RESUMO
Two cAMP signaling compartments centered on adenylyl cyclase (AC) exist in human airway smooth muscle (HASM) cells, one containing ß2-adrenergic receptor AC6 and another containing E prostanoid receptor AC2. We hypothesized that different PDE isozymes selectively regulate cAMP signaling in each compartment. According to RNA-sequencing data, 18 of 24 PDE genes were expressed in primary HASM cells derived from age- and sex-matched donors with and without asthma. PDE8A was the third most abundant of the cAMP-degrading PDE genes, after PDE4A and PDE1A. Knockdown of PDE8A using shRNA evoked twofold greater cAMP responses to 1 µM forskolin in the presence of 3-isobutyl-1-methylxanthine. Overexpression of AC2 did not alter this response, but overexpression of AC6 increased cAMP responses an additional 80%. We examined cAMP dynamics in live HASM cells using a fluorescence sensor. PF-04957325, a PDE8-selective inhibitor, increased basal cAMP concentrations by itself, indicating a significant basal level of cAMP synthesis. In the presence of an AC inhibitor to reduce basal signaling, PF-04957325 accelerated cAMP production and increased the inhibition of cell proliferation induced by isoproterenol, but it had no effect on cAMP concentrations or cell proliferation regulated by prostaglandin E2. Lipid raft fractionation of HASM cells revealed PDE8A immunoreactivity in buoyant fractions containing caveolin-1 and AC5/6 immunoreactivity. Thus, PDE8 is expressed in lipid rafts of HASM cells, where it specifically regulates ß2-adrenergic receptor AC6 signaling without effects on signaling by the E prostanoid receptors 2/4-AC2 complex. In airway diseases such as asthma and chronic obstructive pulmonary disease, PDE8 may represent a novel therapeutic target to modulate HASM responsiveness and airway remodeling.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Adenilil Ciclases/metabolismo , Asma/enzimologia , AMP Cíclico/metabolismo , Músculo Liso/enzimologia , Miócitos de Músculo Liso/enzimologia , Receptores Adrenérgicos beta 2/metabolismo , Sistema Respiratório/enzimologia , 3',5'-AMP Cíclico Fosfodiesterases/genética , Adenilil Ciclases/genética , Remodelação das Vias Aéreas , Asma/genética , Asma/patologia , Asma/fisiopatologia , Estudos de Casos e Controles , Proliferação de Células , Células Cultivadas , Humanos , Microdomínios da Membrana/enzimologia , Microdomínios da Membrana/patologia , Músculo Liso/patologia , Músculo Liso/fisiopatologia , Miócitos de Músculo Liso/patologia , Receptores Adrenérgicos beta 2/genética , Sistema Respiratório/patologia , Sistema Respiratório/fisiopatologia , Sistemas do Segundo Mensageiro , Fatores de TempoRESUMO
Allosteric interactions play vital roles in metabolic processes and signal transduction and, more recently, have become the focus of numerous pharmacological studies because of the potential for discovering more target-selective chemical probes and therapeutic agents. In addition to classic early studies on enzymes, there are now examples of small molecule allosteric modulators for all superfamilies of receptors encoded by the genome, including ligand- and voltage-gated ion channels, G protein-coupled receptors, nuclear hormone receptors, and receptor tyrosine kinases. As a consequence, a vast array of pharmacologic behaviors has been ascribed to allosteric ligands that can vary in a target-, ligand-, and cell-/tissue-dependent manner. The current article presents an overview of allostery as applied to receptor families and approaches for detecting and validating allosteric interactions and gives recommendations for the nomenclature of allosteric ligands and their properties.
Assuntos
Regulação Alostérica/efeitos dos fármacos , Ligantes , Terminologia como Assunto , Humanos , Canais Iônicos/metabolismo , Modelos Químicos , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Seven transmembrane receptors were originally named and characterized based on their ability to couple to heterotrimeric G proteins. The assortment of coupling partners for G protein-coupled receptors has subsequently expanded to include other effectors (most notably the ßarrestins). This diversity of partners available to the receptor has prompted the pursuit of ligands that selectively activate only a subset of the available partners. A biased or functionally selective ligand may be able to distinguish between different active states of the receptor, and this would result in the preferential activation of one signaling cascade more than another. Although application of the "standard" operational model for analyzing ligand bias is useful and suitable in most cases, there are limitations that arise when the biased agonist fails to induce a significant response in one of the assays being compared. In this article, we describe a quantitative method for measuring ligand bias that is particularly useful for such cases of extreme bias. Using simulations and experimental evidence from several κ opioid receptor agonists, we illustrate a "competitive" model for quantitating the degree and direction of bias. By comparing the results obtained from the competitive model with the standard model, we demonstrate that the competitive model expands the potential for evaluating the bias of very partial agonists. We conclude the competitive model provides a useful mechanism for analyzing the bias of partial agonists that exhibit extreme bias.
Assuntos
Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Animais , Arrestinas/metabolismo , Células CHO , Linhagem Celular , Cricetulus , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Ligantes , Receptores Opioides kappa/agonistas , Transdução de Sinais/efeitos dos fármacosRESUMO
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.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Modelos Biológicos , Receptores Acoplados a Proteínas G/metabolismo , Animais , Simulação por Computador , Proteínas de Ligação ao GTP/química , Humanos , Cinética , Ligantes , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química , Transdução de SinaisRESUMO
We investigated how asparagine mutagenesis of conserved aspartic acids in helix 2 (D2.50) and 3 (D3.32) of M1-M4 muscarinic receptors alters the irreversible binding of acetylcholine mustard and BR384 (4-[(2-bromoethyl)methyl-amino]-2-butynyl N-(3-chlorophenyl)carbamate), a nitrogen mustard derivative of McN-A-343 ([4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyl] trimethylammonium chloride). The D2.50N mutation moderately increased the affinity of the aziridinium ions of acetylcholine mustard and BR384 for M2-M4 receptors and had little effect on the rate constant for receptor alkylation. The D3.32N mutation greatly reduced the rate constant for receptor alkylation by acetylcholine mustard but not by BR384, although the affinity of BR384 was reduced. The combination of both mutations (D2.50N/D3.32N) substantially reduced the rate constant for receptor alkylation by BR384 relative to that of wild type and mutant D2.50N and D3.32N receptors. The change in binding affinity caused by the mutations suggests that the D2.50N mutation alters the interaction of acetylcholine mustard with D3.32 of the M1 and M3 receptors but not that of the M4 receptor. BR384 exhibited the converse relationship. The simplest explanation is that acetylcholine mustard and BR384 alkylate at least two residues on M1-M4 receptors and that the D2.50N mutation alters the rate of alkylation of D3.32 relative to another residue, perhaps D2.50 itself.
Assuntos
Acetilcolina/metabolismo , Asparagina/genética , Ácido Aspártico/genética , Mecloretamina/metabolismo , Receptores Muscarínicos/genética , Acetilcolina/análogos & derivados , Animais , Células CHO , Cricetinae , Cricetulus , Cobaias , Humanos , Cinética , Mutagênese , N-Metilescopolamina/metabolismo , Receptores Muscarínicos/química , Receptores Muscarínicos/metabolismoRESUMO
Muscarinic agonists and antagonists are used to treat a handful of gastrointestinal (GI) conditions associated with impaired salivary secretion or altered motility of GI smooth muscle. With regard to exocrine secretion, the major muscarinic receptor expressed in salivary, gastric, and pancreatic glands is the M3 with a small contribution of the M1 receptor. In GI smooth muscle, the major muscarinic receptors expressed are the M2 and M3 with the M2 outnumbering the M3 by a ratio of at least four to one. The antagonism of both smooth muscle contraction and exocrine secretion is usually consistent with an M3 receptor mechanism despite the major presence of the M2 receptor in smooth muscle. These results are consistent with the conditional role of the M2 receptor in smooth muscle. That is, the contractile role of the M2 receptor depends on that of the M3 so that antagonism of the M3 receptor eliminates the response of the M2. The physiological roles of muscarinic receptors in the GI tract are consistent with their known signaling mechanisms. Some so-called tissue-selective M3 antagonists may owe their selectivity to a highly potent interaction with a nonmuscarinic receptor target.
Assuntos
Fármacos Gastrointestinais/farmacologia , Trato Gastrointestinal/efeitos dos fármacos , Agonistas Muscarínicos/farmacologia , Antagonistas Muscarínicos/farmacologia , Sistema Nervoso Parassimpático/efeitos dos fármacos , Receptores Muscarínicos/efeitos dos fármacos , Acetilcolina/metabolismo , Animais , Gastroenteropatias/tratamento farmacológico , Gastroenteropatias/metabolismo , Gastroenteropatias/fisiopatologia , Trato Gastrointestinal/inervação , Trato Gastrointestinal/metabolismo , Humanos , Sistema Nervoso Parassimpático/metabolismo , Sistema Nervoso Parassimpático/fisiopatologia , Receptores Muscarínicos/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
We describe a modification of receptor theory for the estimation of observed affinities (K(obs)) and relative efficacies of orthosteric ligands in functional assays that exhibit constitutive activity. Our theory includes parameters for the fractions of the occupied receptor population in the active (intrinsic efficacy, ε) and inactive (ε(i)) states and analogous parameters for the fractions of the free receptor population in the active (ε(sys)) and inactive (ε(i-sys)) states. The total stimulus represents the summation of the active states of the free and occupied receptor populations. A modified operational model is developed that expresses the response as a logistic function of the total stimulus. This function includes the standard parameters related to affinity and efficacy (K(obs) and τ) as well as a parameter proportional to the activity of the free receptor complex, τ(sys). Two related parameters are proportional to the fraction of the free (τ(i-sys)) and occupied (τ(i)) receptor populations in the inactive state. We show that the estimates of the affinity constants of orthosteric ligands for the active (K(b)) and inactive (K(a)) states of the receptor are equivalent to τK(obs)/τ(sys) and τ(i)K(obs)/τ(i-sys), respectively. We verify our method with computer simulation techniques and apply it to the analysis of M(2) and M(3) muscarinic receptors. Our method is applicable in the analysis of ligand bias in drug discovery programs.
Assuntos
Agonismo Inverso de Drogas , Modelos Biológicos , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/metabolismo , Simulação por Computador , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Inositol/metabolismo , Ligantes , Ligação Proteica/fisiologia , EstereoisomerismoRESUMO
We describe a modification of receptor theory that enables the estimation of relative affinity constants for the inactive state of a G protein-coupled receptor. Our approach includes the traditional parameters of observed affinity (K(obs)) and efficacy (fraction of ligand-receptor complex in the active state, ε) and introduces the concept of the fraction of the ligand-receptor complex in the inactive state (intrinsic inactivity, ε(i)). The relationship between receptor activation and the ligand concentration is known as the stimulus, and the operational model expresses the response as a logistic function of the stimulus. The latter function includes K(obs) and the parameter τ, which is proportional to ε. We introduce the parameter τ(i), which is proportional to ε(i). We have previously shown that the product, K(obs)τ, of one agonist, expressed relative to that of another (intrinsic relative activity, RA(i)), is a relative measure of the affinity constant for the active state of the receptor. In this report, we show that the product, K(obs)τ(i), of one agonist, expressed relative to that of another (intrinsic relative inactivity, RI(i)), is a relative measure of the affinity constant for the inactive state of the receptor. We use computer simulation techniques to verify our analysis and apply our method to the analysis of published data on agonist activity at the M(3) muscarinic receptor. Our method should have widespread application in the analysis of agonist bias in drug discovery programs and in the estimation of a more fundamental relative measure of efficacy (RA(i)/RI(i)).
Assuntos
Modelos Biológicos , Receptor Muscarínico M2/agonistas , Receptor Muscarínico M2/fisiologia , Receptor Muscarínico M3/agonistas , Receptor Muscarínico M3/fisiologia , Animais , Células CHO , Simulação por Computador/estatística & dados numéricos , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Cobaias , Humanos , Camundongos , Preparações Farmacêuticas/metabolismo , Ligação Proteica/efeitos dos fármacos , Receptor Muscarínico M2/metabolismo , Receptor Muscarínico M3/metabolismoRESUMO
Investigating how a test drug alters the reaction of a site-directed electrophile with a receptor is a powerful method for determining whether the drug acts competitively or allosterically, provided that the binding site of the electrophile is known. In this study, therefore, we mutated nucleophilic residues near and within the orthosteric pockets of M(1) and M(2) muscarinic receptors to identify where acetylcholine mustard and 4-[(2-bromoethyl)methyl-amino]-2-butynyl-N-(3-chlorophenyl)carbamate (BR384) bind covalently. BR384 is the nitrogen mustard analog of [4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyl]trimethylammonium chloride (McN-A-343). Mutation of the highly conserved aspartic acid in M(1) (Asp105) and M(2) (Asp103) receptors to asparagine largely prevented receptor alkylation by acetylcholine mustard, although modest alkylation still occurred at M(2) D103N at high concentrations of the mustard. Receptor alkylation by BR384 was also greatly inhibited in the M(1) D105N mutant, but some alkylation still occurred at high concentrations of the compound. In contrast, BR384 rapidly alkylated the M(2) D103N mutant. Its affinity was reduced to one tenth, however. The alkylation of M(2) D103N by BR384 was competitively inhibited by N-methylscopolamine and allosterically inhibited by gallamine. Mutation of a variety of other nucleophilic residues, some in combination with D103N, had little effect on M(2) receptor alkylation by BR384. Our results suggest that BR384 alkylates at least one residue other than the conserved aspartic acid at the ligand-binding site of M(1) and M(2) receptors. This additional residue seems to be located within or near the orthosteric-binding pocket and is not part of the allosteric site for gallamine.
Assuntos
Cloreto de (4-(m-Clorofenilcarbamoiloxi)-2-butinil)trimetilamônio/metabolismo , Acetilcolina/análogos & derivados , Mutagênese Sítio-Dirigida , Receptor Muscarínico M1/metabolismo , Receptor Muscarínico M2/metabolismo , Cloreto de (4-(m-Clorofenilcarbamoiloxi)-2-butinil)trimetilamônio/análogos & derivados , Acetilcolina/química , Acetilcolina/metabolismo , Sítio Alostérico/genética , Animais , Sequência de Bases , Sítios de Ligação/genética , Células CHO , Cricetinae , Cricetulus , Humanos , Mecloretamina/química , Mecloretamina/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida/métodos , Ligação Proteica/genética , Receptor Muscarínico M1/genética , Receptor Muscarínico M2/genéticaRESUMO
We investigated the functional role of a conserved motif, F(x)(6)LL, in the membrane proximal C-tail of the human muscarinic M(1) (hM(1)) receptor. By use of site-directed mutagenesis, several different point mutations were introduced into the C-tail sequence (423)FRDTFRLLL(431). Wild-type and mutant hM(1) receptors were transiently expressed in Chinese hamster ovary cells, and the amount of plasma membrane-expressed receptor was determined by use of intact, whole-cell [(3)H]N-methylscopolamine binding assays. The plasma membrane expression of hM(1) receptors possessing either L430A or L431A or both point mutations was significantly reduced compared with the wild type. The hM(1) receptor possessing a L430A/L431A double-point mutation was retained in the endoplasmic reticulum (ER), and atropine treatment caused the redistribution of the mutant receptor from the ER to the plasma membrane. Atropine treatment also caused an increase in the maximal response and potency of carbachol-stimulated phosphoinositide hydrolysis elicited by the L430A/L431A mutant. The effect of atropine on the L430A/L431A receptor mutant suggests that L(430) and L(431) play a role in folding hM(1) receptors, which is necessary for exit from the ER. Using site-directed mutagenesis, we also identified amino acid residues at the base of transmembrane-spanning domain 1 (TM1), V(46) and L(47), that, when mutated, reduce the plasma membrane expression of hM(1) receptors in an atropine-reversible manner. Overall, these mutagenesis data show that amino acid residues in the membrane-proximal C-tail and base of TM1 are necessary for hM(1) receptors to achieve a transport-competent state.
Assuntos
Membrana Celular/metabolismo , Receptor Muscarínico M1/genética , Receptor Muscarínico M1/metabolismo , Motivos de Aminoácidos , Animais , Atropina/farmacologia , Células CHO , Carbacol/farmacologia , Clonagem Molecular , Sequência Consenso , Cricetinae , Cricetulus , Retículo Endoplasmático/metabolismo , Humanos , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação Puntual , Ligação Proteica , Dobramento de Proteína , TransfecçãoRESUMO
Nalfurafine, a moderately selective kappa opioid receptor (KOR) agonist, is used in Japan for treatment of itch without causing dysphoria or psychotomimesis. Here we characterized the pharmacology of compound 42B, a 3-dehydroxy analogue of nalfurafine and compared with that of nalfurafine. Nalfurafine and 42B acted as full KOR agonists and partial µ opioid receptor (MOR) agonists, but 42B showed much lower potency for both receptors and lower KOR/MOR selectivity, different from previous reports. Molecular modeling revealed that water-mediated hydrogen-bond formation between 3-OH of nalfurafine and KOR accounted for its higher KOR potency than 42B. The higher potency of both at KOR over MOR may be due to hydrogen-bond formation between nonconserved Y7.35 of KOR and their carbonyl groups. Both showed modest G protein signaling biases. In mice, like nalfurafine, 42B produced antinociceptive and antiscratch effects and did not cause conditioned place aversion (CPA) in the effective dose ranges. Unlike nalfurafine, 42B caused motor incoordination and hypolocomotion. As both agonists showed G protein biases, yet produced different effects on locomotor activity and motor incoordination, the findings and those in the literature suggest caution in correlating in vitro biochemical data with in vivo behavior effects. The factors contributing to the disconnect, including pharmacodynamic and pharmacokinetic issues, are discussed. In addition, our results suggest that among the KOR-induced adverse behaviors, CPA can be separated from motor incoordination and hypolocomotion.
Assuntos
Morfinanos , Receptores Opioides kappa/antagonistas & inibidores , Compostos de Espiro , Analgésicos Opioides/farmacologia , Animais , Viés , Camundongos , Morfinanos/farmacologia , Compostos de Espiro/farmacologiaRESUMO
In prior work, we have shown that it is possible to estimate the product of observed affinity and intrinsic efficacy of an agonist expressed relative to that of a standard agonist simply through the analysis of their respective concentration-response curves. In this report, we show analytically and through mathematical modeling that this product, termed intrinsic relative activity (RA(i)), is equivalent to the ratio of microscopic affinity constants of the agonists for the active state of the receptor. We also compared the RA(i) estimates of selected muscarinic agonists with a relative estimate of the product of observed affinity and intrinsic efficacy determined independently through the method of partial receptor inactivation. There was good agreement between these two estimates when agonist-mediated inhibition of forskolin-stimulated cAMP accumulation was measured in Chinese hamster ovary cells stably expressing the human M(2) muscarinic receptor. Likewise, there was good agreement between the two estimates when agonist activity was measured on the ileum from M(2) muscarinic receptor knockout mice, a convenient assay for M(3) receptor activity. The RA(i) estimates of agonists in the mouse ileum were similar to those estimated at the human M(3) receptor with the exception of 4-(m-chlorophenyl-carbamoyloxy)-2-butynyltrimethylammonium (McN-A-343), which is known to be an M(1)- and M(4)-selective muscarinic agonist. Additional experiments showed that the response to McN-A-343 in the mouse ileum included a non-M(3) muscarinic receptor component. Our results show that the RA(i) estimate is a useful receptor-dependent measure of agonist activity and ligand-directed signaling.
Assuntos
Domínios e Motivos de Interação entre Proteínas , Receptor Muscarínico M2/química , Receptor Muscarínico M3/química , Animais , Células CHO , Cricetinae , Cricetulus , Feminino , Humanos , Masculino , Camundongos , Fenômenos FísicosRESUMO
We measured the intrinsic relative activity (RA(i)) of muscarinic agonists to detect possible selectivity for receptor subtypes and signaling pathways. RA(i) is a relative measure of the microscopic affinity constant of an agonist for the active state of a GPCR expressed relative to that of a standard agonist. First, we estimated RA(i) values for a panel of agonists acting at the M(4) muscarinic receptor coupled to three distinct G-protein pathways: G(i) inhibition of cAMP accumulation, G(s) stimulation of cAMP accumulation, and G alpha(15) stimulation of phosphoinositide hydrolysis. Our results show similar RA(i) values for each agonist, suggesting that the same active state of the M(4) receptor triggers the activation of the three G proteins. We also estimated RA(i) values for agonists across M(1) to M(4) muscarinic subtypes stably transfected in Chinese hamster ovary cells. Our results show selectivity of McN-A-343 [4-I-[3-chlorophenyl]carbamoyloxy)-2-butynyltrimethylammnonium chloride] for the M(1) and M(4) subtypes and selectivity of pilocarpine for the M(1) and M(3) subtypes. The other agonists tested lacked marked selectivity among M(1) to M(4) receptors. Finally, we estimated RA(i) values from published literature on M(1), M(2), and M(3) muscarinic responses and obtained results consistent with our own studies. Our results show that the RA(i) estimate is a useful receptor-dependent measure of agonist activity.
Assuntos
Receptor Muscarínico M1/agonistas , Receptor Muscarínico M2/agonistas , Receptor Muscarínico M3/agonistas , Receptor Muscarínico M4/agonistas , Animais , Células CHO , Colforsina/farmacologia , Cricetinae , Cricetulus , AMP Cíclico/fisiologia , Humanos , Cinética , Receptor Muscarínico M1/classificação , Receptor Muscarínico M2/classificação , Receptor Muscarínico M3/classificação , Receptor Muscarínico M4/classificação , Receptores Acoplados a Proteínas G/fisiologia , Proteínas Recombinantes/classificação , Proteínas Recombinantes/metabolismo , Análise de Regressão , TransfecçãoRESUMO
G protein-coupled receptors (GPCRs) can interact with both G proteins and ß-arrestin proteins to propagate different signaling outputs. In some contexts, agonists may drive the receptor to preferentially engage one of these effectors over the other. Such "ligand bias" may present a means to impart pathway-selective signaling downstream of this class of receptors. In cases where physiological responses are mediated by diverse pathways, this could, in part, provide a means to refine GPCR therapeutics. Cell-based signaling assays are used to measure the potential for signaling bias in vitro, and these measures take into account potency, efficacy, and the overall capacity of the assay. However, narrow assay windows sometimes limit the confidence in estimating agonist activity, if a compound performs as a very weakly efficacious partial agonist. This lack of response in an assay hampers the ability to measure and compare potencies, and the degree of separation of an agonist's performance, between two assays. In this chapter, we describe in detail a method for the estimation of the relative activity of a partial agonist and provide a stepwise protocol for calculating bias when this case arises.
Assuntos
Bioensaio/métodos , Modelos Biológicos , Viés , Intervalos de Confiança , Ligantes , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismoRESUMO
We measured the influence of gallamine on the functional responses and binding properties of selected agonists at the M(2) muscarinic receptor and analyzed the data within the context of the allosteric ternary complex model. Our analysis showed that gallamine modified agonist affinity without influencing efficacy. To explain this behavior, we investigated the allosteric ternary complex model at a deeper level of analysis to assess allosterism in terms of the differential affinity of gallamine for ground and active states of the receptor. Our simulations showed that two-state models based on a single orthosteric site for the agonist linked to an allosteric site for gallamine could not account for affinity-only modulation, even if multiple conformations of ground and active states were considered. We also expanded the tandem two-site model (J Biol Chem 275:18836-18844, 2000) within the context of the allosteric ternary complex model and analyzed the resulting hybrid model at the level of receptor states. This model posits that the agonist first binds to a relay site and then shuttles to the activation site to turn on the receptor. If it is assumed that allosterism occurs at the relay site and not the activation site, then this model can account for affinity-only modulation in a manner consistent with the allosteric ternary complex model.
Assuntos
Trietiodeto de Galamina/metabolismo , Modelos Biológicos , Receptor Muscarínico M2/metabolismo , Regulação Alostérica , Animais , Células CHO , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Humanos , Agonistas Muscarínicos/metabolismo , N-Metilescopolamina/metabolismoRESUMO
We determined the functional role of a small domain in the third intracellular loop of the human muscarinic M(1) (hM(1)) receptor. Using site-directed mutagenesis, several mutant hM(1) receptors were made possessing either a deletion or point mutations within the third intracellular loop domain (252)PETPPGRCCRCC(263). Wild-type and mutant hM(1) receptors were transiently expressed in Chinese hamster ovary cells, and the effects of each mutation on radioligand binding, agonist-mediated phosphoinositide hydrolysis, and agonist-induced internalization were determined. The mutant receptors exhibited a modest reduction in affinity for [(3)H]N-methylscopolamine (pK(D) = approximately 9.0) and a moderately increased binding capacity relative to the wild-type receptor. This moderate increase in binding capacity was associated with small increases in the maximal response and potency of carbachol for eliciting phosphoinositide hydrolysis through the mutant receptors (pEC(50) = approximately 5.5) relative to wild-type (pEC(50) = 5.35 +/- 0.05). In contrast, carbachol-induced internalization of mutant hM(1) receptors possessing either C259A/C260A or C262A/C263A or both double point mutations was significantly reduced compared to the wild-type hM(1) receptor. Of the hM(1) receptor mutants tested, those possessing a C262D/C263D double point mutation had the least carbachol-induced internalization. The desensitization and down-regulation of receptors possessing either Cys/Ala or Cys/Asp double point mutations were similar to those observed for the wild-type hM(1) receptor. Collectively, these observations suggest that Cys pairs Cys259/Cys260 and Cys262/Cys263 play an important role in the agonist-induced internalization of hM(1) receptors.
Assuntos
Cisteína/fisiologia , Receptor Muscarínico M1/química , Receptor Muscarínico M1/fisiologia , Animais , Células CHO , Carbacol/farmacologia , Cricetinae , Cricetulus , Cisteína/química , Humanos , Hidrólise , Mutagênese Sítio-Dirigida , N-Metilescopolamina/metabolismo , Fosfatidilinositóis/metabolismo , Estrutura Terciária de Proteína , Receptor Muscarínico M1/agonistas , Receptor Muscarínico M1/antagonistas & inibidores , TransfecçãoRESUMO
We explored the interaction of a nitrogen mustard derivative of acetylcholine with the human M(2) muscarinic receptor expressed in Chinese hamster ovary cells using the muscarinic radioligand, [3H]N-methylscopolamine (NMS). Acetylcholine mustard caused a concentration-dependent, first-order loss of [3H]NMS binding at 37 degrees C, with the half-maximal rate constant occurring at 24 microM and a maximal rate constant of 0.16 min(-1). We examined the effects of various ligands on the rate of alkylation of M(2) receptors by acetylcholine mustard. N-methylscopolamine and 4-(trimethylamino)-2-butynyl-(3-chlorophenyl)carbamate (McN-A-343) competitively slowed the rate of alkylation, whereas the inhibition by gallamine reached a plateau at high concentrations, indicating allosteric inhibition. In contrast, 17-beta-hydroxy-17-alpha-ethynyl-5-alpha-androstano[3,2-beta]-pyrimido[1,2-alpha]benzimidazole (WIN 51708) had no effect. We also measured the inhibition of [3H]NMS binding by acetylcholine mustard at 0 degrees C, conditions under which there is little or no detectable covalent binding. In these experiments, the dissociation constant of the aziridinium ion of acetylcholine mustard was estimated to be 12.3 microM. In contrast, the parent mustard and alcoholic hydrolysis product of acetylcholine mustard were without effect. Our results show that measurement of the effects of ligands on the rate of inactivation of the orthosteric site by a small site-directed electrophile is a powerful method for discriminating competitive inhibition from allosterism.
Assuntos
Acetilcolina/análogos & derivados , Receptor Muscarínico M2/metabolismo , Cloreto de (4-(m-Clorofenilcarbamoiloxi)-2-butinil)trimetilamônio/farmacologia , Acetilcolina/farmacologia , Alquilação , Regulação Alostérica , Androstanos/farmacologia , Animais , Benzimidazóis/farmacologia , Ligação Competitiva , Células CHO , Cricetinae , Cricetulus , Trietiodeto de Galamina/farmacologia , Humanos , N-Metilescopolamina/metabolismoRESUMO
The phenomenon of "ligand-directed signaling" is often considered to be inconsistent with the traditional receptor theory. In this review, I show how the mathematics of the receptor theory can be used to measure the observed affinity and relative efficacy of protean ligands at G protein-coupled receptors. The basis of this analysis rests on the assumption that the fraction of agonist bound in the form of the active receptor-G protein-guanine nucleotide complex is the biochemical equivalent of the pharmacological stimulus. Consequently, this stimulus function is analogous to the current response of a ligand-gated ion channel. Because guanosine triphosphate (GTP) greatly inhibits the formation of the active quaternary complex, even the most efficacious agonists probably only elicit partial receptor activation, and it seems likely that the ceiling of 100% receptor activation is not reached in the intact cell with high intracellular concentrations of GTP. Under these conditions, the maximum of the stimulus function is proportional to the ratio of microscopic affinity constants of the agonist for ground and active states. Ligand-directed signaling depends on the existence of different active states of the receptor with different selectivities for different G proteins or other effectors. This phenomenon can be characterized using classic pharmacological methods. Although not widely appreciated, it is possible to estimate the product of observed affinity and intrinsic efficacy expressed relative to that of another agonist (intrinsic relative activity) through the analysis of the concentration-response curves. No other information is required. This approach should be useful in quantifying agonist activity and in converting the two disparate parameters of potency and maximal response into a single parameter dependent only on the agonist-receptor-effector complex.
Assuntos
Modelos Biológicos , Receptores Acoplados a Proteínas G/agonistas , Transdução de Sinais/efeitos dos fármacos , Animais , Guanosina Trifosfato/metabolismo , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/metabolismo , Ligantes , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Agonists and most natural ligands bind to receptors in their inactive state and quickly induce an active receptor conformation that initiates cell signaling. The active receptor state initiates signaling because of its structural complementariness with coupling proteins that activate signaling pathways, such as G proteins and G protein-coupled receptor kinases. Agonist bias refers to the propensity of an agonist to direct receptor signaling through one pathway relative to another. Thus, if the agonist exhibits much higher affinity for active state 1 compared to active state 2, it will cause a robust activation of receptor coupling protein 1 but not 2, and ultimately, a preferential stimulation of signaling pathway 1. Biased agonists are potentially more selective therapeutic agents because there are numerous cases where the therapeutic and adverse effects of an agonist are mediated by distinct pathways involving G proteins and ß-arrestin. Given the mechanism for agonist bias, the most straightforward approach for quantifying bias involves the estimation of agonist affinity for the inactive receptor state and the active receptor states involved in signaling through different pathways. The approach provides quantitative estimates of the sensitivities of different signaling pathways, enabling one to determine to what extent the observed selectivity is caused by agonist or system bias. In addition, the approach is a powerful adjunct to in silico docking studies and can be applied to in vivo assays, structure-activity relationships, and the analysis of published agonist concentration-response curves.
Assuntos
Receptores Acoplados a Proteínas G/agonistas , Animais , Simulação por Computador , Avaliação Pré-Clínica de Medicamentos , Proteínas de Ligação ao GTP/metabolismo , Humanos , Modelos Biológicos , Receptores Acoplados a Proteínas G/metabolismo , Padrões de ReferênciaRESUMO
We describe a method for estimating the affinities of ligands for active and inactive states of a G protein-coupled receptor (GPCR). Our protocol involves measuring agonist-induced signaling responses of a wild type GPCR and a constitutively active mutant of it under control conditions and after partial receptor inactivation or reduced receptor expression. Our subsequent analysis is based on the assumption that the activating mutation increases receptor isomerization into the active state without affecting the affinities of ligands for receptor states. A means of confirming this assumption is provided. Global nonlinear regression analysis yields estimates of 1) the active (Kact) and inactive (Kinact) receptor-state affinity constants, 2) the isomerization constant of the unoccupied receptor (Kq-obs), and 3) the sensitivity constant of the signaling pathway (KE-obs). The latter two parameters define the output response of the receptor, and hence, their ratio (Kq-obs/KE) is a useful measure of system bias. If the cellular system is reasonably stable and the Kq-obs and KE-obs values of the signaling pathway are known, the Kact and Kinact values of additional agonists can be estimated in subsequent experiments on cells expressing the wild type receptor. We validated our method through computer simulation, an analytical proof, and analysis of previously published data. Our approach provides 1) a more meaningful analysis of structure-activity relationships, 2) a means of validating in silico docking experiments on active and inactive receptor structures and 3) an absolute, in contrast to relative, measure of agonist bias.