G protein-coupled prostaglandin receptor modulates conductive Na+ uptake in lung apical membrane vesicles

1998 ◽  
Vol 274 (4) ◽  
pp. L567-L572
Author(s):  
Somnath Mukhopadhyay ◽  
Asim K. Dutta-Roy ◽  
Gregor K. Fyfe ◽  
Richard E. Olver ◽  
Paul J. Kemp
Keyword(s):  
G Protein ◽  
Protein Function ◽  
Apical Membrane ◽  
Competitive Inhibition ◽  
Rank Order ◽  
Membrane Vesicles ◽  
High Affinity ◽  
High Affinity Binding Site ◽  
Uptake Studies ◽  
G Protein Coupled

Because G protein-regulated cation channels in type II pneumocytes constitute the most likely pathway for alveolar Na+ entry, we explored the hypothesis that a G protein-coupled prostaglandin (PG) E2 receptor controls perinatal lung alveolar Na+ transport. [3H]PGE2binding to the alveolar apical membrane was trypsin sensitive and showed a rank order of competitive inhibition: PGE2 = PGE1 > PGD2 > PGF2α. Kinetic analysis demonstrated both high-affinity [dissociation constant ( K D) = 2.1 ± 0.7 nM; maximal binding (Bmax) = 27 ± 7 fmol/mg protein] and low-affinity ( K D = 28 ± 2 nM; Bmax = 265 ± 29 fmol/mg protein) binding sites. Modulation of high-affinity GTPase activity identified a similar potency order (IC50 = 11 mM for PGF2α vs. 10–50 μM for other PGs), suggesting that the receptors are G protein coupled. Finally, 1 μM PGE2(≈IC25) increased conductive22Na+uptake into membrane vesicles only in the presence of 100 μM intravesicular GTP. The K D value for the high-affinity binding site together with the rank order of PG effect on ligand binding and G protein function places this PG receptor in the EP3 subtype, whereas Na+ uptake studies suggest that it helps maintain perinatal lung Na+homeostasis.

Pharmacological characterization of putative β1-β2-adrenergic receptor heterodimers

2003 ◽  
Vol 81 (2) ◽  
pp. 186-195 ◽  
Author(s):  
Catherine Lavoie ◽  
Terence E Hébert
Keyword(s):  
G Protein ◽  
Receptor Subtypes ◽  
Affinity Binding ◽  
Pharmacological Characterization ◽  
High Affinity Binding ◽  
Hek 293 ◽  
High Affinity ◽  
High Affinity Binding Site ◽  
G Protein Coupled ◽  
Specific Ligand

In the last few years, significant experimental evidence has accumulated showing that many G protein coupled receptors (GPCRs) are structurally and perhaps functionally homodimers. Recently, a number of studies have demonstrated that many GPCRs, notably GABAB, somatostatin, and δ and κ opioid receptors form heterodimers, as well. Based on these observations, we undertook a pharmacological and functional analysis of HEK 293 cells transiently transfected with the β1AR or β2AR or with both subtypes together. High-affinity binding for subtype-specific ligands (betaxolol and xamoterol for the β1AR, and ICI 118,551 and procaterol for the β2AR) was detected in cells expressing the cognate receptors alone with values similar to those reported in the literature. However, a significant portion of these high-affinity interactions were lost when both receptors were expressed together while nonspecific ligands (propranolol and isoproterenol) retained their normal affinities. When competition assays were performed with each subtype-specific ligand in the presence of a constant concentration of the other subtype-specific ligand, the high-affinity binding site was rescued, suggesting that the two receptor subtypes were interacting in a fashion consistent with positive cooperativity. Our data suggest that the β1AR and β2AR can form heterodimers and that these receptors have altered pharmacological properties from the receptor homodimers.Key words: G protein coupled receptor, signalling, G protein, dimerization, heterodimer, adrenergic.

scholarly journals Optimised insert design for improved single-molecule imaging and quantification through CRISPR-Cas9 mediated knock-in

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Abdullah O. Khan ◽  
Carl W. White ◽  
Jeremy A. Pike ◽  
Jack Yule ◽  
Alexandre Slater ◽  
...  
Keyword(s):  
Genome Editing ◽  
G Protein ◽  
Single Molecule ◽  
Protein Function ◽  
Single Molecule Imaging ◽  
Dependent Effect ◽  
Systematic Comparison ◽  
Selection Strategies ◽  
Localisation Microscopy ◽  
G Protein Coupled

Abstract The use of CRISPR-Cas9 genome editing to introduce endogenously expressed tags has the potential to address a number of the classical limitations of single molecule localisation microscopy. In this work we present the first systematic comparison of inserts introduced through CRISPR-knock in, with the aim of optimising this approach for single molecule imaging. We show that more highly monomeric and codon optimised variants of mEos result in improved expression at the TubA1B locus, despite the use of identical guides, homology templates, and selection strategies. We apply this approach to target the G protein-coupled receptor (GPCR) CXCR4 and show a further insert dependent effect on expression and protein function. Finally, we show that compared to over-expressed CXCR4, endogenously labelled samples allow for accurate single molecule quantification on ligand treatment. This suggests that despite the complications evident in CRISPR mediated labelling, the development of CRISPR-PALM has substantial quantitative benefits.

scholarly journals Specific oxylipins enhance vertebrate hematopoiesis via the receptor GPR132

2018 ◽  
Vol 115 (37) ◽  
pp. 9252-9257 ◽  
Author(s):  
Jamie L. Lahvic ◽  
Michelle Ammerman ◽  
Pulin Li ◽  
Megan C. Blair ◽  
Emma R. Stillman ◽  
...  
Keyword(s):  
Fatty Acids ◽  
G Protein ◽  
Cell Lines ◽  
Saturated Fatty Acids ◽  
Receptor Activation ◽  
G Protein Coupled Receptors ◽  
Hydroxy Fatty Acids ◽  
High Affinity ◽  
G Protein Coupled

Epoxyeicosatrienoic acids (EETs) are lipid-derived signaling molecules with cardioprotective and vasodilatory actions. We recently showed that 11,12-EET enhances hematopoietic induction and engraftment in mice and zebrafish. EETs are known to signal via G protein-coupled receptors, with evidence supporting the existence of a specific high-affinity receptor. Identification of a hematopoietic-specific EET receptor would enable genetic interrogation of EET signaling pathways, and perhaps clinical use of this molecule. We developed a bioinformatic approach to identify an EET receptor based on the expression of G protein-coupled receptors in cell lines with differential responses to EETs. We found 10 candidate EET receptors that are expressed in three EET-responsive cell lines, but not expressed in an EET-unresponsive line. Of these, only recombinant GPR132 showed EET-responsiveness in vitro, using a luminescence-based β-arrestin recruitment assay. Knockdown of zebrafish gpr132b prevented EET-induced hematopoiesis, and marrow from GPR132 knockout mice showed decreased long-term engraftment capability. In contrast to high-affinity EET receptors, GPR132 is reported to respond to additional hydroxy-fatty acids in vitro, and we found that these same hydroxy-fatty acids enhance hematopoiesis in the zebrafish. We conducted structure–activity relationship analyses using both cell culture and zebrafish assays on diverse medium-chain fatty acids. Certain oxygenated, unsaturated free fatty acids showed high activation of GPR132, whereas unoxygenated or saturated fatty acids had lower activity. Absence of the carbon-1 position carboxylic acid prevented activity, suggesting that this moiety is required for receptor activation. GPR132 responds to a select panel of oxygenated polyunsaturated fatty acids to enhance both embryonic and adult hematopoiesis.

Opioid receptors in bovine adrenal medulla

1984 ◽  
Vol 62 (10) ◽  
pp. 1284-1291 ◽  
Author(s):  
Michel Dumont ◽  
Simon Lemaire
Keyword(s):  
Binding Site ◽  
G Protein ◽  
Opioid Receptors ◽  
Opioid Peptides ◽  
Affinity Binding ◽  
Endogenous Opioid ◽  
High Affinity Binding ◽  
High Affinity ◽  
High Affinity Binding Site

Using prototypic ligands for each type of opioid receptors (μ, δ, κ, and σ) as well as compounds derived from each class of endogenous opioid peptides (β-endorphin, enkephalins, and dynorphins), we have undertaken the characterization of adrenomedullary opioid binding sites. The specific binding of [3H]etorphine ([3H]ET) to a membrane preparation of bovine adrenal medulla was greatly increased when the incubation temperature was raised from 22 to 37 °C. Characterization of the opioid binding sites was obtained at 37 °C with [3H]ET (nonspecific opioid ligand), [3H]ethylketocyclazocine ([3H]EKC; κ), [3H]dihydromorphine ([3H]DHM; μ), [3H]-[D-Ala2,D-Leu5]enkephalin ([3H]DADLE; δ), and N-[3H]allylnormetazocine ([3H]SKF-10047; σ) in the absence or presence of blocking agents for cross-reacting receptors. [3H]ET had a high affinity binding site (KD = 0.98 nM) with a Bmax of 119 pmol/g protein. All the other opioid compounds showed biphasic saturation curves with KD ranging from 0.6 to 1.29 nM for the high affinity binding site and from 2.49 to 12.1 nM for the low affinity binding site. The opioid μ-receptor was characterized by the high affinity binding site for [3H]DHM (KD = 1.29 nM; Bmax = 38 pmol/g protein). Blockade of the cross-reacting receptor sites for [3H]EKC, [3H]DADLE, and [3H]SKF-10047 revealed the presence of κ (KD = 0.66 nM; Bmax = 12 pmol/g protein), κ2 (benzomorphan site; KD = 11.1 nM; Bmax = 56 pmol/g protein), δ (KD = 0.67 nM; Bmax = 4.7 pmol/g protein), and σ (KD = 4.54 nM; Bmax = 32 pmol/g protein) opioid receptors. The ability of various opioid ligands to displace the binding of [3H]ET indicates a high potency for (−)-(1R,5R,9R,2″S)-5,9-dimethyl-2′-hydroxy-2-tetrahydrofurfuryl-6,7-benzomorphan hydrogen D-tartrate (MR-2034, a κ-opioid ligand; Ki = 6.2 nM), dihydromorphinone (DHMone; Ki = 6.9 nM), oxymorphone (Ki = 8.6 nM), DADLE (Ki high affinity = 8.4 nM) EKC (Ki = 31.8 nM), SKF-10047 (Ki = 75 nM), and opioid agonists/antagonists. trans-(+)-3,4-Dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide methanesulfonate hydrate (U-50,488H), the most specific κ-agonist, was a poor competitor (Ki = 5150 nM). However, the presence of κ-opioid receptors was supported by the ability of U-50,488H to displace [3H]EKC binding (Ki high affinity = 2.5 nM). The relative potency of various endogenous opioid peptides in displacing [3H]ET binding was as follows: β-endorphin [Formula: see text] dynorphin(1-17) > dynorphin(1-13) > [Arg6,Phe7)Met-enkephalin > Met-enkephalin > Leu-enkephalin. In addition, the presence of a high affinity binding site for dynorphin was demonstrated by the high potency of dynorphin (1-13) to displace [3H]EKC binding (Ki high affinity = 2.3 nM). These data provide further insights into the characterization of adrenal opioid receptors and suggest an in situ physiological role for adrenal opioid peptides.

scholarly journals [3H]Adenine is a suitable radioligand for the labeling of G protein-coupled adenine receptors but shows high affinity to bacterial contaminations in buffer solutions

2007 ◽  
Vol 3 (4) ◽  
pp. 347-358 ◽  
Author(s):  
Anke C. Schiedel ◽  
Heiko Meyer ◽  
Bernt B. A. Alsdorf ◽  
Simone Gorzalka ◽  
Hannelore Brüssel ◽  
...  
Keyword(s):  
G Protein ◽  
High Affinity ◽  
Buffer Solutions ◽  
G Protein Coupled ◽  
Adenine Receptors

Molecular basis of G protein-coupled receptor high affinity for gastrin-releasing peptide (GRP)

2003 ◽  
Vol 124 (4) ◽  
pp. A469
Author(s):  
Tomoo Nakagawa ◽  
Jose A. Tapia ◽  
Kenji Tokita ◽  
Samuel Mantey ◽  
Michael Schumann ◽  
...  
Keyword(s):  
G Protein ◽  
Molecular Basis ◽  
G Protein Coupled Receptor ◽  
Gastrin Releasing Peptide ◽  
High Affinity ◽  
G Protein Coupled

scholarly journals II. Regulation of neuropeptide receptors in enteric neurons

1998 ◽  
Vol 274 (5) ◽  
pp. G792-G796
Author(s):  
Karen McConalogue ◽  
Nigel W. Bunnett
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Target Cell ◽  
Biological Effects ◽  
G Protein Coupled Receptors ◽  
Enteric Neurons ◽  
Heterotrimeric G Proteins ◽  
High Affinity ◽  
Neuropeptide Receptors ◽  
G Protein Coupled

Neuropeptides exert their diverse biological effects by interacting with G protein-coupled receptors (GPCRs). In this review we address the question, What regulates the ability of a target cell, in particular a neuron, to respond to a neuropeptide? Available evidence from studies of many GPCRs in reconstituted systems and transfected cell lines indicates that much of this regulation occurs at the level of the receptor and serves to alter the capacity of the receptor to bind ligands with high affinity and to couple to heterotrimeric G proteins. Although some of the knowledge gained from these studies is applicable to the regulation of neuropeptide receptors on neurons, at present there are far more questions than answers.

Multiple carriers for dipeptide transport: carrier-mediated transport of glycyl-L-proline in renal BBMV

1991 ◽  
Vol 261 (4) ◽  
pp. F670-F678
Author(s):  
H. A. Skopicki ◽  
K. Fisher ◽  
D. Zikos ◽  
R. Bloch ◽  
G. Flouret ◽  
...  
Keyword(s):  
Brush Border Membrane ◽  
Competitive Inhibition ◽  
Membrane Vesicles ◽  
The Other ◽  
Lower Affinity ◽  
High Affinity ◽  
Carrier Mediated Transport ◽  
Transport Carrier ◽  
Dipeptide Transport ◽  
Lower Capacity

To determine whether multiple carriers are responsible for luminal uptake of glycyl-L-proline (Gly-Pro) in the renal proximal tubule, transport of Gly-[3H]Pro was measured in brush-border membrane vesicles (BBMV). A Line-weaver-Burk analysis of Michaelis-Menten kinetics revealed the presence of two carriers: a lower affinity, higher capacity carrier (Km = 1.3 x 10(-2) M; Vmax = 4.6 x 10(-8) mol.mg-1.min-1) and a higher affinity, lower capacity carrier (Km = 2.7 x 10(-7) M; Vmax = 7.8 x 10(-13) mol.mg-1.min-1). The dipeptides Gly-Sar, beta Ala-His, and pyroGlu-His competitively inhibited the low-affinity carrier. No effect on the Km or Vmax of Gly-Pro transport in this range was seen in the presence of the dipeptides Gly-Gly or cycloHis-Pro. The high-affinity carrier exhibited a different inhibition spectrum. Competitive inhibition of Gly-Pro transport was demonstrated for the dipeptides Gly-Gly and Gly-Sar. However, none of the other peptides tested above altered Gly-Pro transport in the high-affinity range, including pyroGlu-His, which is transported by a high-affinity carrier. At both low (4 x 10(-8) M) and high (4 x 10(-3) M) concentrations, uptake of Gly-Pro was stimulated in the presence of an inwardly directed H+ gradient but was unaffected by the presence of an inward Na+ gradient. In addition, measurements in the presence of valinomycin and an outwardly directed K+ gradient strongly suggest that H(+)-stimulated uptake at both concentrations is electrogenic.(ABSTRACT TRUNCATED AT 250 WORDS)

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