Characterization of radioligand binding to a transmembrane receptor reconstituted into Lipobeads

Lipobeads are hydrogel beads surrounded by a lipid bilayer membrane and have been developed to act as a cell analogue. The FLAG-tagged M(2) muscarinic receptor was incorporated onto the surface of the Lipobead by incubating pre-Lipobeads with proteoliposomes containing the receptor. Receptors reconstituted onto the surface of the Lipobeads were functional in that they bound the antagonists quinuclidinylbenzilate and scopolamine with characteristic muscarinic affinities. This demonstrates the feasibility of using Lipobeads to study the binding properties of the M(2) muscarinic receptor and offers a promising approach to the study of transmembrane protein biology in general.     

A novel nicotinic acetylcholine receptor antagonist radioligand for PET studies

Using positron emission tomography (PET) with a specific and selective radioligand targeting nicotinic acetylcholine receptor (nAChR) would allow us to better understand various nAChR related CNS disorders. The use of radiolabeled nAChR antagonists would provide a much safer pharmacological profile, avoiding most peripheral side effects that might be generated from radiolabeled nAChR agonists even at the tracer level; thus, PET imaging with nAChR antagonists would facilitate clinical application. A potent and selective nAChR antagonist was labeled and characterized with PET in non-human primates. Its high brain uptake, high signal-to-noise ratio, and high specific binding strongly suggest a great potential to carry out imaging studies in humans. In addition, the use of a C-11 radiotracer would allow us to perform multiple PET studies in the same individual within a short time frame. The presence of an iodine atom in the molecule also allows the possibility to label with radioiodine for SPECT studies.     

Interaction of amiloride with alpha-adrenoreceptors: evidence from radioligand binding studies

We investigated the effect of amiloride on alpha-adrenoreceptors (alpha 1 and alpha 2) using radioligand binding techniques. Amiloride inhibited [3H]yohimbine and [3H]prazosin binding to alpha 2- and alpha 1-adrenoreceptors, respectively, from various tissues in a concentration-dependent manner. Amiloride was approximately 9-12 times more potent in inhibiting [3H]yohimbine binding to alpha 2-adrenoreceptors from rat tissues than from other mammalian tissues. However, it had almost the same potency in inhibiting [3H]prazosin binding to alpha 1-adrenoreceptors from rat as well as other mammalian tissues. Further, in rat tissues, amiloride was approximately 10 times more potent in inhibiting [3H]yohimbine than [3H]prazosin binding. Amiloride inhibited [3H]yohimbine binding noncompetitively and [3H]prazosin binding competitively. The inhibition of [3H]yohimbine and [3H]prazosin binding by amiloride was reversible. Since amiloride has been shown to be an inhibitor of Na+-H+ exchanger protein, we believe that it regulates the alpha 2-adrenoreceptors by binding to Na+ -H+ exchanger protein. Triamterene, a compound similar to amiloride in regard to diuretic effect, had very little effect on [3H]yohimbine and [3H]prazosin binding to rat kidney membranes, suggesting that the alpha-adrenoreceptor antagonistic properties of amiloride are not related to its antikaliuretic effect. The results of the present study suggest that some of the pharmacological actions of amiloride (antihypertensive and diuretic effects) can be explained in part by its regulatory effect on both alpha 1- and alpha 2-adrenoreceptors.     

Locating an antagonist in the 5-HT3 receptor binding site using modeling and radioligand binding

We have used a homology model of the extracellular domain of the 5-HT(3) receptor to dock granisetron, a 5-HT(3) receptor antagonist, into the binding site using AUTODOCK. This yielded 13 alternative energetically favorable models. The models fell into 3 groups. In model type A the aromatic rings of granisetron were between Trp-90 and Phe-226 and its azabicyclic ring was between Trp-183 and Tyr-234, in model type B this orientation was reversed, and in model type C the aromatic rings were between Asp-229 and Ser-200 and the azabicyclic ring was between Phe-226 and Asn-128. Residues located no more than 5 A from the docked granisetron were identified for each model; of 26 residues identified, 8 were found to be common to all models, with 18 others being represented in only a subset of the models. To identify which of the docking models best represents the ligand-receptor complex, we substituted each of these 26 residues with alanine and a residue with similar chemical properties. The mutant receptors were expressed in human embryonic kidney (HEK)293 cells and the affinity of granisetron determined using radioligand binding. Mutation of 2 residues (Trp-183 and Glu-129) ablated binding, whereas mutation of 14 other residues caused changes in the [(3)H]granisetron binding affinity in one or both mutant receptors. The data showed that residues both in and close to the binding pocket can affect antagonist binding and overall were found to best support model B.     

Role of angiotensin II receptor subtypes in porcine basilar artery: functional, radioligand binding, and cell culture studies

We aimed to clarify responsiveness to angiotensin (Ang) II in the porcine basilar artery and the role of Ang II receptor subtypes by functional, radioligand binding, and cell culture studies. Ang II induced more potent contractions in the proximal part than in the distal part of isolated porcine basilar arteries. The contraction induced by Ang II was inhibited by the Ang II type 1 (AT1) receptor antagonist losartan, but the Ang II type 2 (AT2) receptor antagonist PD123319 enhanced it. After removal of the endothelium, the effect of losartan remained but the effect of PD123319 was abolished. The specific binding site of [3H]Ang II on the smooth muscle membrane was inhibited by losartan, but not by PD123319. Stimulation of angiotensin II increased nitric oxide (NO) production in cultured basilar arterial endothelial cells. This production was inhibited by PD123319 and the NO synthase inhibitor L-NG-nitroarginine. These results suggest that the contraction induced by Ang II might be mediated via the activation of AT1 receptors on the basilar arterial smooth muscle cells and be modulated via the activation of AT2 receptors on the endothelial cells, followed by NO production.     

Characterization of a novel low affinity receptor for IFN-gamma on adherent human monocytes by radioligand binding studies and chemical cross-linking

Freshly isolated monocytes in suspension express 2000 to 4000 high affinity receptors for IFN-gamma. Because monocytes change phenotypically as they migrate out of the circulation and adhere to extracellular matrix, modulation of the expression of IFN-gamma receptors may occur. In order to determine if adherence alone modulates the receptor for IFN-gamma, we have studied receptor expression in adherent human peripheral blood monocytes. Elutriation-purified monocytes were allowed to adhere to polystyrene overnight at 37 degrees C. These cells now expressed 1 to 2 x 10(5) low affinity (Ka = 10(8) liters/M) receptors for [125I]rIFN-gamma. Binding to this receptor was specific and saturable. The expression of these receptors occurred rapidly (within 3 h) after adherence and was not inhibited by cycloheximide treatment. Binding to the receptor was abrogated by treating cells with trypsin, but was enhanced after treatment with alkaline protease or proteinase K. mAb against the high affinity receptor did not block binding to the low affinity receptor on adherent cells. The low affinity receptor transduced a signal to the cell as measured by the IFN-gamma-induced enhancement in FcR for human IgG1. The structure of the receptor on adherent cells was investigated by chemical cross-linking techniques. A receptor-[125I]rIFN-gamma complex was observed by SDS-PAGE to have a Mr of 180,000 to 200,000. Reduction of this complex with 2-ME resulted in the loss of the high Mr complex and the appearance of a doublet of lower Mr of 68,000 and 82,000. In contrast, cross-linking of monocytes in suspension yielded a complex of 110,000 to 120,000 Mr, which was unchanged upon reduction. Upon adherence, human monocytes express large numbers of a novel receptor for rIFN-gamma which is capable of stimulating the cell. This receptor appears to be composed of at least two components which are disulfide linked and structurally differs from the high affinity receptor on nonadherent monocytes.     

Tissue slices in radioligand binding assays: studies in brain, pineal and muscle

The use of tissue homogenates in receptor binding assays raises serious questions as to the physiological value of a preparation which examines receptors (binding sites) in disrupted tissue. In order to usefully study the regulatory properties of neurotransmitter receptors under physiological conditions, the necessity for tissue preparations which retain some degree of cellular integrity is clear. We review here the experiments which have utilized intact tissue - largely in the form of thick slices - to perform radioligand binding assays. There are many reports which note marked differences between studies in intact versus broken cell preparations. For example, significant discrepancies in KD and Bmax values are apparent for [3H] quinuclidinyl benzilate (muscarinic) and [3H] ouabain (Na+/K+-ATP ase, sodium pump) sites in brain and muscle respectively. A further example is the well-described stimulatory effect of GABA on benzodiazepine binding sites which is not seen in tissue slices. Other examples are highlighted. For all ligands so far examined, binding to slices is reversible, stereospecific, saturable, displaceable by appropriate drugs and of high affinity (nM). The method developed in our own laboratory is inexpensive, rapid and involves a minimum of tissue preparation. The technique is so simple as to allow many workers to enter this field who would not otherwise have done so. We suggest that metabolically active tissue slices offer the simplest approach to the study of cell-surface receptor regulation in living tissue.     

Histamine type I (H1) receptor radioligand binding studies on normal T cell subsets, B cells, and monocytes

We have documented a single, specific binding site for [3H]pyrilamine on normal human T helper, T suppressor, B cells, and monocytes. The binding of the radioligand to its receptor is reversible with cold H1 antagonist, saturates at 40 to 60 nM, and binding equilibrium is achieved in 2 to 4 min. Using a computer program (Ligand), we calculated the dissociation constants, binding capacities, and numbers of receptors per cell for each of the different cell types. Monocytes were found to have the highest affinity (mean KD +/- SD; 3.8 +/- 4.8 nM) for [3H]pyrilamine, followed by T helper cells (KD = 5.0 +/- 6.6 nM), B cells (KD = 14.2 +/- 2.0 nM), and T suppressor cells (KD = 44.6 +/- 49.4 nM). T suppressor cells were found to express the higher number of H1 receptors per cell (35,697 +/- 15,468), followed by B cells (10,732 +/- 9060), T helper cells (6838 +/- 8167), and monocytes (5589 +/- 2266). The kinetics of binding for this radioligand was carried out in resting and mitogen-stimulated T cells over a 48-hr period. We found that the binding affinity for [3H]pyrilamine increased over the 48-hr period, whereas the number of receptors per T cell was essentially unchanged. In contrast, T cells stimulated with Con A or PHA were shown to have a greater than fourfold increase in the number of receptors per cell, whereas the binding affinity for [3H]pyrilamine decreased over the 48-hr period. Preincubation of T cells with unlabeled histamine before carrying out the radioligand binding assay resulted in a decrease in the binding affinity of the receptors to [3H]pyrilamine, but the number of receptors per cell did not change significantly. Although the function of H1 receptors on T cells, B cells, and monocytes has not been completely defined, this receptor has the potential of playing an important role in modulating the immune response.     

Folate receptor expression in carcinomas and normal tissues determined by a quantitative radioligand binding assay

The folate receptor (FR) is a valuable therapeutic target that is highly expressed on a variety of cancers. The current development of folate-targeted cancer therapies has created the need for quantitating functional FRs in clinical specimens. In this article, we report on the creation of a highly sensitive radioactive binding method for quantitatively measuring FR expression in frozen tissue homogenates. Expression was positive in approximately 89% of human ovarian carcinomas but was negligible in both mucinous ovarian carcinomas and normal ovary. Expression was also significant in carcinomas of the kidney, endometrium, lung, breast, bladder, and pancreas. Normal tissues from humans and six different laboratory species were also analyzed; surprisingly, some interspecies variability in FR expression (especially in kidney, spleen, and lung tissue) was found. Interestingly, normal human lung tissue displayed high expression levels, whereas expression in normal lung of the other species was negligible. However, considering that folate-drug conjugates fail to accumulate in the lungs of patients, the consequence of this finding was not considered to be of clinical concern. Overall, this new methodology is reliable for determining functional FR expression levels in tissues, and it could possibly be a useful clinical test to determine patient candidacy for FR-targeted therapeutics.     

Development of an efficient and selective radioligand for bradykinin B1 receptor occupancy studies

We have developed an efficient and selective radioligand, the [³⁵S]-radiolabeled dihydroquinoxalinone derivative, 4, for an ex vivo receptor occupancy assay in transgenic rats over-expressing the human bradykinin B₁ receptor.     

Structural studies of receptor binding by cholera toxin mutants.

The wide range of receptor binding affinities reported to result from mutations at residue Gly 33 of the cholera toxin B-pentamer (CTB) has been most puzzling. For instance, introduction of an aspartate at this position abolishes receptor binding, whereas substitution by arginine retains receptor affinity despite the larger side chain. We now report the structure determination and 2.3-A refinement of the CTB mutant Gly 33-->Arg complexed with the GM1 oligosaccharide, as well as the 2.2-A refinement of a Gly 33-->Asp mutant of the closely related Escherichia coli heat-labile enterotoxin B-pentamer (LTB). Two of the five receptor binding sites in the Gly 33-->Arg CTB mutant are occupied by bound GM1 oligosaccharide; two other sites are involved in a reciprocal toxin:toxin interaction; one site is unoccupied. We further report a higher resolution (2.0 A) determination and refinement of the wild-type CTB:GM1 oligosaccharide complex in which all five oligosaccharides are seen to be bound in essentially identical conformations. Saccharide conformation and binding interactions are very similar in both the CTB wild-type and Gly 33-->Arg mutant complexes. The protein conformation observed for the binding-deficient Gly 33-->Asp mutant of LTB does not differ substantially from that seen in the toxin:saccharide complexes. The critical nature of the side chain of residue 33 is apparently due to a limited range of subtle rearrangements available to both the toxin and the saccharide to accommodate receptor binding. The intermolecular interactions seen in the CTB (Gly 33-->Arg) complex with oligosaccharide suggest that the affinity of this mutant for the receptor is close to the self-affinity corresponding to the toxin:toxin binding interaction that has now been observed in crystal structures of three CTB mutants.     

Determination of radioligand specific activity using competition binding assays.

Radioligand binding assays are routinely utilized in laboratories throughout the world to study receptors and their related binding sites, carrier proteins, and enzymes. To accurately estimate equilibrium binding parameters, such as the equilibrium dissociation constant (Kd) and maximal number of binding sites (Bmax), the investigator must know the correct value of the specific activity of the radioligand. If the specific activity is overestimated the Kd and Bmax values will be underestimated, while underestimation of the specific activity results in an overestimation of the Kd and Bmax. The present communication describes a simple and rapid method for determining the specific activity of a radioligand using homologous competition binding assays. Performing the competition assays at two or more different concentrations of the radioligand allows the specific activity to be determined from the IC50 values without the need of analytical methods to quantify minute amounts of the radioligand. In addition to providing the specific activity, use of this method estimates the Kd for the radioligand. This method was utilized to determine the specific activity and Kd for two blockers of the dopamine uptake carrier, [3H]GBR-12935 and [3H]-CFT, which share a common binding site in the striatum.     

Behavioral and radioligand binding evidence for irreversible dopamine receptor blockade by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline

N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), an irreversible alpha adrenergic antagonist, also acts as a potent and longlasting in vivo antagonist of D-2 dopamine receptors. Rats given EEDQ 3-10 mg/kg i.p. exhibit catalepsy and greatly reduced apomorphine-induced stereotypy, behavioral effects associated with D-2 dopamine receptor blockade. These effects are apparent up to 4 days after drug administration, with scores returning to control level by day 7. In vitro receptor binding assays of striatal membrane preparations from these animals using the radioligand 3H-spiroperidol directly demonstrate that EEDQ is a potent D-2 dopamine receptor antagonist, revealing the apparent basis of the behavioral effects of EEDQ. This antagonism proceeds via a reduction in D-2 receptor Bmax, with no change in the observed KD for 3H-spiroperidol, and is resistant to extensive washing of the membrane preparation after in vivo EEDQ exposure. These observations suggest that EEDQ inhibition of D-2 receptors is irreversible. Administration of behaviorally active doses of EEDQ effect a reduction of 50-85% in D-2 receptor number. Recovery of this loss roughly parallels recovery of normal catalepsy and apomorphine stereotypy scores. These doses of EEDQ also reduce binding of 3H-flupentixol to D-1 and 3H-dopamine to D-3 type dopaminergic binding sites, putative dopamine receptors with no known behavioral correlates. Recovery of D-1 and D-3 binding also occurs with a similar timecourse. Because of the apparent covalent nature of its interaction with dopamine receptors and because of its activity after peripheral administration, EEDQ may prove useful in the study of the function and turnover of dopamine receptors.     

Characterization of human and rodent native and recombinant adenosine A2B receptors by radioligand binding studies

Adenosine A_2B receptors of native human and rodent cell lines were investigated using [³H]PSB-298 [(8-{4-[2-(2-hydroxyethylamino)-2-oxoethoxy]phenyl}-1-propylxanthine] in radioligand binding studies. [³H]PSB-298 showed saturable and reversible binding. It exhibited a K_D value of 60 ± 1 nM and limited capacity (B_max = 3.511 fmol per milligram protein) at recombinant human adenosine A_2B receptors expressed in human embryonic kidney cells (HEK-293). The addition of sodium chloride (100 mM) led to a threefold increase in the number of binding sites recognized by the radioligand. The curve of the agonist 5′-N-ethylcarboxamidoadenosine (NECA) was shifted to the right in the presence of NaCl, while the curve of the antagonist PSB-298 was shifted to the left, indicating that PSB-298 may be an inverse agonist at A_2B receptors. Adenosine A_2B receptors were shown to be the major adenosine A₂ receptor subtype on the mouse neuroblastoma x rat glioma hybrid cell line NG108-15 cells. Binding studies at rat INS-1 cells (insulin secreting cell line) demonstrated that [³H]PSB-298 is a selective radioligand for adenosine A_2B binding sites in this cell line.     

The use of site-directed mutagenesis, transient transfection, and radioligand binding

Receptor-ligand interactions have traditionally been evaluated using a number of biochemical techniques including radioligand binding, photoaffinity labeling, crosslinking, and chemical modification. In modern biochemistry, these approaches have largely been superseded by site-directed mutagenesis in the study of protein function, owing in part to a better understanding of the chemical properties of oligonucleotides and to the ease with which mutant clones can now be generated. The Altered Sites II in vitro Mutagenesis System from the Promega Corporation employs oligonucleotides containing two mismatches to introduce specific nucleotide substitutions in the nucleic acid sequence of a target DNA. One of these mismatches will alter the primary sequence of a given protein, whereas the second will give rise to a silent restriction site that is used to screen for mutants. Transient transfection of tsA201 cells with mutant cDNA constructs using calcium phosphate as a carrier for plasmid DNA permits expression of recombinant receptors that can be characterized using radioligand binding assays. In this article, we focus on site-directed mutagenesis, heterologous expression in eukaryotic cells, and radioligand binding as a methodology to enable the characterization of receptor-ligand interactions.     

Competitive binding studies with multiple sites: Effects arising from depletion of the free radioligand

Apparently anomalous behaviour arises if a radiolabelled drug and a non-radioactive drug compete for binding to a membrane-bound receptor when (a) there is severe depletion of the radiolabelled drug and (b) the non-radioactive drug binds to a heterogeneous population of binding sites. In extreme cases, binding of the non-radioactive drug to the sites of high affinity can be obscured completely. This phenomenon is illustrated by the binding of carbachol to muscarinic receptors, estimated by inhibition of a radiolabelled antagonist.     

Orphanin FQ/nociceptin receptor binding studies

A review of the binding studies performed on the receptor (ORL) for Orphanin FQ/Nociceptin is presented. Binding studies have been conducted using a variety of receptor sources: cell lines expressing the cloned receptor, cell lines endogenously expressing the receptor, and brain and other tissue from several different species. Binding studies of opioids, new ligands and antagonists at the ORL receptor are briefly discussed. Saturation, competition and binding kinetic experiments, and the effects of buffer composition are reviewed. There are numerous instances of conflicting data in published reports on OFQ; the basis for these disparities is as yet undetermined. This review endeavors to compile the results and conditions employed in binding studies as an aid to current and new researchers in this field. In an attempt to explain binding disparities, we have determined that Orphanin/Nociceptin binds to glass fiber filtermats in a "specific" manner; these new data are presented.     

Chemoreception in hydra: specific binding of glutathione to a membrane fraction.

Specific binding of reduced [35S]glutathione (GSH) was measured using a crude membrane fraction of Hydra vulgaris (attenuata). The specific binding shows both rapid displaceable and nondisplaceable components. Rapid displaceable binding accounted for 20% of the total specific binding. Data from saturation binding experiments indicates half maximal total specific binding occurs at 2 microM GSH which is similar to reported EC50 values from behavioral experiments. Calcium is required for displaceable binding of GSH to the putative receptor. Oxidized glutathione (GSSG), an antagonist of the GSH-activated feeding response, and S-methylglutathione (GSM), an agonist of the feeding response, inhibit the binding of radiolabeled GSH to the putative receptor. Glutamate, a putative competitive antagonist of the GSH-activated feeding response in hydra, does not inhibit the specific binding of radiolabeled GSH to the receptor and must therefore block the feeding response by a mechanism other than competitive inhibition.