia. Pregnancy-associated changes were largely comparable in PlGF−/− and wild-type dams. They included expanded sc fat compartments and adipocyte hypertrophy, whereas adipose expression of key angiogenesis/adipogenesis (vascular endothelial growth factor receptor-1, peroxisome proliferator-activated receptor-γ2) and thermogenesis (β3-adrenergic receptors, peroxisome proliferator-activated receptor-γ coactivator-1α, and UCP-1) genes was down-regulated; circulating insulin levels gradually increased during pregnancy. In conclusion, reduced adipose vascularization in PlGF−/− mice impairs adaptive thermogenesis in favor of energy storage, thereby promoting insulin resistance and hyperinsulinemia. Pregnancy adds to these changes by PlGF-independent mechanisms. Disturbed intraadipose dynamics is a novel mechanism to explain metabolic changes in late pregnancy in general and preeclamptic pregnancy in particular.

scholarly journals N-terminal fatty acylation of the α-subunit of the G-protein Gi1: only the myristoylated protein is a substrate for palmitoylation

1994 ◽  
Vol 303 (3) ◽  
pp. 697-700 ◽  
Author(s):  
F Galbiati ◽  
F Guzzi ◽  
A I Magee ◽  
G Milligan ◽  
M Parenti
Keyword(s):  
Palmitic Acid ◽  
G Protein ◽  
Fatty Acyl ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Α Subunit ◽  
Metabolic Labelling ◽  
Fatty Acylation ◽  
Absolute Requirement ◽  
Cdna Construct

The alpha-subunit of the G-protein Gi1 carries two fatty acyl moieties covalently bound to its N-terminal region: myristic acid is linked to glycine-2 and palmitic acid is linked to cysteine-3. Using site-directed mutagenesis on a cDNA construct of alpha i1 we have generated an alpha i1-G2A mutant, carrying alanine instead of glycine at position 2, and alpha i1-C3S mutant, in which serine replaced cysteine-3 and a double mutant with both substitutions (alpha i1-G2A/C3S). These constructs were individually expressed by transfection in Cos-7 cells, and incorporation of fatty acids into the various mutants was compared with wild-type alpha i1 monitoring metabolic labelling with [3H]palmitate or [3H]myristate. The disruption of the palmitoylation site in alpha i1-C3S did not influence myristoylation, whereas prevention of myristoylation in alpha i1-G2A also abolished palmitoylation. Co-translational myristoylation is thus an absolute requirement for alpha i1 to be post-translationally palmitoylated. The non-palmitoylated alpha i1-C3S showed reduced membrane binding to the same extent as the non-myristoylated/non-palmitoylated alpha i1-G2A and alpha i1-G2A/C3S mutants, indicating that the attachment of palmitic acid is necessary for proper interaction with the membrane.

scholarly journals An engineered decoy receptor for SARS-CoV-2 broadly binds protein S sequence variants

2021 ◽  
Vol 7 (8) ◽  
pp. eabf1738 ◽  
Author(s):  
Kui K. Chan ◽  
Timothy J. C. Tan ◽  
Krishna K. Narayanan ◽  
Erik Procko
Keyword(s):  
In Vitro Selection ◽  
Protein S ◽  
Saturation Mutagenesis ◽  
Wild Type ◽  
Host Cell Membrane ◽  
Decoy Receptor ◽  
Decoy Receptors ◽  
Binding Surface ◽  
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.

Sevoflurane but not propofol provided dual effects of cell survival in human neuroblastoma SH-SY5Y cells

2021 ◽  
Vol 18 ◽  
Author(s):  
Xue Gao ◽  
Xiu Wang ◽  
Lei Zhang ◽  
Ge Liang ◽  
Rachel Mund ◽  
...  
Keyword(s):  
Cell Survival ◽  
Prolonged Exposure ◽  
Calcium Influx ◽  
Cell Damage ◽  
Cytosolic Calcium ◽  
Extracellular Calcium ◽  
Mitochondrial Calcium ◽  
General Anesthetics ◽  
Wild Type ◽  
Human Neuroblastoma

Background: We have hypothesized that the most commonly used intravenous (propofol) and inhalational (sevoflurane) general anesthetics affect cell survival concentration and duration dependently with different potency associated with their differential potency to affect intracellular calcium homeostasis. Methods: Human neuroblastoma SH-SY5Y cells stably transfected with either wild type or M146L mutant human presenilin 1 were cultured and exposed to equipotent of propofol or sevoflurane. Cell viability, cytosolic and mitochondrial calcium were measured. Results: Sevoflurane but not propofol, at clinically relevant concentrations and durations, promoted cell survival. Prolonged exposure (24 hours) of 1% sevoflurane resulted in significant cell damage in both types of cells. Both sevoflurane and propofol had significantly higher cell response rates to the elevation of cytosolic calcium or mitochondrial calcium in the presence of extracellular calcium. With the contribution of calcium influx, sevoflurane but not equipotent 1 MAC propofol, caused a significantly greater increase in peak and overall calcium in Alzheimer’s mutation cell than in wild type cells, but significantly more increase in overall mitochondrial calcium concentrations in wild type than mutation cells. In the absence of extracellular calcium influx, sevoflurane, but not propofol, caused more significant elevations of overall mitochondrial calcium concentration in mutation cells than control cells. Conclusion: Calcium influx contributed to the general anesthetics mediated elevation of cytosolic or mitochondrial calcium, which is especially true for propofol. Sevoflurane has a greater potency to either promote or inhibit cell survival than propofol, which may be associated with its ability to affect cytosolic or mitochondrial calcium.

Activation and inhibition of erythropoietin receptor function: role of receptor dimerization

1994 ◽  
Vol 14 (6) ◽  
pp. 3535-3549
Author(s):  
S S Watowich ◽  
D J Hilton ◽  
H F Lodish
Keyword(s):  
Signal Transduction ◽  
Growth Hormone Receptor ◽  
Initial Step ◽  
Inhibitory Effect ◽  
Erythropoietin Receptor ◽  
Interface Region ◽  
Wild Type ◽  
Receptor Dimerization ◽  
Dimer Interface ◽  
Constitutively Active

Members of the cytokine receptor superfamily have structurally similar extracellular ligand-binding domains yet diverse cytoplasmic regions lacking any obvious catalytic domains. Many of these receptors form ligand-induced oligomers which are likely to participate in transmembrane signaling. A constitutively active (factor-independent) mutant of the erythropoietin receptor (EPO-R), R129C in the exoplasmic domain, forms disulfide-linked homodimers, suggesting that the wild-type EPO-R is activated by ligand-induced homodimerization. Here, we have taken two approaches to probe the role EPO-R dimerization plays in signal transduction. First, on the basis of the crystal structure of the ligand-bound, homodimeric growth hormone receptor (GH-R) and sequence alignment between the GH-R and EPO-R, we identified residues of the EPO-R which may be involved in intersubunit contacts in an EPO-R homodimer. Residue 129 of the EPO-R corresponds to a residue localized to the GH-R dimer interface region. Alanine or cysteine substitutions were introduced at four other residues of the EPO-R predicted to be in the dimer interface region. Substitution of residue E-132 or E-133 with cysteine renders the EPO-R constitutively active. Like the arginine-to-cysteine mutation at position 129 in the exoplasmic domain (R129C), E132C and E133C form disulfide-linked homodimers, suggesting that constitutive activity is due to covalent dimerization. In the second approach, we have coexpressed the wild-type EPO-R with inactive mutants of the receptor missing all or part of the cytosolic domain. These truncated receptors have a dominant inhibitory effect on the proliferative action of the wild-type receptor. Taken together, these results strengthen the hypothesis that an initial step in EPO- and EPO-R-mediated signal transduction is ligand-induced receptor dimerization.

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

2017 ◽  
Vol 474 (11) ◽  
pp. 1879-1895 ◽  
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.

Abstract 5501: Reduced Coronary Reserve as a Mechanism in Beta-Adrenergic Receptor Mediated Cardiomyopathy and its Rescue by Adenylyl Cyclase Type 5 Knockout

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Shumin Gao ◽  
Lin Yan ◽  
Chull Hong ◽  
Xin Zhao ◽  
Li Chen ◽  
...  
Keyword(s):  
Adenylyl Cyclase ◽  
Mouse Models ◽  
Adrenergic Receptors ◽  
Adrenergic Receptor ◽  
Potential Mechanism ◽  
Wild Type ◽  
Coronary Reserve ◽  
Small Heart ◽  
Cardinal Feature ◽  
Adenylyl Cyclase Type 5

Reduced coronary reserve (CR) is a cardinal feature of human cardiomyopathy, but it has not been studied extensively in mouse models of cardiomyopathy, potentially because of difficulties inherent to measuring CR in such a small heart. The first goal of this study was to determine if reduced CR is a potential mechanism involved in a transgenic (Tg) mouse model of cardiomyopathy, induced by cardiac overexpression of beta 2 -adrenergic receptors (beta 2 -AR Tg). Older beta 2 -AR Tg mice (16–17 months, n=7) developed cardiomyopathy, as reflected by decreased LV ejection fraction (LVEF, 50±7%), and increased fibrosis (5.2±0.4%) and apoptosis (3.8±0.6%). CR was obtained by measuring LV coronary blood flow at baseline and with adenosine (160 microg/kg/min) induced hyperemia using high resolution ultrasound (Vevo 770). CR was significantly decreased in beta 2 -AR Tg (1.8±0.2) compared with wild type (WT) (3.0±0.3, n=6). The next goal was to determine if the rescue of the beta 2 -AR Tg cardiomyopathy resulted in a rescue of reduced CR. Since adenylyl cyclase type 5 knockout (AC5 KO) mice are protected against catecholamine stress and the development of heart failure (HF), and exhibit enhanced CR (3.7±0.4, n=5), we hypothesized that mating these mice with beta 2 -AR Tg might rescue the cardiomyopathy. Older (16–17 months, n=4) bigenic mice (beta 2 -AR Tg x AC5 KO) demonstrated a rescue of cardiomyopathy, as reflected by normalized LVEF (71±1%) and levels of apoptosis (0.11±0.01%) and fibrosis (1.09±0.33%), similar to WT. In these mice, CR was also normalized (3.4±0.6). Reduced CR in beta 2 -AR Tg cardiomyopathy is similar to that observed in large mammalian models, and in patients with cardiomyopathy. Thus, this mechanism is important to consider in mouse models of cardiomyopathy, in general, and also in particular for the mechanism of the rescue of the beta 2 -AR Tg cardiomyopathy by AC5 KO.

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

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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