Agonist-specific, high-affinity binding epitopes are contributed by an arginine in the N-terminus of the human oxytocin receptor

The effects of the peptide hormone oxytocin (OT) are mediated by the oxytocin receptor, which is a member of the G-protein-coupled receptor family. Defining differences between the binding of agonists and antagonists to the OTR, at the molecular level, is of fundamental importance to understanding OTR activation and to rational drug design. Previous reports have indicated that the N-terminus of the OTR is required for OT binding. The aim of this study was to identify which individual residues within the N-terminal domain of the human OTR provided these OT binding epitopes. A series of truncated OTRs and mutant receptor constructs with systematic alanine substitution were characterized with respect to their pharmacological profile and intracellular signaling capability. Although a number of residues within the OTR will be required for optimal OT-OTR interaction, our data establish that Arg(34) within the N-terminal domain contributes to high-affinity OT binding. Removal of Arg(34) by truncation or substitution resulted in a 2000-fold decrease in OT affinity. In addition, we show that the arginyl at this locus is required for high-affinity binding of agonists in general. However, the importance of Arg(34) is restricted to agonist interaction with the OTR, as it was not required for binding peptide antagonist or non-peptide antagonist. It is noteworthy that the corresponding Arg in the related rat V(1a) vasopressin receptor is also required for high-affinity agonist binding. This study defines, at the molecular level, the role of the N-terminus of the OTR in high-affinity agonist binding and identifies a key residue for this function.     

A single residue (arg46) located within the N-terminus of the V1a vasopressin receptor is critical for binding vasopressin but not peptide or nonpeptide antagonists

A fundamental issue in molecular endocrinology is to define how agonist:receptor interaction differs from antagonist:receptor interaction. The vasopressin V1a receptor (V1aR) is a member of a subfamily of related G protein-coupled receptors that are activated by the hormone AVP or related peptides. The N-terminus of the V1aR has recently been shown to be critical for binding agonists but not antagonists. Using a combination of N-terminally truncated constructs and alanine-scanning mutagenesis, individual residues that provide these agonist-specific binding epitopes have now been identified in this study. Our data establish that a single residue, Arg46, is critical for AVP binding to the V1aR. Systematic substitution revealed that Arg was required at this locus and could not be substituted by Lys, Glu, Leu, or Ala. In contrast, antagonist binding (cyclic or linear, peptide or nonpeptide) was unaffected. Disruption of Arg46 also resulted in defective intracellular signaling. Arginine is conserved at this locus in all members of the neurohypophysial peptide hormone receptor family cloned to date, indicative of a fundamental role in receptor function. In addition to Arg46, the residues Leu42, Gly43, Asp45 form a patch contributing to AVP binding. This study provides molecular insight into the role of the V1aR N-terminus and key differences between agonist and antagonist binding requirements.     

An arginyl in the N-terminus of the V1a vasopressin receptor is part of the conformational switch controlling activation by agonist.

Defining how the agonist-receptor interaction differs from that of the antagonist-receptor and understanding the mechanisms of receptor activation are fundamental issues in cell signalling. The V1a vasopressin receptor (V1aR) is a member of a family of related G-protein coupled receptors that are activated by neurohypophysial peptide hormones, including vasopressin (AVP). It has recently been reported that an arginyl in the distal N-terminus of the V1aR is critical for binding agonists but not antagonists. To determine specific features required at this locus to support high affinity agonist binding and second messenger generation, Arg46 was substituted by all other 19 encoded amino acids. Our data establish that there is an absolute requirement for arginyl, as none of the [R46X]V1aR mutant constructs supported high affinity agonist binding and all 19 had defective signalling. In contrast, all of the mutant receptors possessed wildtype binding for both peptide and nonpeptide antagonists. The ratio of Ki to EC50, an indicator of efficacy, was increased for all substitutions. Consequently, although [R46X]V1aR constructs have a lower affinity for agonist, once AVP has bound all 19 are more likely than the wildtype V1aR to become activated. Therefore, in the wildtype V1aR, Arg46 constrains the inactive conformation of the receptor. On binding AVP this constraint is alleviated, promoting the transition to active V1aR. Our findings explain why arginyl is conserved at this locus throughout the evolutionary lineage of the neurohypophysial peptide hormone receptor family of G-protein coupled receptors.     

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.     

Dynamic interaction of human vasopressin/oxytocin receptor subtypes with G protein-coupled receptor kinases and protein kinase C after agonist stimulation

Examination of the structure of [Arg(8)]-vasopressin receptors (AVPRs) and oxytocin receptors (OTRs) suggests that G protein-coupled receptor kinases (GRKs) and protein kinase C (PKC) are involved in their signal transduction. To explore the physical association of AVPRs and OTRs with GRKs and PKC, wild types and mutated forms of these receptor subtypes were stably expressed as green fluorescent protein fusion proteins and analyzed by fluorescence, immunoprecipitation, and immunoblotting. Addition of a C-terminal GFP tag did not interfere with ligand binding, internalization, and signal transduction. After agonist stimulation, PKC dissociated from the V(1)R, did not associate with the V(2)R, but associated with the V(3)R and the OTR. After AVP stimulation, only GRK5 briefly associated with AVPRs following a time course that varied with the receptor subtype. No GRK associated with the OTR. Exchanging the V(1)R and V(2)R C termini altered the time course of PKC and GRK5 association. Deletion of the V(1)R C terminus resulted in no PKC association and a ligand-independent sustained association of GRK5 with the receptor. Deletion of the GRK motif prevented association and reduced receptor phosphorylation. Thus, agonist stimulation of AVP/OT receptors leads to receptor subtype-specific interactions with GRK and PKC through specific motifs present in the C termini of the receptors.     

A Gly/Ala switch contributes to high affinity binding of benzoxazinone-based non-peptide oxytocin receptor antagonists

Non-peptide antagonists of the oxytocin receptor (OTR) have been developed to prevent pre-term labour. The benzoxazinone-based antagonists L-371,257 and L-372,662 display pronounced species-dependent pharmacology with respect to selectivity for the OTR over the V(1a) vasopressin receptor. Examination of receptor sequences from different species identified Ala(318) in helix 7 of the human OTR as a candidate discriminator required for high affinity binding. The mutant receptor [A318G]OTR was engineered and characterised using ligands representing many different chemical classes. Of all the ligands investigated, only the benzoxazinone-based antagonists had decreased affinity for [A318G]OTR. Molecular modelling revealed that Ala(318) provides a direct hydrophobic contact with a methoxy group of L-371,257 and L-372,662.     

Hybrid peptide-small molecule oxytocin analogs are potent and selective agonists of the oxytocin receptor

Oxytocin (OT) is a peptide hormone agonist of the oxytocin receptor (OTR) that has been proposed as a therapeutic to treat a number of social and emotional disorders in addition to its current clinical use to induce labor and treat postpartum bleeding. OT is administered intravenously and intranasally rather than orally, in part because its low passive permeability causes low oral bioavailability. Non-peptidic OTR agonists have also been reported, but none with the exquisite potency of the peptide based agonists. In this report, we describe the OTR agonist activity and exposed polarity of a set of truncated OT analogs as well as hybrid peptide-small molecule analogs of OT. Examples of both truncated analogs and peptide-small molecule hybrid analogs are potent and selective OTR agonists. Hybrid agonist 13, which is 232?Da smaller than OT, still retains subnanomolar potency, full agonist activity, and selectivity over V1a. While these compounds were designed to address the low permeability of OT and other full length analogs, we found that reduction in molecular weight and the removal or replacement of the three amino acid tail of OT did not have a significant effect on passive permeability.     

Analysis of interactions responsible for vasopressin binding to human neurohypophyseal hormone receptors-molecular dynamics study of the activated receptor-vasopressin-G(alpha) systems.

Vasopressin (CYFQNCPRG-NH(2), AVP) is a semicyclic endogenous peptide, which exerts a variety of biological effects in mammals. The main physiological roles of AVP are the regulation of water balance and the control of blood pressure and adrenocorticotropin hormone (ACTH) secretion, mediated via three different subtypes of vasopressin receptors: V1a, V1b and V2 receptors (V1aR, V1bR and V2R, respectively). They are the members of the class A, G-protein-coupled receptors (GPCRs). AVP also modulates several behavioral and social functions. In this study, the interactions responsible for AVP binding to vasopressin V1a and V2 receptors versus the closely related oxytocin ([I3,L8]AVP, OT) receptor (OTR) have been investigated. Three-dimensional models of the activated receptors were constructed using multiple sequence alignment, followed by homology modeling using the complex of activated rhodopsin with Gt(alpha) C-terminal peptide of transducin MII-Gt(338-350) prototype as a template. AVP was docked into the receptor-G(alpha) systems. The three lowest-energy pairs of receptor-AVP-G(alpha) (two complexes per each receptor) were selected. The 1-ns unconstrained molecular dynamics (MD) of complexes embedded into the fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) lipid bilayer was conducted in the AMBER 7.0 force field. Six relaxed receptor-AVP-G(alpha) models were obtained. The residues responsible for AVP binding to vasopressin receptors have been identified and a different mechanism of AVP binding to V2R than to V1aR has been proposed.     

Pyridobenzodiazepines: a novel class of orally active, vasopressin V2 receptor selective agonists

Our efforts in seeking low molecular weight agonists of the antidiuretic peptide hormone arginine vasopressin (AVP) have led to the identification of the clinical candidate WAY-151932 (VNA-932). Further exploration of the structural requirements for agonist activity has provided another class of potent, orally active, non-peptidic vasopressin V2 receptor selective agonists exemplified by the 5,11-dihydro-pyrido[2,3-b][1,5]benzodiazepine as a candidate for further development.     

Identification of a novel V1-type AVP receptor based on the molecular recognition theory.

BACKGROUND: The molecular recognition theory predicts that binding domains of peptide hormones and their corresponding receptor binding domains evolved from complementary strands of genomic DNA, and that a process of selective evolutionary mutational events within these primordial domains gave rise to the high affinity and high specificity of peptide hormone-receptor interactions observed today in different peptide hormone-receptor systems. Moreover, this theory has been broadened as a general hypothesis that could explain the evolution of intermolecular protein-protein and intramolecular peptide interactions. MATERIALS AND METHODS: Applying a molecular cloning strategy based on the molecular recognition theory, we screened a rat kidney cDNA library with a vasopressin (AVP) antisense oligonucleotide probe, expecting to isolate potential AVP receptors. RESULTS: We isolated a rat kidney cDNA encoding a functional V1-type vasopressin receptor. Structural analysis identified a 135 amino acid-long polypeptide with a single transmembrane domain, quite distinct from the rhodopsin-based G protein-coupled receptor superfamily. Functional analysis of the expressed V1-type receptor in Cos-1 cells revealed AVP-specific binding, AVP-specific coupling to Ca2+ mobilizing transduction system, and characteristic V1-type antagonist inhibition. CONCLUSIONS: This is the second AVP receptor cDNA isolated using AVP antipeptide-based oligonucleotide screening, thus providing compelling evidence in support of the molecular recognition theory as the basis of the evolution of this peptide hormone-receptor system, as well as adds molecular complexity and diversity to AVP receptor systems.     

Investigation of mechanism of desmopressin binding in vasopressin V2 receptor versus vasopressin V1a and oxytocin receptors: molecular dynamics simulation of the agonist-bound state in the membrane-aqueous system

The vasopressin V2 receptor (V2R) belongs to the Class A G protein-coupled receptors (GPCRs). V2R is expressed in the renal collecting duct (CD), where it mediates the antidiuretic action of the neurohypophyseal hormone arginine vasopressin (CYFQNCPRG-NH2, AVP). Desmopressin ([1-deamino, 8-D]AVP, dDAVP) is strong selective V2R agonist with negligible pressor and uterotonic activity. In this paper, the interactions responsible for binding of dDAVP to vasopressin V2 receptor versus vasopressin V1a and oxytocin receptors has been examined. Three-dimensional activated models of the receptors were constructed using the multiple sequence alignment and the complex of activated rhodopsin with Gt(alpha) C-terminal peptide of transducin MII-Gt(alpha) (338-350) prototype (Slusarz, R.; Ciarkowski, J. Acta Biochim Pol 2004 51, 129-136) as a template. The 1-ns unconstrained molecular dynamics (MD) of receptor-dDAVP complexes immersed in the fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) membrane model was conducted in an Amber 7.0 force field. Highly conserved transmembrane residues have been proposed as being responsible for V2R activation and G protein coupling. Molecular mechanism of the dDAVP binding has been suggested. The internal water molecules involved in an intricate network of the hydrogen bonds inside the receptor cavity have been identified and their role in the stabilization of the agonist-bound state proposed.     

Vasopressin antisense peptide interactions with the V1 receptor

The molecular recognition hypothesis, that peptide ligands and their receptor binding sites are encoded by complementary nucleotide sequences, was tested for arginine vasopressin (AVP) and its V1 receptor. Binding of [125I] [d(CH2)5,Sar7]AVP (a selective V1 vasopressin antagonist radioligand) or [3H]AVP to rat liver plasma membranes was inhibited by peptides known to bind to V1 receptors but not by the AVP complementary peptide (Ser-Ser-Trp-Ala-Val-Leu-Glu-Val-Ala) (PVA). Rabbit anti-PVA antibodies were nonimmunoreactive with any protein in rat liver membranes or in a partially purified preparation from rat liver containing reconstitutable vasopressin binding activity. Furthermore, there was no suppression of the AVP pressor effect by PVA in vivo using a rat blood pressure bioassay. These findings do not support the hypothesis that the V1 receptor binding site is encoded by the antisense DNA strand to AVP.     

The N-terminal juxtamembrane segment of the V1a vasopressin receptor provides two independent epitopes required for high-affinity agonist binding and signaling

It is fundamentally important to define how agonist-receptor interaction differs from antagonist-receptor interaction. The V1a vasopressin receptor (V1aR) is a member of the neurohypophysial hormone subfamily of G protein-coupled receptors. Using alanine-scanning mutagenesis of the N-terminal juxtamembrane segment of the V1aR, we now establish that Glu54 (1.35) is critical for arginine vasopressin binding. The mutant [E54A]V1aR exhibited decreased arginine vasopressin affinity (1700-fold) and disrupted signaling, but antagonist binding was unaffected. Mutation of Glu54 had an almost identical pharmacological effect as mutation of Arg46, raising the possibility that agonist binding required a mutual interaction between Glu54 and Arg46. The role of these two charged residues was investigated by 1) substituting Glu54; 2) inserting additional Glu/Arg in transmembrane helix (TM) 1; 3) repositioning the Glu/Arg in TM1; and 4) characterizing the reciprocal mutant [R46E/E54R]V1aR. We conclude that 1) the positive/negative charges need to be precisely positioned in this N terminus/TM1 segment; and 2) Glu54 and Arg46 function independently, providing two discrete epitopes required for high-affinity agonist binding and signaling. This study explains why Glu and Arg, part of an -R(X3)L/V(X3)E(X3)L- motif, are conserved at these loci throughout this G protein-coupled receptor subfamily and provides molecular insight into key differences between agonist and antagonist binding requirements.     

Tyr115 is the key residue for determining agonist selectivity in the V1a vasopressin receptor.

Using a three-dimensional model of G protein-coupled receptors (GPCR), we have previously succeeded in docking the neurohypophysial hormone arginine-vasopressin (AVP) into the V1a receptor. According to this model, the hormone is completely embedded in the transmembrane part of the receptor. Only the side chain of the Arg residue at position 8 projects outside the transmembrane core of the receptor and possibly interacts with a Tyr residue located in the first extracellular loop at position 115. Residue 8 varies in the two natural neurohypophysial hormones, AVP and oxytocin (OT); similarly, different residues are present at position 115 in the different members of the AVP/OT receptor family. Here we show that Arg8 is crucial for high affinity binding of AVP to the rat V1a receptor. Moreover, when Tyr115 is replaced by an Asp and a Phe, the amino acids naturally occurring in the V2 and in the OT receptor subtypes, the agonist selectivity of the V1a receptor switches accordingly. Our results indicate that the interaction between peptide residue 8 and the receptor residue at position 115 is not only crucial for agonist high affinity binding but also for receptor selectivity.     

Two small molecule agonists of glucagon-like peptide-1 receptor modulate the receptor activation response differently

The glucagon-like peptide-1 receptor (GLP-1R) is a target for type 2 diabetes treatment. Due to the inconvenience of peptide therapeutics, small-molecule GLP-1R agonists have been studied. Compound 2 (6,7-dichloro-2-methylsulfonyl-2-N-tert-butylaminoquinoxaline) and compound B (4-(3-(benzyloxy)phenyl)-2-(ethylsulfinyl)-6-(trifluoromethyl)pyrimidine) have been described as small molecule, ago-allosteric modulators of GLP-1R. However, their modes of action at the GLP-1R have not been elucidated. Thus, in this study, we compared the mechanisms of action between these two compounds. When compound 2 was treated with endogenous or exogenous peptide agonists (GLP-1 and exenatide) or fragments of peptide agonists (GLP-1(9-36), Ex3, Ex4, and Ex5), the response curve of these peptide agonists shifted left without a change in maximum efficacy. In contrast, compound B potentiated the response and increased maximum efficacy. However, N-terminal truncated orthosteric antagonists including Ex7, Ex9, and Ex10, augmented the response of compound 2 at the GLP-1R but did not alter compound B activity. Intriguingly, when we co-treated compound 2 with compound B in CHO cells expressing full-length hGLP-1R or N-terminal extracellular domain-truncated GLP-1R, the activation of both types of receptors increased additively, implying that the N-terminus of the receptor is not involved in the modulation by compound agonists. We confirmed that these two compounds increased calcium influx by different patterns in CHO cells expressing GLP-1R. Taken together, our findings suggest that compounds 2 and B have different modes of action to activate GLP-1R. Further study to identify the putative binding sites will help in the discovery of orally available GLP-1R agonists.     

Characterization of rodent liver and kidney AVP receptors: pharmacologic evidence for species differences.

Radioligand binding studies with [3H]vasopressin (AVP) were used to determine the affinities of AVP receptor agonists and antagonists for mouse liver and kidney plasma membrane preparations. Both membrane preparations exhibited one class of high-affinity binding site. AVP ligand binding inhibition studies confirmed that mouse liver binding sites belong to the V1A subtype while kidney binding sites belong to the V2 receptor subtype. The affinity of each ligand for mouse V1A receptors was very similar to that for rat V1A receptors, showing differences in Ki values of less than 3-fold. In contrast, several peptide (d(CH2)5Tyr(Me)AVP) and nonpeptide (OPC-21268 and SR 49059) ligands had different affinities for mouse and rat kidney V2 receptors, with differences in Ki values ranging from 14- to 17-fold. These results indicate that mouse and rat kidney V2 receptors show significant pharmacologic differences.     

Identification of functional domains in the formyl peptide receptor-like 1 for agonist-induced cell chemotaxis

Formyl peptide receptor-like 1 (FPRL1) is a seven transmembrane domain, G protein-coupled receptor that interacts with a variety of exogenous and host-derived agonists. In order to identify domains crucial for ligand recognition by FPRL1, we used chimeric receptors with segments in FPRL1 replaced by corresponding amino acid sequences derived from the prototype formyl peptide receptor FPR. The chimeric receptors were stably transfected into human embryonic kidney epithelial cells and the capacity of the cells to migrate in response to formyl peptide receptor agonists was evaluated. Our results showed that multiple domains in FPRL1 are involved in the receptor response to chemotactic agonists with the sixth transmembrane domain and the third extracellular loop playing a prominent role. Interestingly, the N-terminus and a segment between the fourth transmembrane domain and the third intracellular loop of FPRL1 are important for receptor interaction with a 42 amino acid amyloid beta peptide (Abeta42), an Alzheimer's disease-associated FPRL1 agonist, but not with MMK-1, a synthetic FPRL1 agonist, suggesting that diverse agonists may use different domains in FPRL1. Considering the potential importance of FPRL1 in inflammation and neurodegenerative diseases, the identification of functional domains in this receptor will provide valuable information for the design of specific receptor antagonists.     

Arginine vasopressin (AVP) replacement of helper cell requirement in IFN-gamma production. Evidence for a novel AVP receptor on mouse lymphocytes

Arginine vasopressin (AVP), a nine-amino acid neurohypophyseal hormone, is capable of replacing the helper cell requirement for IFN-gamma production by Lyt-2+ mouse splenic lymphocytes. We present data here showing that the AVP helper signal occurs via interaction with a novel R on splenic lymphocytes and involves primarily the N-terminal six-amino acid cyclic ring (pressinoic acid) with the C-terminal three-amino acid end of AVP playing a minor role. Pressinoic acid was capable of providing help at concentrations similar to those of AVP, whereas oxytocin and isoleucine pressinoic acid were 10- and 100-fold less effective, respectively. Isoleucine pressinoic acid has the same structure as pressinoic acid except for the substitution of isoleucine for phenylalanine in position 3 of the sequence. Consistent with the function data, R binding competitions with splenic lymphocyte membrane preparations showed that AVP and pressinoic acid competed similarly with [3H]AVP, whereas oxytocin and isoleucine pressinoic acid were much less effective competitors. Further characterization of the AVP lymphocyte R was performed using AVP analogues having well defined agonist and antagonist activities on either V1 (vasopressor) R or V2 (antidiuretic) R. The AVP helper signal was blocked by the V1 antagonist [d(CH2)1(5) Tyr(methyl)]AVP but not by another V1 antagonist, [d(CH2)1(5)D-Tyr(ethyl)2Val4]AVP. Both V1-R antagonists were able to block [3H]AVP binding to the V1-R on liver cells, whereas only the V1 antagonist that blocked AVP help was able to compete effectively for the spleen AVP-R. Neither a V2 agonist nor a V2 antagonist had any effect on AVP help in IFN-gamma production. These data strongly indicate the presence of a novel AVP-R on spleen lymphocytes, which is related to the classic V1-R on liver cell membranes.     

Identification of a domain in the delta subunit (S238-V264) of the alpha4beta3delta GABAA receptor that confers high agonist sensitivity

We have expressed the alpha4beta3delta and alpha4beta3gamma2L subtypes of the rat GABAA receptor in Xenopus oocytes and have investigated their agonist activation properties. GABA was a more potent agonist of the alpha4beta3delta receptor (EC50 approximately 1.4 micromol/L) than of the alpha4beta3gamma2L subtype (EC50 approximately 27.6 micromol/L). Other GABAA receptor agonists (muscimol, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol, imidazole-4-amino acid) displayed similar subtype selectivity. The structural determinants underlying these differences have been investigated by co-expressing chimeric delta/gamma2L subunits with alpha4 and beta3 subunits. A stretch of amino acids in the delta subunit, S238-V264, is shown to play an important role in determining both agonist potency and the efficacies of full or partial agonists. This segment includes transmembrane domain 1 and the short intracellular loop that leads to the second transmembrane domain. The effects of the competitive antagonists, bicuculline and SR95531, and the channel blocker, picrotoxin, were not significantly affected by the incorporation of chimeric subunits. As the delta and gamma2L subunits have not been previously implicated directly in agonist binding, we suggest that the effects are likely to arise from changes in the transduction mechanisms that link agonist binding to channel activation.     

Demonstration of distinct agonist and antagonist conformations of the A1 adenosine receptor

A1 adenosine receptor-binding subunits can be visualized using high affinity antagonist and agonist photoaffinity radioligands. In the present study, we examined whether agonists and antagonists bind to the same receptor-binding subunit and if agonists and antagonists induce different conformational states of the receptor in intact membranes. It was demonstrated that several agonist and antagonist photoaffinity receptor-binding subunit. When the agonist and antagonist photoaffinity labeled peptides were denatured and subjected to partial peptide map analysis using a two-dimensional gel electrophoresis system similar peptide fragments were generated from each specifically labeled protein. This suggests that both classes of ligand label and incorporate into the same binding subunit. Proteolytic digestions of agonist- and antagonist-occupied receptors in native intact membranes revealed distinct and different peptide fragments depending on whether the ligand was an agonist or an antagonist. Manipulation of incubation conditions to perturb ligand-receptor interactions alter the pattern of peptide fragments generated with each specific protease. These data suggest that agonist and antagonist photoaffinity probes interact with an incorporate into the same binding subunit but that agonist binding is associated with a unique and detectable receptor conformation.