Trends in Pharmacological Sciences
PrinciplesCalculating receptor number from binding experiments using same compound as radioligand and competitor
Abstract
Saturation experiments using increasing concentrations of .radioligand are commonly used to determine receptor number and affinity, but this protocol is not feasible in all situations. Alternatively, competitive binding experiments are often performed in which binding of a single concentration of .radioligand is competed for by multiple concentrations of the same unlabelled ligand, but the analysis of such data has been difficult. Antonio DeBlasi and colleagues present here a simple method for calculating receptor number and affinity from competitive binding data. This experimental protocol is useful when a single class of binding site is present, but is often not able to detect the presence of two classes of site.
References (3)
- P.J. Munson
Methods Emymol.
(1983)
Cited by (248)
Molecular and in vivo phenotyping of missense variants of the human glucagon receptor
2022, Journal of Biological ChemistryNaturally occurring missense variants of G protein–coupled receptors with loss of function have been linked to metabolic disease in case studies and in animal experiments. The glucagon receptor, one such G protein–coupled receptor, is involved in maintaining blood glucose and amino acid homeostasis; however, loss-of-function mutations of this receptor have not been systematically characterized. Here, we observed fewer glucagon receptor missense variants than expected, as well as lower allele diversity and fewer variants with trait associations as compared with other class B1 receptors. We performed molecular pharmacological phenotyping of 38 missense variants located in the receptor extracellular domain, at the glucagon interface, or with previously suggested clinical implications. These variants were characterized in terms of cAMP accumulation to assess glucagon-induced Gαs coupling, and of recruitment of β-arrestin-1/2. Fifteen variants were impaired in at least one of these downstream functions, with six variants affected in both cAMP accumulation and β-arrestin-1/2 recruitment. For the eight variants with decreased Gαs signaling (D63ECDN, P86ECDS, V96ECDE, G125ECDC, R2253.30H, R3085.40W, V3686.59M, and R3787.35C) binding experiments revealed preserved glucagon affinity, although with significantly reduced binding capacity. Finally, using the UK Biobank, we found that variants with wildtype-like Gαs signaling did not associate with metabolic phenotypes, whereas carriers of cAMP accumulation-impairing variants displayed a tendency toward increased risk of obesity and increased body mass and blood pressure. These observations are in line with the essential role of the glucagon system in metabolism and support that Gαs is the main signaling pathway effecting the physiological roles of the glucagon receptor.
GLP-2 and GIP exert separate effects on bone turnover: A randomized, placebo-controlled, crossover study in healthy young men
2019, BoneCitation Excerpt :Sigmoid curves were fitted logistically with a Hill slope of 1.0. Calculations of Ki values were based on the Cheng Prussoffs formula [29]. The study protocol was approved by the Scientific Ethical Committee of the Capital Region of Denmark (protocol no. H-16047626) and the Danish Data Protection Agency (journal no. SUND-2017-21), and registered at ClinicalTrials.gov (NCT03159741).
Glucagon-like peptide-2 (GLP-2) and glucose-dependent insulinotropic polypeptide (GIP) both inhibit bone resorption in humans but the underlying mechanisms are poorly understood. In vitro, GLP-2 activates the GIP-receptor (GIPR).
Based on in vitro studies, we hypothesized that the antiresorptive effect of GLP-2 was mediated through the GIPR. This was tested using the selective GIPR-antagonist GIP(3-30)NH2.
The study was a randomized, single-blinded, placebo-controlled, crossover study conducted at Hvidovre University Hospital, Denmark.
Eight healthy young men were included and studied on four study days: GIP (200 μg), GLP-2 (800 μg), GIP(3-30)NH2 (800 pmol/kg/min) + GLP-2 (800 μg), and placebo.
The main outcomes were bone resorption measured as collagen type 1 C-terminal telopeptide (CTX) and bone formation measured as procollagen type 1 N-terminal propeptide (P1NP).
CTX (mean ± SEM) significantly decreased after both GIP (to 55.3 ± 6.3% of baseline at t = 90 min) and GLP-2 (to 60.5 ± 5.0% of baseline at t = 180 min). The maximal reduction in CTX after GIP(3-30)NH2 + GLP-2 (to 63.2 ± 3.1% of baseline) did not differ from GLP-2 alone (p = 0.95) nor did net AUC0–240 (−6801 ± 879%*min vs −6027 ± 648%*min, p = 0.56). At t = 30 min, GIP significantly (p < 0.0001) increased P1NP to 115.1 ± 2.2% of baseline compared with 103.1 ± 1.5% after placebo. Both GLP-2 and GIP(3-30)NH2 + GLP-2 significantly (p < 0.0001) decreased P1NP to 91.3 ± 1.1% and 88.1 ± 3.0% of baseline, respectively (at t = 45 min) compared with placebo.
GIPR antagonism did not inhibit the GLP-2-induced reduction in bone resorption (CTX) in healthy young men. In contrast to GLP-2, GIP increased P1NP despite decreasing CTX indicating an uncoupling of bone resorption from formation. Thus, GLP-2 and GIP seem to exert separate effects on bone turnover in humans.
Use of virus-like particles as a native membrane model to study the interaction of insulin with the insulin receptor
2019, Biochimica et Biophysica Acta - BiomembranesThere is emerging evidence of the utility of virus-like particles (VLPs) as a novel model for the study of receptor-ligand interactions in a native plasma membrane environment. VLPs consist of a viral core protein encapsulated by portions of the cell membrane with membrane proteins and receptors expressed in their native conformation. VLPs can be generated in mammalian cells by transfection with the retroviral core protein (gag). In this study, we used Chinese hamster ovary (CHO T10) cells stably overexpressing the insulin receptor (IR) to generate IR bearing VLPs. The diameter and size uniformity of VLPs were estimated by dynamic light scattering and morphological features examined by scanning electron microscopy. The presence of high affinity IR on VLPs was demonstrated by competitive binding assays (KD: 2.3 ± 0.4 nM, n = 3), which was similar to that on the parental CHO T10 cells (KD: 2.1 ± 0.4 nM, n = 3). We also report that increases or decreases in membrane cholesterol content by treatment with methyl-β-cyclodextrin (MBCD) or cholesterol pre-loaded methyl-β-cyclodextrin (cMBCD), respectively, substantially decreased insulin binding (> 30%) to both VLPs and cells, and we speculate this is due to a change in receptor disposition. We suggest that this novel finding of decreases in insulin binding in response to changes in membrane cholesterol content may largely account for the unexplained decreases in insulin signalling events previously reported elsewhere. Finally, we propose VLPs as a viable membrane model for the study of insulin-IR interactions in a native membrane environment.
Human GIP(3-30)NH<inf>2</inf> inhibits G protein-dependent as well as G protein-independent signaling and is selective for the GIP receptor with high-affinity binding to primate but not rodent GIP receptors
2018, Biochemical PharmacologyGIP(3-30)NH2 is a high affinity antagonist of the GIP receptor (GIPR) in humans inhibiting insulin secretion via G protein-dependent pathways. However, its ability to inhibit G protein-independent signaling is unknown. Here we determine its action on arrestin-recruitment and receptor internalization in recombinant cells. As GIP is adipogenic, we evaluate the inhibitory actions of GIP(3-30)NH2 in human adipocytes. Finally, we determine the receptor selectivity of GIP(3-30)NH2 among other human and animal GPCRs.
cAMP accumulation and β-arrestin 1 and 2 recruitment were studied in transiently transfected HEK293 cells and real-time internalization in transiently transfected HEK293A and in HEK293A β-arrestin 1 and 2 knockout cells. Furthermore, human subcutaneous adipocytes were assessed for cAMP accumulation following ligand stimulation. Competition binding was examined in transiently transfected COS-7 cells using human 125I-GIP(3–30)NH2. The selectivity of human GIP(3-30)NH2 was examined by testing for agonistic and antagonistic properties on 62 human GPCRs.
Human GIP(3-30)NH2 inhibited GIP(1-42)-induced cAMP and β-arrestin 1 and 2 recruitment on the human GIPR and Schild plot analysis showed competitive antagonism with a pA2 and Hill slope of 16.8 nM and 1.11 ± 0.02 in cAMP, 10.6 nM and 1.15 ± 0.05 in β-arrestin 1 recruitment, and 10.2 nM and 1.06 ± 0.05 in β-arrestin 2 recruitment. Efficient internalization of the GIPR was dependent on the presence of either β-arrestin 1 or 2. Moreover, GIP(3-30)NH2 inhibited GIP(1-42)-induced internalization in a concentration-dependent manner and notably also inhibited GIP-mediated signaling in human subcutaneous adipocytes. Finally, the antagonist was established as GIPR selective among 62 human GPCRs being species-specific with high affinity binding to the human and non-human primate (Macaca fascicularis) GIPRs, and low affinity binding to the rat and mouse GIPRs (Kd values of 2.0, 2.5, 31.6 and 100 nM, respectively).
In conclusion, human GIP(3–30)NH2 is a selective and species-specific GIPR antagonist with broad inhibition of signaling and internalization in transfected cells as well as in human adipocytes.
Validation of a Na<sup>+</sup>-shift binding assay for estimation of the intrinsic efficacy of ligands at the A<inf>2A</inf> adenosine receptor
2017, Journal of Pharmacological and Toxicological MethodsDetermination of the intrinsic efficacy of ligands at the A2A receptor is important for selecting drug candidates, e.g. in the case of inflammatory diseases where agonists are searched for or in Parkinson disease (antagonists).
Three functional binding assays were compared with up to seven ligands with different efficacies: the GTP-shift method based on the decrease of affinity observed with agonists when GTP is added to the competition binding assay; the Ki ratio method based on the different affinity states of the receptor when using an agonist or antagonist radioligand and the Na+-shift assay based on the difference of affinity of agonists when tested in a medium containing a divalent cation (50 mM MgCl2) favoring the G protein coupled agonist-receptor complex or sodium (100 mM NaCl) as negative allosteric modulator.
The Na+-shift assay proposed herein successfully discriminated the full agonists CGS21680, NECA and adenosine (IC50 ratio = 13–14) from the weak inverse agonists ZM241385 and IBMX (IC50 ratio = 0.85) and the partial agonists LUF5834 and regadenoson (IC50 ratios equal to 3 and 10, respectively).
We conclude that the Na+-shift assay proposed herein for the A2A receptors has been validated and represents a rapid, economic and efficient functional binding assay to be used in a drug development program for early estimation of the intrinsic efficacy of hits.
Purification of Functional CB<inf>1</inf> and Analysis by Site-Directed Fluorescence Labeling Methods
2017, Methods in EnzymologyThe human cannabinoid receptor, CB1, has been difficult to purify in a functional form, hampering structural and biophysical studies. Here, we present our approaches for obtaining pure, detergent solubilized, functional CB1. We also discuss our site-directed fluorescence labeling (SDFL) methods for identifying different structural changes that CB1 can undergo upon binding different cannabinoid ligands.
To identify optimal CB1 constructs for these studies (those with the best expression levels, solubility in detergent and function), we first screened various CB1-green fluorescent protein chimeras in a mammalian expression system. Once identified, we then tagged the best candidates with the 1D4 epitope (the C-terminus of rhodopsin) and purified them using a single-step immunoaffinity process. The resulting, highly pure proteins retain their ability to activate G-protein, and are ~ 85% functional, as assessed by radioligand binding studies.
The SDFL studies involve introducing single cysteine residues at key places in the receptor, then labeling them with a small fluorophore, bimane. The spectral properties of the bimane probe are then monitored before and after addition of cannabinoid ligands. Changes in fluorescence of the attached probe indicate regions of the receptor undergoing conformational changes upon ligand binding.
Together, these approaches set the stage for a deeper understanding of the structure and function of CB1. Access to pure, functional CB1 makes subsequent structural studies possible (such as crystallography and single-particle EM analysis), and the SDFL studies enable a better structural and mechanistic understanding of this key receptor and the dynamic changes it undergoes during activation and attenuation.