Presence Of GDP Research Articles

Development of a FUT8 Inhibitor with Cellular Inhibitory Properties.

Core fucosylation is catalyzed by α-1,6-fucosyltransferase (FUT8), which fucosylates the innermost GlcNAc of N-glycans. Given the association of FUT8 with various diseases including cancer, selective FUT8 inhibitors applicable to in vivo or cell-based systems are highly sought-after. Here, we report the discovery of a compound that selectively inhibits FUT8 in cell-based assays. High-throughput screening revealed a FUT8-inhibiting pharmacophore, and further structural optimization yielded an inhibitor with a KD of 49 nM. Notably, this binding occurs only in the presence of GDP (a product of the enzymatic reaction catalyzed by FUT8). Mechanistic studies suggested that this inhibitor generates a highly reactive naphthoquinone methide derivative at the binding site in FUT8, which subsequently reacts with FUT8. Furthermore, prodrug derivatization of this inhibitor improved its stability, enabling suppression of core fucose expression and subsequent EGFR and T-cell signaling in cell-based assays, paving the way for the development of drugs targeting core fucosylation.

The role of resource rent in shaping CO2 emissions in BRICS countries: A panel data approach

This study investigates the impact of resource rent on CO2 emissions in the presence of GDP, renewable energy consumption, and research and development spending for BRICS nations from 1990 to 2020. Employing quantile regression approach, this study finds that resource rents are responsible for rising CO2 emissions in BRCIS countries. However, there exists a threshold level of resource rents beyond which the nature of the relationship between variables changes. Since, the extraction of natural resources requires a significant amount of energy, it may result in excessive levels of energy consumption as well as the reckless discharge of waste chemicals into the water, land, and air. We find that the impact of GDP on CO2 emissions is greater for nations with a low level of CO2 emissions and is lower for nations with relatively high CO2 emissions. The findings of the research paper highlight several policy implications for the BRICS nations to reduce their CO2 emissions. Policymakers should prioritize investment in renewable energy sources such as wind, solar, and hydroelectric power and encourage research and development spending in renewable energy technologies. The study encourages channelizing resources revenue for a sustainable environment and development.

Innovation for renewable energy and energy related greenhouse gases: Evaluating the role of green finance

Environmental degradation is one of the many unintended consequences of industrialization, which has resulted in misery for humans, and biodiversity around the world. These environmental concerns require consideration in order to gain a better understanding of how to design policy around climate change. In this regard, a number of deals have been reached with the objective of reducing pollutants in the environment so as to keep an average temperature that below 2 degrees Celsius. In order to effectively reduce greenhouse gas emissions, nations all over the world have been putting more of their attention on investments in R&D for renewable energy, often known as green financing. This study explores the influence of green finance in the presence of GDP, human capital, and economic risk on GHG emissions in G7 countries for the period of 1990–2020. We find that green finance and economic risk abates GHG emission intensity. It is interesting that the impact of green finance is stronger for countries with high emissions level and lower for low emitter countries. Human capital and GDP increase GHG emissions. The results of the present study have important consequence for G7 nations because majority of these countries have been facing high oil demand. Policies to enhance green financing during the period of high energy demand are vital. It is imperative that policies be put in place to maintain the development in green financing.

Financial Development and Health Outcomes: Do Financial Globalization Matter in Selected Asian Economies?

The importance of health is well documented in the development economics literature because of its increasing effects on economic growth in the long-run. Financial development and financial globalization are essential resources for health. This study examines the role of financial development and financial globalization in the rapid rise of life expectancy in China, India, and Japan by using the annual data covering the period of 1991–2019. The ARDL bounds testing approach confirm the long-run relationship between financial development, financial globalization, and life expectancy in the presence of GDP, health expenditure, and the internet. The long-run findings indicate that financial development positively affects life expectancy by 0.599% in China. The novel findings also indicate that financial globalization positively affects life expectancy by 1.247% in Japan and 1.121% in India. Our findings offer new empirical insights to policymakers crucial to improving life expectancy in China, India, and Japan.

Probing the Molecular Basis of Cofactor Affinity and Conformational Dynamics of Mycobacterium tuberculosis Elongation Factor Tu: An Integrated Approach Employing Steered Molecular Dynamics and Umbrella Sampling Simulations.

The emergence of multidrug-resistant and extensively drug-resistant tuberculosis strains is the reason that the infectious tuberculosis pathogen is still the most common cause of death. The quest for new antitubercular drugs that can fit into multidrug regimens, function swiftly, and overcome the ever-increasing prevalence of drug resistance continues. The crucial role of MtbEF-Tu in translation and trans-translation processes makes it an excellent target for antitubercular drug design. In this study, the primary sequence of MtbEF-Tu was used to model the three-dimensional structures of MtbEF-Tu in the presence of GDP ("off" state) and GTP ("on" state). The binding free energy computed using both the molecular mechanics/Poisson-Boltzmann surface area and umbrella sampling approaches shows that GDP binds to MtbEF-Tu with an ∼2-fold affinity compared to GTP. The steered molecular dynamics (SMD) and umbrella sampling simulation also shows that the dissociation of GDP from MtbEF-Tu in the presence of Mg2+ is a thermodynamically intensive process, while in the absence of Mg2+, the destabilized GDP dissociates very easily from the MtbEF-Tu. Naturally, the dissociation of Mg2+ from the MtbEF-Tu is facilitated by the nucleotide exchange factor EF-Ts, and this prior release of magnesium makes the dissociation process of destabilized GDP easy, similar to that observed in the umbrella sampling and SMD study. The MD simulations of MtbEF-Tu's "on" state conformation in the presence of GTP reveal that the secondary structure of switch-I and Mg2+ coordination network remains similar to its template despite the absence of identity in the conserved region of switch-I. On the other hand, the secondary structure in the conserved region of the switch-I of MtbEF-Tu unwinds from a helix to a loop in the presence of GDP. The major conformational changes observed in switch-I and the movement of Thr64 away from Mg2+ mainly reflect essential conformational changes to make the shift of MtbEF-Tu's "on" state to the "off" state in the presence of GDP. These obtained structural and functional insights into MtbEF-Tu are pivotal for a better understanding of structural-functional linkages of MtbEF-Tu, and these findings may serve as a basis for the design and development of MtbEF-Tu-specific inhibitors.

FUT8-Directed Core Fucosylation of N-glycans Is Regulated by the Glycan Structure and Protein Environment

FUT8 is an essential α-1,6-fucosyltransferase that fucosylates the innermost GlcNAc of N-glycans, a process called core fucosylation. In vitro, FUT8 exhibits substrate preference for the biantennary complex N-glycan oligosaccharide (G0), but the role of the underlying protein/peptide to which N-glycans are attached remains unclear. Here, we explored the FUT8 enzyme with a series of N-glycan oligosaccharides, N-glycopeptides, and an Asn-linked oligosaccharide. We found that the underlying peptide plays a role in fucosylation of paucimannose (low mannose) and high-mannose N-glycans but not for complex-type N-glycans. Using saturation transfer difference (STD) NMR spectroscopy, we demonstrate that FUT8 recognizes all sugar units of the G0 N-glycan and most of the amino acid residues (Asn-X-Thr) that serve as a recognition sequon for the oligosaccharyltransferase (OST). The largest STD signals were observed in the presence of GDP, suggesting that prior FUT8 binding to GDP-β-l-fucose (GDP-Fuc) is required for an optimal recognition of N-glycans. We applied genetic engineering of glycosylation capacities in CHO cells to evaluate FUT8 core fucosylation of high-mannose and complex-type N-glycans in cells with a panel of well-characterized therapeutic N-glycoproteins. This confirmed that core fucosylation mainly occurs on complex-type N-glycans, although clearly only at selected glycosites. Eliminating the capacity for complex-type glycosylation in cells (KO mgat1) revealed that glycosites with complex-type N-glycans when converted to high mannose lost the core Fuc. Interestingly, however, for erythropoietin that is uncommon among the tested glycoproteins in efficiently acquiring tetra-antennary N-glycans, two out of three N-glycosites obtained Fuc on the high-mannose N-glycans. An examination of the N-glycosylation sites of several protein crystal structures indicates that core fucosylation is mostly affected by the accessibility and nature of the N-glycan and not by the nature of the underlying peptide sequence. These data have further elucidated the different FUT8 acceptor substrate specificities both in vitro and in vivo in cells, revealing different mechanisms for promoting core fucosylation.

Influence of a Ser111-phosphorylation on Rab1b GTPase conformational dynamics studied by advanced sampling simulations.

Rab GTPases constitute the largest branch of the Ras protein superfamily that regulate intra-cellular membrane trafficking. Their signaling activity is mediated by the transition between an active GTP-bound state and an inactive GDP-bound state. In the inactive state the switch I and II segments adopt largely disordered flexible conformations, whereas in the active state these regions are in well-defined conformations. The switch I and II states are central for recognition of Rab GTPases by interacting partners. Phosphorylation of the Rab1b-GTPase at residue Ser111 (pS111) results in modulation of the signaling activity due to alterations of the protein interaction interface and also due to modulation of the conformational flexibility. We have studied the flexibility of native and pS111-Rab1b in complex with GTP or GDP using extensive Molecular Dynamics (MD) simulations and an advanced sampling method called DIhedral Angle-biasing potential Replica-Exchange Molecular dynamics (DIA-REMD). The DIA-REMD method promotes backbone and side chain dihedral transitions along a series of replica simulations in selected protein segments and through exchanges also improves sampling in an unbiased reference simulation. Application to the Rab1b system results in significantly enhanced sampling of different switch I/II conformational states in the GDP-bound Rab1b state. The pS111 modification is found to reduce the conformational flexibility even in the presence of GDP, which may influence signaling activities. The stabilizing effect can be attributed to the formation of additional surface salt bridges between Arg-residues and pS111 not present in the native structure. The DIA-REMD method could be a valuable approach for studying also other signaling proteins that contain flexible segments.

Characterization of binding kinetics of A2AR to Gαs protein by surface plasmon resonance

Because of their surface localization, G protein-coupled receptors (GPCRs) are often pharmaceutical targets as they respond to a variety of extracellular stimuli (e.g., light, hormones, small molecules) that may activate or inhibit a downstream signaling response. The adenosine A2A receptor (A2AR) is a well-characterized GPCR that is expressed widely throughout the human body, with over 10 crystal structures determined. Truncation of the A2AR C-terminus is necessary for crystallization as this portion of the receptor is long and unstructured; however, previous work suggests shortening of the A2AR C-terminus from 412 to 316 amino acids (A2AΔ316R) ablates downstream signaling, as measured by cAMP production, to below that of constitutive full-length A2AR levels. As cAMP production is downstream of the first activation event—coupling of G protein to its receptor—investigating that first step in activation is important in understanding how the truncation effects native GPCR function. Here, using purified receptor and Gαs proteins, we characterize the association of A2AR and A2AΔ316R to Gαs with and without GDP or GTPγs using surface plasmon resonance (SPR). Gαs affinity for A2AR was greatest for apo-Gαs, moderately affected in the presence of GDP and nearly completely ablated by the addition of GTPγs. Truncation of the A2AR C-terminus (A2AΔ316R) decreased the affinity of the unliganded receptor for Gαs by ∼20%, suggesting small changes to binding can greatly impact downstream signaling.

Structural insights into G domain dimerization and pathogenic mutation of OPA1.

The fusion of mammalian inner mitochondrial membranes (IMMs) is mediated by dynamin-like GTPase OPA1. Mutations in human OPA1 cause optic atrophy, but the molecular basis for membrane fusion and pathogenesis is not clear. Here, we determined the crystal structure of the minimal GTPase domain (MGD) of human OPA1. A three-helix bundle (HB) domain including two helices extending from the GTPase (G) domain and the last helix of OPA1 tightly associates with the G domain. In the presence of GDP and BeF3-, OPA1-MGD forms a dimer, the interface of which is critical for the maintenance of mitochondrial morphology. The catalytic core of OPA1 possesses unique features that are not present in other dynamin-like proteins. Biochemical experiments revealed that OPA1-MGD forms nucleotide-dependent dimers, which is important for membrane-stimulated GTP hydrolysis, and an N-terminal extension mediates nucleotide-independent dimerization that facilitates efficient membrane association. Our results suggest a multifaceted assembly of OPA1 and explain the effect of most OPA1 mutations on optic atrophy.

Hesperidin depolarizes the pacemaker potentials through 5-HT4 receptor in murine small intestinal interstitial cells of Cajal

ABSTRACT Hesperidin, a citrus flavonoid, can exert numerous beneficial effects on human health. Interstitial cells of Cajal (ICC) are pacemaker cells in the gastrointestinal (GI) tract. In the present study, we investigated potential effects of hesperidin on pacemaker potential of ICC in murine small intestine and GI motility. A whole-cell patch-clamp configuration was used to record pacemaker potential in ICC, and GI motility was investigated in vivo by recording gastric emptying (GE) and intestinal transit rate (ITR). Hesperidin depolarized pacemaker potentials of ICC in a dose-dependent manner. Pre-treatment with methoctramine or 4-DAMP did not inhibit hesperidin-induced pacemaker potential depolarization. Neither a 5-HT3 receptor antagonist (Y25130) nor a 5-HT7 receptor antagonist (SB269970) reduced the effect of hesperidin on ICC pacemaker potential, whereas the 5-HT4 receptor antagonist RS39604 was found to inhibit this effect. In the presence of GDP–β–S, hesperidin-induced pacemaker potential depolarization was inhibited. Moreover, in the presence of U73122 and calphostin C, hesperidin did not depolarize pacemaker potentials. Furthermore, hesperidin accelerated GE and ITR in vivo. These results imply that hesperidin depolarized ICC pacemaker potential via 5-HT4 receptors, G protein, and PLC/PKC dependent pathways and that it increased GI motility. Therefore, hesperidin may be a promising novel drug to regulate GI motility.

Complex formation between the Escherichia coli [NiFe]-hydrogenase nickel maturation factors.

The biosynthesis of the dinuclear metal cluster at the active sites of the [NiFe]-hydrogenase enzymes is a multi-step process executed by a suite of accessory proteins. Nickel insertion during maturation of Escherichia coli [NiFe]-hydrogenase 3 is achieved by the metallochaperones HypA, SlyD and the GTPase HypB, but how these proteins cooperate to ensure nickel delivery is not known. In this study, the complexes formed between the individual purified proteins were examined by using several methods. Size exclusion chromatography (SEC) indicated that SlyD and HypB interact primarily in a 1:1 complex. The affinity of HypB-SlyD was measured by using surface plasmon resonance, which revealed a KD of 24 ± 10nM in the absence of nucleotide and an interaction several fold tighter in the presence of GDP. A ternary complex between all three proteins was not detected, and instead SlyD blocked the interaction of HypA with HypB in competitive binding experiments. Furthermore, cross-linking experiments suggest a weak interaction between HypA and SlyD, which is not detectable by SEC. Electrochemical analysis confirmed each of the pairwise interactions and that the relative affinities of these complexes are on the order of HypB-SlyD > HypB-HypA > HypA-SlyD. These results indicate a hierarchy of interactions, as opposed to a single multiprotein complex, and provide insight into the nickel delivery process during hydrogenase enzyme maturation.

Biochemical analysis of GTPase FlhF which controls the number and position of flagellar formation in marine Vibrio

FlhF controls the number and position of the polar flagellar formation of Vibrio species. FlhF, is a paralog of FtsY, a GTPase acting in the Sec membrane transport system of bacteria, and localizes at the cell pole. Mutations in the conserved GTPase motif of FlhF lost polar localization capability and flagellar formation. Vibrio FlhF has not, until now, been purified as soluble protein. Here, we report that addition of MgCl2 and GTP or GDP at the step of cell lysis greatly improved the solubility of FlhF, allowing us to purify it in homogeneity. Purified FlhF showed GTPase activity only in the presence of FlhG. Of twelve FlhF GTPase motif mutants showing reduced function, eleven were recovered as precipitate after the cell disruption. The E440K substitution could be purified and showed no GTPase activity even in the presence of FlhG. Interestingly an FlhF substitution in the putative catalytic residue for GTP hydrolysis, R334A, allowed normal flagellar formation although GTPase activity of FlhF was completely abolished. Furthermore, size exclusion chromatography of purified FlhF revealed that it forms dimers in the presence of GTP but exists as monomer in the presence of GDP. We speculate that the GTP binding allows FlhF to dimerize and localize at the pole where it initiates flagellar formation, and the GDP-bound form diffuses as monomer.

High concentrations of GTP induce conformational changes in the essential bacterial GTPase EngA and enhance its binding to the ribosome.

EngA is a conserved bacterial GTPase involved in ribosome biogenesis. While essential in bacteria, EngA does not have any human orthologue and can thus be an interesting target for new antibacterial compounds. EngA is the only known GTPase bearing two G domains, making unique its catalytic cycle and the induced modulation of its conformation and interaction with the ribosome. We have investigated nucleotide-induced conformational changes in EngA in order to unveil their role in ribosome binding. SAXS and limited proteolysis were used to probe EngA conformational changes, and revealed a change in protein structure and a distinct rate of proteolysis induced by GTP. Structure analysis showed that the conformation adopted in solution in the presence of GTP does not match any known EngA structure, while the SAXS data measured in the presence of GDP are in perfect agreement with two crystal structures (i.e. 2HGJ and 4DCU). Combination of mass spectrometry and N-terminal sequencing for the analysis of the fragmentation pattern upon proteolytic cleavage gave insights into which regions become more or less accessible in the different nucleotide-bound states. Interactions studies confirmed a stronger binding of EngA to the bacterial ribosome in the presence of GTP and suggest that the induced change in conformation of EngA plays a key role for ribosome binding.

The Chloroplast Tubulin Homologs FtsZA and FtsZB from the Red Alga Galdieria sulphuraria Co-assemble into Dynamic Filaments

FtsZ is a homolog of eukaryotic tubulin and is present in almost all bacteria and many archaea, where it is the major cytoskeletal protein in the Z ring, required for cell division. Unlike some other cell organelles of prokaryotic origin, chloroplasts have retained FtsZ as an essential component of the division machinery. However, chloroplast FtsZs have been challenging to study because they are difficult to express and purify. To this end, we have used a FATT tag expression system to produce as soluble proteins the two chloroplast FtsZs from Galdieria sulphuraria, a thermophilic red alga. GsFtsZA and GsFtsZB assembled individually in the presence of GTP, forming large bundles of protofilaments. GsFtsZA also assembled in the presence of GDP, the first member of the FtsZ/tubulin superfamily to do so. Mixtures of GsFtsZA and GsFtsZB assembled protofilament bundles and hydrolyzed GTP at a rate approximately equal to the sum of their individual rates, suggesting a random co-assembly. GsFtsZA assembly by itself in limiting GTP gave polymers that remained stable for a prolonged time. However, when GsFtsZB was added, the co-polymers disassembled with enhanced kinetics, suggesting that the GsFtsZB regulates and enhances disassembly dynamics. GsFtsZA-mts (where mts is a membrane-targeting amphipathic helix) formed Z ring-like helices when expressed in Escherichia coli Co-expression of GsFtsZB (without an mts) gave co-assembly of both into similar helices. In summary, we provide biochemical evidence that GsFtsZA assembles as the primary scaffold of the chloroplast Z ring and that GsFtsZB co-assembly enhances polymer disassembly and dynamics.

The structure of a small GTPaseRhoA in complex with PDZRhoGEF and the inhibitor HL47.

To study the structure of a small GTPaseRhoA in complex with PDZRhoGEF and the inhibitor HL47, and to provide an easier template for R&D of RhoA inhibitor. Our initial attempts to obtain a binary complex of RhoA with the inhibitor HL47 were unsuccessful probably due to the presence of GDP. By targeting a ternary complex involving the RhoA-specific guanine nucleotide exchange factor PDZRhoGEF, we eliminated GDP and obtained a 2.3Å structure of the RhoA-PDZRhoGEF-inhibitor HL47 ternary complex. This structure provides a new template for target-based pharmaceutical design against RhoA.

Occupation of nucleotide in the binding pocket is critical to the stability of Rab11A

The Ras superfamily of small G proteins is a family of guanosine triphosphatases (GTPases) and each GTPase has conserved amino acid sequences in the enzymatic active site that are responsible for specific interactions with GDP and GTP molecules. Rab GTPases, which belong to the Ras superfamily, are key regulators of intracellular vesicle trafficking via the recruitment of effector molecules. Here, we purified wild type, active mutant and inactive mutant of Rab11A. In this process, we found that the inactive mutant (Rab11A S25N) had low stability compared with wild type and other mutants. Further analysis revealed that the stability of Rab11A S25N is dependent on the occupation of GDP in the nucleotide binding pocket of the protein. We found that the stability of Rab11A S25N is affected by the presence of GDP, not other nucleotides, and is independent of pH or salt in FPLC buffer. Our results provide a better understanding of how GTPase can be stable under in vitro conditions without effector proteins and how proper substrate/cofactor coordination is crucial to the stability of Rab11A. Successful purification and proposed purification methods will provide a valuable guide for investigation of other small GTPase proteins.

A Kinetic Model of GPCRs: Analysis of G protein Activity, Occupancy, Coupling and Receptor‐State Affinity Constants

G protein-coupled receptors, upon agonist binding, accelerate the exchange of GDP for GTP in G proteins coupled to them. The activated G protein interacts with effector proteins to mediate diverse biological functions. We present a kinetic, quaternary complex model, based on a system of coupled linear first-order differential equations that accounts for GTPase activity, ligand binding to receptor, and guanine nucleotide binding to G protein. We solved the model numerically to predict the extents of G protein activation, receptor occupancy by ligand, and receptor coupling that result from varying the ligand concentration, presence of GDP and/or GTP, the ratio of G protein to receptor, and the equilibrium constants governing receptor pre-coupling and constitutive activity. We also simulated responses downstream from G protein activation using a transducer function. Our model shows that agonist-induced G protein activation can occur with either a net decrease or increase in total receptor-G protein coupling. We also demonstrate that affinity constants of the ligand for both the active and inactive states of the receptor can be derived to a close approximation from analysis of simulated responses downstream from receptor activation. Finally, we show that the concentration of GDP greatly affects the potency of agonists for stimulating the binding of non-hydrolyzable GTP analogs. Our analysis relates to agonist-stimulated [35S]GTPγS binding measurements and the interpretation of receptor-G protein interactions using fluorescence techniques. Finally, it validates our prior methods for estimating the active state affinity constants of ligands.

A kinetic model of GPCRs: analysis of G protein activity, occupancy, coupling and receptor-state affinity constants

Context: G protein-coupled receptors are vital macromolecules for a wide variety of physiological processes. Upon agonist binding, these receptors accelerate the exchange of GDP for GTP in G proteins coupled to them. The activated G protein interacts with effector proteins to implement downstream biological functions. Objective: We present a kinetic, quaternary complex model, based on a system of coupled linear first-order differential equations, which accounts for the binding attributes of the ligand, receptor, G protein and two types of guanine nucleotide (GDP and GTP) as well as for GTPase activity. Methods: We solved the model numerically to predict the extents of G protein activation, receptor occupancy by ligand and receptor coupling that result from varying the ligand concentration, presence of GDP and/or GTP, the ratio of G protein to receptor and the equilibrium constants governing receptor pre-coupling and constitutive activity. We also simulated responses downstream from G protein activation using a transducer function. Results: Our model shows that agonist-induced G protein activation can occur with either a net decrease or increase in total receptor-G protein coupling. In addition, we demonstrate that affinity constants of the ligand for both the active and inactive states of the receptor can be derived to a close approximation from analysis of simulated responses downstream from receptor activation. Discussion and conclusion: The latter result validates our prior methods for estimating the active state affinity constants of ligands, and our results on receptor coupling have relevance to studies investigating receptor-G protein interactions using fluorescence techniques.

The Structural Basis of FtsY Recruitment and GTPase Activation by SRP RNA

The universally conserved signal recognition particle (SRP) system mediates the targeting of membrane proteins to the translocon in a multistep process controlled by GTP hydrolysis. Here we present the 2.6 Å crystal structure of the GTPase domains of the E. coli SRP protein (Ffh) and its receptor (FtsY) in complex with the tetraloop and the distal region of SRP-RNA, trapped in the activated state in presence of GDP:AlF4. The structure reveals the atomic details of FtsY recruitment and, together with biochemical experiments, pinpoints G83 as the key RNA residue that stimulates GTP hydrolysis. Insertion of G83 into the FtsY active site orients a single glutamate residue provided by Ffh (E277), triggering GTP hydrolysis and complex disassembly at the end of the targeting cycle. The complete conservation of the key residues of the SRP-RNA and the SRP protein implies that the suggested chemical mechanism of GTPase activation is applicable across all kingdoms.

Ligands Raise the Constraint That Limits Constitutive Activation in G Protein-coupled Opioid Receptors

Using a cell-free bioluminescence resonance energy transfer strategy we compared the levels of spontaneous and ligand-induced receptor-G protein coupling in δ (DOP) and μ (MOP) opioid receptors. In this assay GDP can suppress spontaneous coupling, thus allowing its quantification. The level of constitutive activity was 4-5 times greater at the DOP than at the MOP receptor. A series of opioid analogues with a common peptidomimetic scaffold displayed remarkable inversions of efficacy in the two receptors. Agonists that enhanced coupling above the low intrinsic level of the MOP receptor were inverse agonists in reducing the greater level of constitutive coupling of the DOP receptor. Yet the intrinsic activities of such ligands are identical when scaled over the GDP base line of both receptors. This pattern is in conflict with the predictions of the ternary complex model and the "two state" extensions. According to this theory, the order of spontaneous and ligand-induced coupling cannot be reversed if a shift of the equilibrium between active and inactive forms raises constitutive activation in one receptor type. We propose that constitutive activation results from a lessened intrinsic barrier that restrains spontaneous coupling. Any ligand, regardless of its efficacy, must enhance this constraint to stabilize the ligand-bound complexed form.