RESUMEN
The human histamine H(1) receptor (H(1)R) is a prototypical G protein-coupled receptor and an important, well characterized target for the development of antagonists to treat allergic conditions. Many neuropsychiatric drugs are also known to potently antagonize this receptor, underlying aspects of their side effect profiles. We have used the cell-based receptor selection and amplification technology assay to further define the clinical pharmacology of the human H(1)R by evaluating >130 therapeutic and reference drugs for functional receptor activity. Based on this screen, we have reported on the identification of 8R-lisuride as a potent stereospecific partial H(1)R agonist (Mol Pharmacol 65:538-549, 2004). In contrast, herein we report on a large number of varied clinical and chemical classes of drugs that are active in the central nervous system that display potent H(1)R inverse agonist activity. Absolute and rank order of functional potency of these clinically relevant brain-penetrating drugs may possibly be used to predict aspects of their clinical profiles, including propensity for sedation.
Asunto(s)
Fármacos del Sistema Nervioso Central/farmacología , Antagonistas de los Receptores Histamínicos H1/farmacología , Receptores Histamínicos H1/efectos de los fármacos , Animales , Células COS , Chlorocebus aethiops , Clonación Molecular , Agonistas de los Receptores Histamínicos/farmacología , Humanos , Metilhistaminas/farmacología , Ratones , Células 3T3 NIH , Pirilamina/farmacologíaRESUMEN
Little research has focused on experiences of volunteers for people living with HIV/AIDS but other research on caregivers for this population illustrates the stress often experienced. This study identifies stressors experienced by volunteers for people with HIV/AIDS, and determines the types of social support needed to help volunteers cope with the stressors identified. Six volunteers were interviewed at a small non-profit organization in Appalachia providing such services. Participants indicated that providing services for people with HIV/AIDS is a rewarding experience; however, stress and frustration played a large role in their experiences with the organization. Further, the results of this study indicate that emotional and informational social support may help volunteers cope with stressors associated with providing services for people with HIV/AIDS.
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Agotamiento Profesional/prevención & control , Estrés Psicológico/prevención & control , Voluntarios/psicología , Infecciones por VIH/psicología , Humanos , Apoyo SocialRESUMEN
Drugs that antagonize D2-like receptors are effective antipsychotics, but the debilitating movement disorder side effects associated with these drugs cannot be dissociated from dopamine receptor blockade. The "atypical" antipsychotics have a lower propensity to cause extrapyramidal symptoms (EPS), but the molecular basis for this is not fully understood nor is the impact of inverse agonism upon their clinical properties. Using a cell-based functional assay, we demonstrate that overexpression of Galphao induces constitutive activity in the human D2-like receptors (D2, D3, and D4). A large collection of typical and atypical antipsychotics was profiled for activity at these receptors. Virtually all were D2 and D3 inverse agonists, whereas none was D4 inverse agonist, although many were potent D4 antagonists. The inverse agonist activity of haloperidol at D2 and D3 receptors could be reversed by mesoridazine demonstrating that there were significant differences in the degrees of inverse agonism among the compounds tested. Aripiprazole and the principle active metabolite of clozapine NDMC [8-chloro-11-(1-piperazinyl)-5H-dibenzo [b,e] [1,4] diazepine] were identified as partial agonists at D2 and D3 receptors, although clozapine itself was an inverse agonist at these receptors. NDMC-induced functional responses could be reversed by clozapine. It is proposed that the low incidence of EPS associated with clozapine and aripiprazole used may be due, in part, to these partial agonist properties of NDMC and aripiprazole and that bypassing clozapine blockade through direct administration of NDMC to patients may provide superior antipsychotic efficacy.
Asunto(s)
Antipsicóticos/metabolismo , Clozapina/metabolismo , Receptores de Dopamina D2/agonistas , Receptores de Dopamina D3/agonistas , Receptores de Dopamina D4/metabolismo , Animales , Haloperidol/farmacología , Humanos , Ratones , Células 3T3 NIH , Pergolida/farmacología , Plásmidos , Proteínas RGS/metabolismo , TransfecciónRESUMEN
RATIONALE: Clozapine is a unique antipsychotic, with efficacy against positive symptoms in treatment-resistant schizophrenic patients, and the ability to improve cognition and treat the negative symptoms characteristic of this disease. Despite its unique clinical actions, no specific molecular mechanism responsible for these actions has yet been described. OBJECTIVES AND METHODS: To comprehensively profile a large library of neuropsychiatric drugs, including most antipsychotics, at human monoamine receptors using R-SAT, an in vitro functional assay. RESULTS: Profiling revealed that N-desmethylclozapine (NDMC), the principal metabolite of clozapine, but not clozapine itself, is a potent and efficacious muscarinic receptor agonist, a molecular property not shared by any other antipsychotic. To further explore the role of NDMC muscarinic receptor agonist properties in mediating the physiological actions of clozapine, systemically administered NDMC was found to stimulate the phosphorylation of mitogen-activated protein kinase (MAP kinase) in mouse CA1 hippocampal neurons, an effect that was blocked by scopolamine, confirming central M1 muscarinic receptor agonist activity in vivo. Lastly, an analysis of clozapine and NDMC serum levels in schizophrenic patients indicated that high NDMC/clozapine ratios better predicted improvement in cognitive functioning and quality of life than the levels of either compound alone. CONCLUSIONS: The muscarinic receptor agonist activities of NDMC are unique among antipsychotics, and provide a possible molecular basis for the superior clinical effects of clozapine pharmacotherapy.
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Clozapina/análogos & derivados , Clozapina/farmacología , Agonistas Muscarínicos/farmacología , Receptor Muscarínico M1/agonistas , Animales , Células CHO , Cricetinae , Relación Dosis-Respuesta a Droga , Hipocampo/efectos de los fármacos , Hipocampo/fisiología , Humanos , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Receptor Muscarínico M1/fisiologíaRESUMEN
The human histamine H1 receptor (H1R) is an important, well characterized target for the development of antagonists to treat allergic conditions. Many neuropsychiatric drugs are known to potently antagonize the H1R, thereby producing some of their side effects. In contrast, the tolerability and potential therapeutic utility of H1R agonism is currently unclear. We have used a cell-based functional assay to evaluate known therapeutics and reference drugs for H1R agonist activity. Our initial functional screen identified three ergot-based compounds possessing heretofore-unknown H1R agonist activity. 8R-lisuride demonstrated potent agonist activity in various assays including receptor selection and amplification technology, inositol phosphate accumulation, and activation of nuclear factor-kappaB with pEC50 values of 8.1, 7.9, and 7.9, respectively, and with varying degrees of efficacy. Based on these assays, 8R-lisuride is the most potent stereospecific partial agonist for the human H1R yet reported. Investigation of the residues involved in histamine and lisuride binding, using H1R mutants and molecular modeling, have revealed that although these ligands are structurally different, the lisuride-binding pocket in the H1R closely corresponds to the histamine-binding pocket. The discovery of a potent stereospecific partial H1R agonist provides a valuable tool to further characterize this important therapeutic target in vitro.
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Agonistas de los Receptores Histamínicos/farmacología , Lisurida/farmacología , Receptores Histamínicos H1/metabolismo , Células 3T3 , Animales , Sitios de Unión , Clonación Molecular , Análisis Mutacional de ADN , Ergolinas/farmacología , Células HeLa , Humanos , Lisurida/química , Ratones , Modelos Moleculares , Conformación Molecular , Agonistas de Receptores de Serotonina/farmacologíaRESUMEN
The G-protein-coupled receptor (GPCR) superfamily is one of the largest classes of proteins in mammalian genomes. GPCRs mediate diverse physiological functions and are the targets of >50% of all clinical drugs. The sequencing of the human genome and large-scale polymorphism discovery efforts have established an abundant source of single nucleotide polymorphisms (SNPs), particularly those that result in a change in the encoded amino acids (cSNPs), many are of which in GPCRs. Although the majority of these cSNPs are assumed not to be disease-causing (nDCs), experimental data on their functional impact are lacking. Here, we have computationally analyzed the distribution of 454 cSNPs within the GPCR gene family and have found that disease-causing cSNPs (DCs) are overrepresented, whereas nDCs are underrepresented or neutral in transmembrane and extracellular loop domains, respectively. This finding reflects the relative importance of these domains to GPCR function and implies different biological characteristics for the two sets of human polymorphisms.
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Proteínas de Unión al GTP/metabolismo , Familia de Multigenes , Polimorfismo Genético , Receptores de Superficie Celular/genética , Humanos , Receptores de Superficie Celular/metabolismoRESUMEN
The pharmacology of histamine H(3) receptors suggests the presence of distinct receptor isoforms or subtypes. We herein describe multiple, functionally distinct, alternatively spliced isoforms of the human H(3) receptor. Combinatorial splicing at three different sites creates at least six distinct receptor isoforms, of which isoforms 1, 2, and 4, encode functional proteins. Detailed pharmacology on isoforms 1 (unspliced receptor), and 2 (which has an 80 amino acid deletion within the third intracellular loop of the protein) revealed that both isoforms displayed robust responses to a series of known H(3) agonists, while all agonists tested displayed increased potency at isoform 2 relative to isoform 1. Histamine, N(alpha)-methylhistamine, and R(-) and S(+)-alpha-methylhistamine are 16-23-fold more potent, while immepip and imetit are three to fivefold more potent. Antagonist experiments revealed a rank order of potency at both isoforms of clobenpropit>iodophenpropit>thioperamide, and these drugs are fivefold less potent at isoform 2 than isoform 1. To further explore the pharmacology of H(3) receptor function, we screened 150 clinically relevant neuropsychiatric drugs for H(3) receptor activity, and identified a small number of antipsychotics that possess significant antagonist activity.
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Metilhistaminas/farmacología , Receptores Histamínicos H3/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , Cobayas , Humanos , Datos de Secuencia Molecular , Isoformas de Proteínas/efectos de los fármacos , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Empalme del ARN , Ratas , Receptores Histamínicos H3/efectos de los fármacos , Receptores Histamínicos H3/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
We have used a cell-based functional assay to define the pharmacological profiles of a wide range of central nervous system active compounds as agonists, competitive antagonists, and inverse agonists at almost all known monoaminergic G-protein-coupled receptor (GPCR) subtypes. Detailed profiling of 40 antipsychotics confirmed that as expected, most of these agents are potent competitive antagonists of the dopamine D2 receptor. Surprisingly, this analysis also revealed that most are potent and fully efficacious 5-hydroxytryptamine (5-HT)2A receptor inverse agonists. No other molecular property was shared as universally by this class of compounds. Furthermore, comparisons of receptor potencies revealed that antipsychotics with the highest extrapyramidal side effects (EPS) liability are significantly more potent at D2 receptors, the EPS-sparing atypical agents had relatively higher potencies at 5-HT2A receptors, while three were significantly more potent at 5-HT2A receptors. Functional high-throughput screening of a diverse chemical library identified 530 ligands with inverse agonist activity at 5-HT2A receptors, including several series of compounds related to known antipsychotics, as well as a number of novel chemistries. An analog of one of the novel chemical series, AC-90179, was pharmacologically profiled against the remaining monoaminergic GPCRs and found to be a highly selective 5-HT2A receptor inverse agonist. The behavioral pharmacology of AC-90179 is characteristic of an atypical antipsychotic agent.
Asunto(s)
Antipsicóticos/farmacología , Receptores de Serotonina/efectos de los fármacos , Agonistas de Receptores de Serotonina/farmacología , Animales , Conducta Animal/efectos de los fármacos , Clonación Molecular , Evaluación Preclínica de Medicamentos , Proteínas de Unión al GTP/metabolismo , Amplificación de Genes , Movimientos de la Cabeza/efectos de los fármacos , Masculino , Ratones , Actividad Motora/efectos de los fármacos , Relación Estructura-Actividad Cuantitativa , Ratas , Ratas Sprague-Dawley , Receptor de Serotonina 5-HT2A , Reflejo de Sobresalto/efectos de los fármacosRESUMEN
Pilocarpine has been used to lower intraocular pressure (IOP) in glaucoma patients for more than 100 years. Since the identification of five muscarinic receptor subtypes, there has been an interest in separating the IOP-lowering effects from the ocular side effects of pupil constriction and lens accommodation. However, all these actions seem to be mediated by the M3 receptor. A novel muscarinic receptor agonist, AGN 199170, that has no activity on the M3 subtype was compared to pilocarpine in a monkey glaucoma model. This compound lowered IOP suggesting that muscarinic agonists targeted at muscarinic receptors other than the M3 subtype may be able to selectively lower IOP.
Asunto(s)
Glaucoma/tratamiento farmacológico , Presión Intraocular/efectos de los fármacos , Agonistas Muscarínicos/farmacología , Agonistas Muscarínicos/uso terapéutico , Pilocarpina/farmacología , Isoformas de Proteínas/metabolismo , Receptores Muscarínicos/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Macaca fascicularisRESUMEN
The calcium-sensing receptor (CaR) belongs to family C of the G-protein-coupled receptor superfamily. To date 14 activating mutations in CaR showing increased sensitivity to Ca(2+) have been identified in humans with autosomal dominant hypocalcemia. Four of these activating mutations are found in the Ala(116)-Pro(136) region of CaR, indicating that this part of the receptor is particularly sensitive to mutation-induced activation. This region was subjected to random saturation mutagenesis, and 219 mutant receptor clones were isolated and screened pharmacologically in a high throughput screening assay. Selected mutants were characterized further in an inositol phosphate assay. The vast majority of the mutants tested displayed an increased affinity for Ca(2+). Furthermore, 21 of the mutants showed increased basal activity in the absence of agonist. This constitutive activity was not diminished when the mutations were transferred to a chimeric receptor Ca/1a consisting of the amino-terminal domain of the CaR and the 7 transmembrane and intracellular domains of the metabotropic glutamate receptor mGluR1a. CPCCOEt, a noncompetitive antagonist acting at the 7 transmembrane domain of mGluR1a, suppressed the elevated basal response of the constitutively activated Ca/1a mutants demonstrating inverse agonist activity of CPCCOEt. Taken together, our results demonstrate that the Ala(116)-Pro(136) region is of key importance for the maintenance of the inactive conformation of CaR.
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Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas de Unión al GTP/metabolismo , Ratones , Datos de Secuencia Molecular , Mutagénesis , Conformación Proteica , Receptores Sensibles al Calcio , Receptores de Superficie Celular/genética , Transducción de Señal , Relación Estructura-ActividadAsunto(s)
Seguridad Computacional , Registros Médicos , Ética Médica , Informática Médica , PrivacidadRESUMEN
Ras and rac are each members of the superfamily of monomeric GTPases and both function as molecular switches to link cell-surface signals to intracellular responses. Using a novel assay of cellular proliferation called R-SAT (Receptor Selection and Amplification Technology), we examined the roles of ras and rac in mediating the proliferative responses to a variety of cell-surface receptors. Activated, wild-type and dominant-negative mutants of rac and ras were tested for their effects on cellular proliferation either alone or in combination with receptors. Activated rac (rac Q61L, henceforth rac*) and ras (ras G12V, henceforth ras*) each induced strong proliferative responses. Dominant-negative rac (rac T17N, henceforth rac(-)) dramatically suppressed proliferative responses to G-protein coupled receptors (GPCR's) including the m5 muscarinic receptor and the alpha1B adrenergic receptor. In contrast, rac(-) had little or no effect upon responses to the tyrosine kinase receptor TrkC, and only partially suppressed responses to the Janus kinase (JAK/STAT) linked granulocyte macrophage colony stimulating factor (GM-CSF) receptor. Dominant-negative ras (ras T17N, henceforth ras(-)) blocked the proliferative responses to all of the tested receptors. Compared to rac(-) and ras(-), wild-type rac and ras had only modest effects on the tested receptors. Overall these results demonstrate that rac mediates the proliferative effects of G-protein coupled receptors through a pathway that is distinct from the proliferative signaling pathway utilized by tyrosine kinase linked and JAK-linked receptors.
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GTP Fosfohidrolasas/fisiología , Proteínas de Unión al GTP/genética , Receptores de Superficie Celular/metabolismo , Células 3T3 , Animales , División Celular/genética , Ratones , Receptor Muscarínico M5 , Receptores Adrenérgicos beta 1/metabolismo , Receptores Muscarínicos/metabolismo , Transducción de Señal , Proteínas de Unión al GTP racRESUMEN
We have completed a systematic search of the intracellular loops of a muscarinic acetylcholine receptor for domains that govern G-protein coupling. A unique feature of the second intracellular (i2) loop was an ordered cluster of residues where diverse substitutions cause constitutive activation. A second group of residues in i2 was identified where mutations compromised receptor/G-protein coupling. The residues of each group alternate and are spaced three to four positions apart, suggesting an alpha-helical structure where these groups form opposing faces of the helix. We propose that the constitutively activating face normally constrains the receptor in the "off-state," while the other face couples G-proteins in the "on-state." Therefore, the i2 loop functions as the switch enabling G-protein activation.
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Proteínas de Unión al GTP/metabolismo , Estructura Secundaria de Proteína , Receptores Muscarínicos/química , Receptores Muscarínicos/fisiología , Células 3T3 , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Línea Celular , Biblioteca de Genes , Humanos , Cinética , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Puntual , Pliegue de Proteína , Receptor Muscarínico M5 , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , TransfecciónRESUMEN
G-protein-coupled receptors spontaneously switch between active and inactive conformations. Agonists stabilize the active conformation, whereas antagonists stabilize the inactive conformation. In a systematic search for residues that participate in receptor function, several regions of the m5 muscarinic receptor were randomly mutated and tested for their functional properties. Mutations spanning one face of transmembrane 6 (TM6) were found to induce high levels of receptor activity in the absence of agonists (constitutive activity). The same face of TM6 contained several residues crucial for receptor activation by agonists and one residue identified as a contact site for both agonists and antagonists. In addition, one mutation induced agonist-like responses from the receptor when exposed to classical antagonists. These results suggest that TM6 is a switch that defines the activation state of the receptor, and that ligand interactions with TM6 stabilize the receptor in either an active or an inactive conformation.
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Receptores Muscarínicos/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Atropina/metabolismo , Sitios de Unión , Carbacol/metabolismo , Ligandos , Ratones , Datos de Secuencia Molecular , Agonistas Muscarínicos/metabolismo , Antagonistas Muscarínicos/metabolismo , Mutagénesis Sitio-Dirigida , Conformación Proteica , Estructura Secundaria de Proteína , Receptores Muscarínicos/genéticaRESUMEN
The synthesis and SAR of a series of (Z)-(+/-)-1-azabicyclo[2.2. 1]heptan-3-one, O-(3-aryl-2-propynyl)oximes are described. The biochemistry and pharmacology of 24Z (PD 142505) and its enantiomers are highlighted. 24Z is functionally an m1-selective muscarinic agonist. Efficacy and m1 selectivity reside in the R enantiomer, (R)-24Z (CI-1017).
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Compuestos Bicíclicos Heterocíclicos con Puentes/síntesis química , Diseño de Fármacos , Agonistas Muscarínicos/síntesis química , Oximas/síntesis química , Receptores Muscarínicos/efectos de los fármacos , Células 3T3 , Inhibidores de Adenilato Ciclasa , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/toxicidad , Células CHO , Cricetinae , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/toxicidad , Humanos , Fosfatos de Inositol/biosíntesis , Masculino , Memoria/efectos de los fármacos , Ratones , Agonistas Muscarínicos/metabolismo , Agonistas Muscarínicos/farmacología , Agonistas Muscarínicos/toxicidad , Oximas/metabolismo , Oximas/farmacología , Oximas/toxicidad , Ratas , Receptor Muscarínico M1 , Receptores Muscarínicos/metabolismo , Estereoisomerismo , Relación Estructura-Actividad , TransfecciónRESUMEN
Using random saturation mutagenesis, we have previously identified the amino acids K439, A440, and A441 in the C-terminus of the third intracellular loop (Ci3) of the m5 muscarinic receptor as being critical for G-protein coupling [Burstein, E. S., Spalding, T. A., Hill-Eubanks, D., and Brann, M. R. (1995) J. Biol. Chem. 270, 3141-3146]. In the present study, we have constructed a series of point mutants at each of these residues and characterized their functional phenotypes in order to define the structure/function relationships of each of these residues for G-protein coupling. Although a wide variety of substitutions were tolerated at K439, most caused significant increases in the EC50 of carbachol and decreases in the maximum response (Rmax). Only other basic residues were well tolerated (<10-fold increase in EC50, >70% of wild type). Acidic substitutions had the largest effects, reducing Rmax to under 20% of wild type. At A440, only the conservative substitution threonine was well tolerated. Substitutions by hydrophobic, polar, and basic residues caused 10-80-fold increases in EC50 values and in many cases also significantly reduced Rmax (<70% of wild type). In contrast, at A441 mutations selectively affected EC50 but not Rmax values. Previously we identified I216, Y217, T220, and R223 as the residues in the N-terminus of the i3 loop of m5 (Ni3) that are critical for G-protein coupling [Burstein, E. S., Spalding, T. S., and Brann, M. R. (1996) J. Biol. Chem. 271, 2882-2885]. To investigate whether there were additive contributions of Ni3 and Ci3 to G-protein coupling, the functional responses of two double mutants, R223E/K439E and Y217S/A441T, were evaluated. Though these mutations were tolerated individually, both double mutant receptors produced almost indetectable responses. Little or no changes in expression levels or ligand binding properties were detected, suggesting the observed effects were caused primarily by changes receptor/G-protein coupling. We conclude that K439 participates in G-protein activation through an ionic mechanism, that A440 fulfills a structural role forming part of the G-protein coupling pocket, and that A441 contributes to receptor affinity for G-proteins. We propose that the third intracellular loop forms a G-protein coupling pocket comprised of a positively charged "lip" and a hydrophobic core.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Receptores Muscarínicos/química , Células 3T3 , Alanina/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Animales , Carbacol/metabolismo , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/fisiología , Lisina/genética , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Insercional , Reacción en Cadena de la Polimerasa , Estructura Secundaria de Proteína , Receptor Muscarínico M5 , Receptores Muscarínicos/genética , Receptores Muscarínicos/metabolismo , Receptores Muscarínicos/fisiología , Relación Estructura-ActividadRESUMEN
The m5 muscarinic acetylcholine receptor was constitutively activated by a wide range of amino acid substitutions at a residue (serine 465) that is positioned at the junction of the sixth transmembrane domain and the extracellular loop. Of 13 substitutions tested, 11 produced significant increases in constitutive activity. Replacement of serine 465 with large (phenylalanine and valine) or basic residues (arginine and lysine) increased the constitutive activity of the receptor to between 55 and 110% of the maximum response of the wild-type receptor to the agonist carbachol. Other substitutions (e.g., cysteine and leucine) increased the constitutive activity to an intermediate level (30%), while small and acidic residues (glycine, aspartate, and glutamate) caused small or insignificant increases. The increase in the constitutive activity of each mutant receptor correlated with an increase in the potency of carbachol in both binding and functional assays, with the most constitutively activated receptors showing a 40-fold decrease in the EC50 of carbachol. The negative antagonist atropine bound to and reversed the constitutive activity of all mutant receptors with equal potency. These data were fitted to a two-state model of receptor function. The data are consistent with the primary effect of substitutions to serine 465 being to selectively destabilize the inactive state of the receptor, thus favoring formation of the active state in the absence of agonists. Our data strongly support this two-state model of receptor function and identify a critical role of this domain in the activation of muscarinic receptors.
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Receptores Muscarínicos/metabolismo , Células 3T3 , Animales , Atropina/farmacología , Carbacol/farmacología , Ratones , Agonistas Muscarínicos/farmacología , Mutagénesis Sitio-Dirigida , Fenotipo , Unión Proteica , Ensayo de Unión Radioligante , Receptores Muscarínicos/efectos de los fármacos , Receptores Muscarínicos/genéticaRESUMEN
We have examined the effects of raising G protein concentration on the pharmacology of a series of agonist and antagonist ligands at the m1, m3, and m5 muscarinic subtypes using a functional assay. Overexpression of G(alpha q) induced constitutive activity of these receptors. The constitutive activity was reversed completely by every muscarinic antagonist tested, which indicates that they are all negative antagonists (inverse agonists). The potencies of antagonists for reversing G protein-induced activity and agonist-induced activity were identical, suggesting the same mechanism of action. Overexpression of G(alpha q) increased the potencies of every tested agonist and the efficacies of all partial agonists. The fold-gains in potency were positively correlated with ligand efficacy with the most efficacious agonists displaying the greatest potency gains. In addition, the efficacies of partial agonists approached those of full agonists. Constitutive activity of receptors has been explained by allosteric models in which receptors exist in spontaneous equilibrium between active and inactive conformations that are stabilized by agonists and antagonists, respectively. In this context, drug efficacy and potency are interrelated because they both depend on the same parameters, namely the absolute and relative affinities of a compound for receptors in active and inactive states and the ratio and concentrations of receptors in active and inactive states. All of our data are consistent with this model, in which raising G protein levels favors formation of the active conformation of receptors. Based on our findings, regulation of G protein concentration may be an important means of controlling receptor activity in vivo. These results define the functional relationship between G protein levels and muscarinic receptor pharmacology.
Asunto(s)
Atropina/farmacología , Carbacol/farmacología , Proteínas de Unión al GTP/metabolismo , Receptores Muscarínicos/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , LigandosRESUMEN
The proliferative and transforming properties of m2 and m5 muscarinic acetylcholine receptors and a series of wild-type, chimeric, and mutant G proteins were measured alone or in combination in NIH 3T3 cells to determine which G proteins mediate these signals and to what extent these signals can be influenced by changing the stoichiometry of receptors and G proteins. Responses were measured using the focus-forming assay and a novel assay called R-SAT (Receptor Selection and Amplification Technology) in which proliferative responses are monitored using a reporter gene. Individually, GTPase-deficient mutants (*) of G alpha q and G alpha 12, wild-type G alpha q, and m5 were active in R-SAT. G alpha 12* and m5 also induced focus formation. m2 was inactive in both assays. The ability of m5 to induce foci was significantly reduced by coexpression of G alpha q*. Synergistic effects of receptor/ G protein combinations were not observed in focus-forming assays but were readily detected by R-SAT. Coexpression of G alpha q with m5 induced constitutive activity in R-SAT and increased the potency of agonists at m5 by 90-fold. G alpha q also evoked agonist-dependent responses from m2 but not constitutive activity. Agonist potency was increased 10-fold at m2 and decreased 15-fold at m5 when these receptors were coexpressed with G alpha qi5, a chimeric G protein containing the five C-terminal residues of G alpha i2, compared with coexpression with G alpha q. Both G alpha q and G alpha qi5 had biphasic effects on the proliferative responses to m5 and m2, respectively, inhibiting responses at high agonist concentrations. Coexpression of G alpha 12 or G alpha 12i5 had no effect on the concentration-response relationships of m5, but both elicited weak responses from m2. We conclude that although G alpha 12 is a more potent oncogene, G alpha q transduces m5-driven cellular responses. The demonstrations that proliferative responses can be elicited from a nonmitogenic receptor by altering the type and concentration of available G proteins and that constitutive responses can be induced by G proteins imply that both the magnitude and type of receptor-initiated signal can be regulated at the level of G proteins in vivo.