Agonists and Antagonists Recognize Different but Overlapping Populations of A1 Adenosine Receptors: Modulation of Receptor Number by MgCl2, Solubilization, and Guanine Nucleotides

A1 selective agonist and antagonist radioligands bind to the same A1 adenosine receptor binding subunit, as documented by photoaffinity labelling and partial peptide maps. In this study we document that although these radioligands recognize the same A1 adenosine receptor (A1AR), they recognize different numbers of A1ARs in bovine brain membranes, with agonist number being greater than antagonist number. Neither addition of guanine nucleotides nor removal of Mg2+ ions enhanced antagonist binding in membranes. On solubilization, agonists still recognized a greater number of A1ARs but addition of guanine nucleotides or removal of Mg2+ substantially increased the number of receptors detected with antagonist radioligands. The effects of Mg2+ and guanine nucleotides were not additive, suggesting that formation of a "low agonist-receptor-G protein state" by either modulating agent was sufficient to alter the receptor conformation such that it could be recognized by antagonist. These studies suggest that a proportion of the "precoupled A1AR-G protein complex" in membranes are in a conformation that cannot be recognized by antagonists and that membrane constraints are such that ions or guanine nucleotides cannot sufficiently modulate the conformation to allow it to recognize antagonists. On removal of membrane structure by solubilization, these constraints are removed.     

Photoaffinity labeling of the beta-adrenergic receptor

A new photoactive beta-adrenergic antagonist, p-azidobenzylcarazolol (pABC) has been synthesized by combining a carbazole moiety with a p-azido-benzyl substituent. The compound has been labeled with tritium to a specific activity of 26 Ci/mmol. In frog erythrocyte membranes, [3H]p-azido-benzylcarazolol binds to the beta-adrenergic receptor with the expected beta 2 specificity and with high affinity (KD congruent to 100 +/- 10 pM). Unlabeled p-azido-benzylcarazolol can irreversibly inactivate the [3H]dihydroalprenolol-binding activity of frog erythrocyte membranes in a photodependent manner which can be prevented by beta-adrenergic agents. Incubation of frog erythrocyte membranes or digitonin-solubilized preparations of these membranes or digitonin-solubilized preparations of these membranes which had been enriched in beta-adrenergic receptors by a Sepharose-alprenolol chromatography step led to covalent incorporation of radioactivity into a Mr = 58,000 peptide. Specific incorporation of [3H]pABC into the Mr = 58,000 peptide could be prevented by both beta-adrenergic agonists and antagonists. This peptide has previously been purified and shown to contain the beta-adrenergic receptor-binding site (Shorr, R. G. L., Lefkowitz, R. J., and Caron, M. G. (1981) J. Biol. Chem. 256, 5820-5826). Thus, photoaffinity labeling of the beta-adrenergic receptor protein directly identifies the same hormone-binding subunit as has been isolated by conventional purification techniques.     

Mapping of the acetylcholine binding site of the nicotinic acetylcholine receptor: [3H]nicotine as an agonist photoaffinity label

The agonist [3H]nicotine was used as a photoaffinity label for the acetylcholine binding sites on the Torpedo nicotinic acetylcholine receptor (AChR). [3H]nicotine binds at equilibrium with Keq = 0.6 microM to the agonist binding sites. Irradiation with 254-nm light of AChR-rich membranes equilibrated with [3H]nicotine resulted in covalent incorporation into the alpha- and gamma-subunits, which was inhibited by agonists and competitive antagonists but not by noncompetitive antagonists. Inhibition of labeling by d-tubocurarine demonstrated that the alpha-subunit was labeled via both agonist sites but the gamma-subunit was labeled only via the site that binds d-tubocurarine with high affinity. Within the alpha-subunit, 93% of the labeling was contained within a 20-kDa Staphylococcus aureus V8 proteolytic fragment beginning at Ser-173. Sequence analysis of this peptide indicated that approximately 80% of the incorporation was into Tyr-198, approximately 13% was into Cys-192, and approximately 7% was into Tyr-190. Chymotryptic digestion of the alpha-subunit confirmed that Tyr-198 was the principal amino acid labeled by [3H]nicotine. This confirmation required a novel radio-sequencing strategy employing omicron-phthalaldehyde, since the efficiency of photolabeling was low (approximately 1.0%) and the labeled chymotryptic peptide was not isolated in sufficient quantity to be identified by mass. [3H]Nicotine, which is the first photoaffinity agonist used, labels primarily Tyr-198 in contrast to competitive antagonist affinity labels, which label primarily Tyr-190 and Cys-192/Cys-193.     

N-linked oligosaccharides are responsible for rat striatal dopamine D2 receptor heterogeneity

The glycoprotein nature of the binding subunit of the dopamine D2 receptor in rat striatum has been examined by photoaffinity labeling receptor preparations with N-(p-azido-m-[125I]iodophenethyl)spiperone followed by treatment of crude membrane receptor or receptor fractions isolated from sodium dodecyl sulfate (SDS) polyacrylamide gels with endo- and exoglycosidases. The major photoaffinity labeled protein migrates as a heterogeneous species on 10% SDS polyacrylamide gels and ranges from 130,000 to 75,000 relative molecular mass (Mr). This heterogeneity can be explained by glycosylation of the receptor by complex-type N-linked oligosaccharides. Three fractions of labeled receptor were isolated from SDS polyacrylamide gels over a range of 130,000 to 75,000 Mr; after digestion with peptide-N4-[N-acetyl-beta-glucosaminyl] asparagine amidase, all fractions yielded a single peptide approximately 40,000 Mr. Treatment of photoaffinity labeled membranes with alpha-mannosidase was without effect. The dopamine D2 receptor appears to contain substantial amounts of sialic acid as treatment of photoaffinity labeled membranes with neuraminidase increased the receptor mobility on SDS polyacrylamide gels to a species of 50,000-54,000 Mr. Treatment of the receptor with neuraminidase followed by endo-alpha-N-acetylgalactosaminidase did not change the electrophoretic migration pattern from that seen after neuraminidase treatment alone, suggesting that the binding peptide contains no serine- or threonine-linked oligosaccharides. A smaller binding peptide of approximately 31,000 Mr is also apparent in crude photoaffinity labeled membranes. This material also contains N-linked oligosaccharide. Complete removal of N-linked oligosaccharide from the dopamine D2 receptor did not change the rank order potency of agonist and antagonist compounds to compete for [3H]spiperone binding to crude membrane fractions. The dopamine D2 receptor represents a highly glycosylated neural receptor.     

Photoaffinity labeling of A1-adenosine receptors

The ligand-binding subunit of the A1-adenosine receptor has been identified by photoaffinity labeling. A photolabile derivative of R-N6-phenylisopropyladenosine, R-2-azido-N6-p-hydroxyphenylisopropyladenosine (R-AHPIA), has been synthesized as a covalent specific ligand for A1-adenosine receptors. In adenylate cyclase studies with membranes of rat fat cells and human platelets, R-AHPIA has adenosine receptor agonist activity with a more than 60-fold selectivity for the A1-subtype. It competes for [3H]N6-phenylisopropyladenosine binding to A1-receptors of rat brain membranes with a Ki value of 1.6 nM. After UV irradiation, R-AHPIA binds irreversibly to the receptor, as indicated by a loss of [3H]N6-phenylisopropyladenosine binding after extensive washing; the Ki value for this photoinactivation is 1.3 nM. The p-hydroxyphenyl substituent of R-AHPIA can be directly radioiodinated to give a photoaffinity label of high specific radioactivity (125I-AHPIA). This compound has a KD value of about 1.5 nM as assessed from saturation and kinetic experiments. Adenosine analogues compete for 125I-AHPIA binding to rat brain membranes with an order of potency characteristic for A1-adenosine receptors. Dissociation curves following UV irradiation at equilibrium demonstrate 30-40% irreversible specific binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the probe is photoincorporated into a single peptide of Mr = 35,000. Labeling of this peptide can be blocked specifically and stereoselectively by adenosine receptor agonists and antagonists in a manner which is typical for the A1-subtype. The results indicate that 125I-AHPIA identifies the ligand-binding subunit of the A1-adenosine receptor, which is a peptide with Mr = 35,000.     

Photoaffinity labeling of the acetylcholine binding sites on the nicotinic receptor by an aryldiazonium derivative

p-(Dimethylamino)benzenediazonium fluoroborate (DDF) behaves, in the dark, as a reversible competitive antagonist of the electrical response of Electrophorus electricus electroplaque to acetylcholine and of the acetylcholine-gated single-channel currents recorded in the C2 mouse cell line. This chemically stable but highly photoreactive compound binds irreversibly to the acetylcholine receptor when irradiated by visible light. In vivo, it irreversibly blocks the postsynaptic response of E. electricus electroplaque to agonists. In vitro, it reduces the alpha-bungarotoxin-binding capacity of acetylcholine receptor rich membrane fragments prepared from Torpedo marmorata electric organ. Once reversibly bound to the T. marmorata acetylcholine receptor, this ligand can be selectively photodecomposed by an energy-transfer reaction involving a tryptophan residue(s) of the protein. By use of reagent concentrations that are below the dissociation constant at equilibrium, up to 60% of the agonist-binding sites are covalently labeled. Under these conditions the alpha subunit of the acetylcholine receptor is preferentially labeled, and this labeling is partially prevented by agonists or competitive antagonists. This protective effect is substantially increased by prior incubation with phencyclidine, a compound known to prevent the binding of DDF at the level of the high-affinity site for noncompetitive blockers [Kotzyba-Hibert, F., Langenbuch-Cachat, J., Jaganathen, J., Goeldner, M. P., & Hirth, C. G. (1985) FEBS Lett. 182, 297-301]. The incorporation of about one molecule of label in an agonist/competitive antagonist protectable manner per alpha-bungarotoxin-binding site suffices to fully block alpha-bungarotoxin binding to the membrane-bound receptor. Thus, DDF behaves as a monovalent photoaffinity label of the acetylcholine-binding site.     

Photoaffinity Labeling of Benzodiazepine Receptors in Rat Brain with Flunitrazepam Alters the Affinity of Benzodiazepine Receptor Agonist But Not Antagonist Binding

Abstract: Recently, it was proposed that β-carbolines interact with a subset of benzodiazepine (BZD) binding sites in mouse brain. This postulate was based upon evidence showing changes in binding properties of the BZD receptor following photoaffinity labeling of membranes with flunitrazepam (FLU). Under conditions in which 80% of specific [³H]diazepam binding was lost in photolabeled membranes, specific [³H]propyl β-carboline-3-carboxylate ([³H]PCC) binding was spared. In this study, the binding of the BZD antagonists [³H]PCC, [³H]Ro15 1788 and [³H]CGS 8216 was examined in rat brain membranes following photoaffinity labeling with FLU. No significant changes in the apparent K_D and small reductions in the B_max of ³H antagonist binding were observed. However, in the same membranes, up to 89% of specific [³H]FLU binding was lost. When [³H]PCC (0.05 nM) was used to label the receptors in control and photolabeled membranes, the ability of BZD receptor agonists to inhibit [³H]PCC binding was greatly diminished in the photolabeled membranes. In contrast, the potency of BZD antagonists remained the same in both control and treated membranes. Based upon PCC/[³H]Ro15 1788 competition experiments, the ability of PCC to discriminate between BZD receptor subtypes was unaffected by photoaffinity labeling of cortical membranes. Overall, these findings suggest that β-carbolines do not interact with a subset of BZD binding sites per se, but may be a consequence of the differential interaction of BZD agonists and antagonists with BZD binding sites that have been photoaffinity labeled with FLU. A possible mechanism underlying this phenomenon is discussed. The ability of photolabeled membranes to differentiate between BZD agonists and antagonists provides a potential screen for agonist and antagonist activity in compounds that interact with the BZD receptor.     

Photoaffinity labeling of the D2-dopamine receptor using a novel high affinity radioiodinated probe

The ligand binding subunit of the D2 subtype of the dopamine receptor has been identified by photoaffinity labeling. In order to develop a specific covalent receptor probe, an analogue of the potent D2 selective antagonist spiperone, N-(p-aminophenethyl)spiperone (NAPS) has been synthesized. The aminophenethyl substituent of NAPS can be radioiodinated to theoretical specific radioactivity (2,175 Ci/mmol) and then the arylamine group converted to an arylazide to yield a photosensitive probe [( 125I]N3-NAPS). In rat striatal membranes, the nonradiolabeled azide probe (N3-NAPS) binds to the receptor with high affinity (KD congruent to 1.6 +/- 0.05 nM) and upon photoactivation irreversibly decreases the number of available receptors in these membranes as measured by [3H]spiperone binding. More importantly, however, incubation of rat striatal membranes with [125I]N3-NAPS leads to the photodependent covalent incorporation of the probe into a peptide of Mr = 94,000 as assessed by autoradiography of gels after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Labeling of this Mr = 94,000 peptide can be blocked specifically and stereoselectively by dopaminergic antagonists such as (+)- and (-)-butaclamol but not by non-dopaminergic antagonists. Moreover, dopaminergic agonists also attenuate the covalent labeling of this peptide with an order of potency which is typically D2-dopaminergic. Therefore, the specificity of [125I]N3-NAPS labeling of the Mr = 94,000 peptide suggests that this peptide represents the ligand binding subunit of the D2-dopamine receptor.     

Localization of the strychnine binding site on the 48-kilodalton subunit of the glycine receptor

Amino acid residues that participate in antagonist binding to the strychnine-sensitive glycine receptor (GlyR) have been identified by selectively modifying functional groups with chemical reagents. Moreover, a region directly involved with strychnine binding has been localized in the 48-kDa subunit of this receptor by covalent labeling and proteolytic mapping. Modification of tyrosyl or arginyl residues promotes a marked decrease of specific [3H]strychnine binding either to rat spinal cord plasma membranes or to the purified GlyR incorporated into phospholipid vesicles. Occupancy of the receptor by strychnine, but not by glycine, completely protects from the inhibition caused by chemical reagents. Furthermore, these tyrosine- or arginine-specific reagents decrease the number of binding sites (Bmax) for [3H]strychnine binding without affecting the affinity for the ligand (Kd). These observations strongly suggest that such residues are present at, or very close to, the antagonist binding site. In order to localize the strychnine binding domain within the GlyR, purified and reconstituted receptor preparations were photoaffinity labeled with [3H]strychnine. The radiolabeled 48-kDa subunit was then digested with specific chemical proteolytic reagents, and the peptides containing the covalently bound radioligand were identified by fluorography after gel electrophoresis. N-Chlorosuccinimide treatment of [3H]strychnine-labeled 48K polypeptide yielded a single labeled peptide of Mr approximately 7300, and cyanogen bromide gave a labeled peptide of Mr 6200.(ABSTRACT TRUNCATED AT 250 WORDS)     

GnRH analogues--agonists and antagonists

GnRH analogues have achieved widespread clinical use for the control of reproduction in animals. Over 2000 analogues of GnRH have been developed and tested over the last 30 years. Paradoxical anti-fertility effects are seen when the more potent agonists are delivered continuously to animals. The evaluation of agonist potency depends largely on the model used and wide varying potencies are reported for the same agonist. The design of analogues has centered on improving the receptor-binding and subsequent activation for agonists. Antagonists have been produced with strong receptor binding but without activation. Deslorelin is classified as a superagonist, with a potency perhaps 100 times that of GnRH. The interactions between agonist potency, dose and duration of treatment largely determine whether pro- or anti-fertility effects are induced. Due to the peptide nature of the synthetic analogues oral administration and potential gastrointestinal enzymatic degradation poor bioavailability results necessitating a parenteral delivery system. Some GnRH antagonists have been associated with significant histamine release, inhibiting their widespread use. More recently, antagonists have been developed that avoid this side effect without compromising potency. However the GnRH antagonist development has lagged behind that of the agonists, in part related to their high cost of production. In conclusion, GnRH agonists have achieved widespread clinical use in animals for controlling reproduction in either pro- or anti-fertility roles, yet antagonist development has been slower.     

Probing the salmeterol binding site on the beta 2-adrenergic receptor using a novel photoaffinity ligand, [(125)I]iodoazidosalmeterol.

Salmeterol is a long-acting beta2-adrenergic receptor (beta 2AR) agonist used clinically to treat asthma. In addition to binding at the active agonist site, it has been proposed that salmeterol also binds with very high affinity at a second site, termed the "exosite", and that this exosite contributes to the long duration of action of salmeterol. To determine the position of the phenyl ring of the aralkyloxyalkyl side chain of salmeterol in the beta 2AR binding site, we designed and synthesized the agonist photoaffinity label [(125)I]iodoazidosalmeterol ([125I]IAS). In direct adenylyl cyclase activation, in effects on adenylyl cyclase after pretreatment of intact cells, and in guinea pig tracheal relaxation assays, IAS and the parent drug salmeterol behave essentially the same. Significantly, the photoreactive azide of IAS is positioned on the phenyl ring at the end of the molecule which is thought to be involved in exosite binding. Carrier-free radioiodinated [125I]IAS was used to photolabel epitope-tagged human beta 2AR in membranes prepared from stably transfected HEK 293 cells. Labeling with [(125)I]IAS was blocked by 10 microM (-)-alprenolol and inhibited by addition of GTP gamma S, and [125I]IAS migrated at the same position on an SDS-PAGE gel as the beta 2AR labeled by the antagonist photoaffinity label [125I]iodoazidobenzylpindolol ([125I]IABP). The labeled receptor was purified on a nickel affinity column and cleaved with factor Xa protease at a specific sequence in the large loop between transmembrane segments 5 and 6, yielding two peptides. While the control antagonist photoaffinity label [125I]IABP labeled both the large N-terminal fragment [containing transmembranes (TMs) 1-5] and the smaller C-terminal fragment (containing TMs 6 and 7), essentially all of the [125I]IAS labeling was on the smaller C-terminal peptide containing TMs 6 and 7. This direct biochemical evidence demonstrates that when salmeterol binds to the receptor, its hydrophobic aryloxyalkyl tail is positioned near TM 6 and/or TM 7. A model of IAS binding to the beta 2AR is proposed.     

Interactions of agonists and antagonists with beta-adrenergic receptors on intact L6 muscle cells

A binding assay has been developed to characterize beta-adrenergic receptors on intact L6 muscle cells. The affinity of beta-adrenergic receptors for the radioligand iodohydroxybenzylpindolol (IHYP) was the same in membrane preparations and in intact cells when determined by either equilibrium binding or kinetic analysis. The number of specific IHYP binding sites per cell was approximately the same on intact cells as on membranes. The pharmacological properties of antagonists indicated that the receptors on intact cells were identical to those on membranes. However, the beta-adrenergic receptors on intact cells had a 100-400 fold lower affinity at equilibrium for the agonist isoproterenol than did beta-adrenergic receptors on membranes. This low affinity of the receptor for agonists as measured by inhibition of radioligand binding in intact cells has also been observed in C6 (2) and S49 (3) cells. Our results suggest that beta receptors on intact cells after a 1 minute incubation was similar to the KD value for isoproterenol measured in membranes at equilibrium in the presence of GTP. After 1-2 minutes of exposure to a low concentration of agonist, binding of IHYP was no longer inhibited. These results suggest that agonists rapidly convert the beta receptors on intact cells to a state which has a low affinity for agonists. The affinity of the receptor for antagonists did not change during the incubation.     

Covalent labeling of the acetylcholine receptor from Torpedo electric tissue with the channel blocker [3H]triphenylmethylphosphonium by ultraviolet irradiation

The lipophilic cation [3H]triphenylmethylphosphonium, frequently used as a voltage sensor in membrane systems, binds reversibly to a site different from the acetylcholine binding site. This is concluded from the different pH dependences of the binding of these two ligands. Furthermore [3H]triphenylmethylphosphonium, previously identified as a channel blocker, can be covalently incorporated into acetylcholine receptor-rich membranes from Torpedo electric tissue by UV irradiation of the receptor-ligand complex. In the absence of effector, predominantly the alpha-polypeptide chains (Mr 40000) of the receptor protein are labeled by the radioactive ligand. The agonist carbamoylcholine strongly stimulates the labeling, but it directs the label predominantly to the delta- and beta-polypeptide chains. The antagonist D-tubocurarine and the virtually irreversible competitive antagonist alpha-bungarotoxin have qualitatively the same effect as the agonist carbamoylcholine. Significant differences were obtained with receptor-rich membranes prepared from Torpedo marmorata and Torpedo californica: No agonist- or antagonist-stimulated reaction was observed with the latter. The results are interpreted as an indication of a rearrangement of the receptor's quaternary structure caused by cholinergic effector binding preceding discrimination between agonists and antagonists.     

Mammalian beta-adrenergic receptors. Structural differences in beta 1 and beta 2 subtypes revealed by peptide maps

Photoaffinity labeling techniques using p-azido-m-[125I]iodobenzylcarazolol have recently demonstrated that both the beta 1- and beta 2-adrenergic receptor-binding subunits from mammalian tissues including heart, lung, and erythrocytes reside on peptides of Mr approximately equal to 62,000-64,000. In this study, a two-dimensional gel electrophoresis method for peptide mapping was used to investigate and compare the structure of beta 1 - and beta 2-adrenergic receptor subtypes. When the photoaffinity labeled Mr approximately equal to 62,000 peptides from the beta 2-adrenergic receptors of rat lung and erythrocyte are subjected to simultaneous proteolysis using Staphylococcus aureus V8 proteinase or papain, exactly the same peptide fragments are generated from each subunit. In contrast, when the Mr approximately equal to 62,000 peptide containing the beta 1-adrenergic receptor-binding subunit derived from the rat heart is proteolyzed simultaneously with the Mr approximately equal to 62,000 peptide containing the beta 2-adrenergic receptors from either lung or erythrocyte, the peptide fragments generated are distinctly different. Peptide maps of beta 1-adrenergic receptors from the myocardial tissue of different species (pig versus rat) yield slightly different maps while the maps derived from the beta 2-adrenergic receptors of hamster lung and rat lung or erythrocytes reveal no interspecies differences. These data suggest: 1) alterations in the primary structure of the beta-adrenergic receptor may be responsible for the pharmacological specificities characteristic of beta 1- and beta 2-adrenergic receptor subtypes; and 2) alterations in the primary structure of similar beta-adrenergic receptor subtypes across different species may relate to the magnitude of their phylogenetic differences.     

Peptide mapping of the insulin-binding site of the 130-kDa subunit of the insulin receptor by means of a novel cleavable radioactive photoprobe

A radioactive photoaffinity probe for the insulin receptor was prepared by derivatizing insulin at its B29 lysine with a novel crosslinking reagent having a cleavable azo linkage. Insulin receptors purified from human placental membranes were photoaffinity labeled with this probe. The photolabeled receptor was treated with dithionite to cleave the azo linkage, thereby removing the insulin ligand and transferring the radioactivity to the receptor protein. The radioactive labeled subunit was isolated and digested with elastase for peptide mapping and separation by high performance liquid chromatography. Results obtained indicated that it will be feasible to use this new photoaffinity probe to obtain radioactive peptides representing the insulin-binding site(s) on the receptor subunit.     

Conserved aromatic residues in the transmembrane region VI of the V1a vasopressin receptor differentiate agonist vs. antagonist ligand binding.

Despite their opposite effects on signal transduction, the nonapeptide hormone arginine-vasopressin (AVP) and its V1a receptor-selective cyclic peptide antagonist d(CH2)5[Tyr(Me)2]AVP display homologous primary structures, differing only at residues 1 and 2. These structural similarities led us to hypothesize that both ligands could interact with the same binding pocket in the V1a receptor. To determine receptor residues responsible for discriminating binding of agonist and antagonist ligands, we performed site-directed mutagenesis of conserved aromatic and hydrophilic residues as well as nonconserved residues, all located in the transmembrane binding pocket of the V1a receptor. Mutation of aromatic residues of transmembrane region VI (W304, F307, F308) reduced affinity for the d(CH2)5[Tyr(Me)2]AVP and markedly decreased affinity for the unrelated strongly hydrophobic V1a-selective nonpeptide antagonist SR 49059. Replacement of these aromatic residues had no effect on AVP binding, but increased AVP-induced coupling efficacy of the receptor for its G protein. Mutating hydrophilic residues Q108, K128 and Q185 in transmembrane regions II, III and IV, respectively, led to a decrease in affinity for both agonists and antagonists. Finally, the nonconserved residues T333 and A334 in transmembrane region VII, controlled the V1a/V2 binding selectivity for both nonpeptide and cyclic peptide antagonists. Thus, because conserved aromatic residues of the V1a receptor binding pocket seem essential for antagonists and do not contribute at all to the binding of agonists, we propose that these residues differentiate agonist vs. antagonist ligand binding.     

Microscopic kinetics and energetics distinguish GABA(A) receptor agonists from antagonists

Although agonists and competitive antagonists presumably occupy overlapping binding sites on ligand-gated channels, these interactions cannot be identical because agonists cause channel opening whereas antagonists do not. One explanation is that only agonist binding performs enough work on the receptor to cause the conformational changes that lead to gating. This idea is supported by agonist binding rates at GABA(A) and nicotinic acetylcholine receptors that are slower than expected for a diffusion-limited process, suggesting that agonist binding involves an energy-requiring event. This hypothesis predicts that competitive antagonist binding should require less activation energy than agonist binding. To test this idea, we developed a novel deconvolution-based method to compare binding and unbinding kinetics of GABA(A) receptor agonists and antagonists in outside-out patches from rat hippocampal neurons. Agonist and antagonist unbinding rates were steeply correlated with affinity. Unlike the agonists, three of the four antagonists tested had binding rates that were fast, independent of affinity, and could be accounted for by diffusion- and dehydration-limited processes. In contrast, agonist binding involved additional energy-requiring steps, consistent with the idea that channel gating is initiated by agonist-triggered movements within the ligand binding site. Antagonist binding does not appear to produce such movements, and may in fact prevent them.     

Determinants of antagonist binding at the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit, GluR-D. Role of the conserved arginine 507 and glutamate 727 residues.

Previous structural and mutagenesis studies indicate that the invariant alpha-amino and alpha-carboxyl groups of glutamate receptor agonists are engaged in polar interactions with oppositely charged, conserved arginine and glutamate residues in the ligand-binding domain of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor. To examine the role of these residues (R507 and E727 in the GluR-D subunit) in the discrimination between agonists and antagonists, we analyzed the ligand-binding properties of homomeric GluR-D and its soluble ligand-binding domain with mutations at these positions. Filter-binding assays using [3H]AMPA, an agonist, and [3H]Ro 48-8587, a high-affinity antagonist, as radioligands revealed that even a conservative mutation at R507 (R507K) resulted in the complete loss of both agonist and antagonist binding. In contrast, a negative charge at position 727 was necessary for agonist binding, whereas the isosteric mutation, E727Q, abolished all agonist binding but retained high-affinity binding for [3H]Ro 48-8587, displaceable by 7,8-dinitroquinoxaline-2,3-dione. Competition binding studies with antagonists representing different structural classes in combination with ligand docking experiments suggest that the role of E727 is antagonist-specific, ranging from no interaction to weak electrostatic interactions involving indirect and direct hydrogen bonding with the antagonist molecule. These results underline the importance of ion pair interaction with E727 for agonist activity and suggest that an interaction with R507, but not with E727, is essential for antagonist binding.     

Photoaffinity cross-linked A1 adenosine receptor-binding subunits. Homologous glycoprotein expression by different tissues

Mammalian A1 adenosine receptor-binding peptides can be visualized by covalently labeling them with the photoaffinity cross-linking ligand N6-2-(4-amino-3-[125I] iodophenyl)ethyladenosine followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/autoradiography. The proteins comprising the A1 adenosine receptor-binding subunit of rat brain and fat migrate with Mr 38,000. In this study, the glycoproteins representing the radiolabeled A1 adenosine receptor-binding subunit expressed in each of these tissues (brain and fat) were compared through the use of peptide mapping and exo- and endoglycosidase treatments. Peptide mapping studies with several enzymes demonstrate that the protein component of the radiolabeled A1 adenosine receptor-binding subunit is conserved between different tissues. Both labeled receptor peptides demonstrate a sensitivity to neuraminidase as evidenced by increased mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggesting that the receptors contain complex-type carbohydrate chains. Insensitivity to alpha-mannosidase suggests a lack of high mannose-type carbohydrate chains. Deglycosylation of the labeled receptor-binding subunits with endoglycosidase F results in a single labeled polypeptide of Mr 32,000 for both systems. These data suggest that the A1 adenosine receptor-binding subunits expressed in the rat brain and fat are similar glycoproteins as evidenced by similar overall molecular weights, identical peptide maps, and equivalent responses to endo- and exoglycosidase treatment.     

Identification of amino acids in the nicotinic acetylcholine receptor agonist binding site and ion channel photolabeled by 4-[(3-trifluoromethyl)-3H-diazirin-3-yl]benzoylcholine,a novel photoaffinity antagonist

[(3)H]4-[(3-trifluoromethyl)-3H-diazirin-3-yl]benzoylcholine (TDBzcholine) was synthesized and used as a photoaffinity probe to map the orientation of an aromatic choline ester within the agonist binding sites of the Torpedo nicotinic acetylcholine receptor (nAChR). TDBzcholine acts as a nAChR competitive antagonist that binds at equilibrium with equal affinity to both agonist sites (K(D) approximately 10 microM). Upon UV irradiation (350 nm), nAChR-rich membranes equilibrated with [(3)H]TDBzcholine incorporate (3)H into the alpha, gamma, and delta subunits in an agonist-inhibitable manner. The specific residues labeled by [(3)H]TDBzcholine were determined by N-terminal sequence analysis of subunit fragments produced by enzymatic cleavage and purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and/or reversed-phase high-performance liquid chromatography. For the alpha subunit, [(3)H]TDBzcholine photoincorporated into alphaCys-192, alphaCys-193, and alphaPro-194. For the gamma and delta subunits, [(3)H]TDBzcholine incorporated into homologous leucine residues, gammaLeu-109 and deltaLeu-111. The photolabeling of these amino acids suggests that when the antagonist TDBzcholine occupies the agonist binding sites, the Cys-192-193 disulfide and Pro-194 from the alpha subunit Segment C are oriented toward the agonist site and are in proximity to gammaLeu-109/deltaLeu-111 in Segment E, a conclusion consistent with the structure of the binding site in the molluscan acetylcholine binding protein, a soluble protein that is homologous to the nAChR extracellular domain.