Analysis of Agonism and Inverse Agonism in Functional Assays with Constitutive Activity: Estimation of Orthosteric Ligand Affinity Constants for Active and Inactive Receptor States

We describe a modification of receptor theory for the estimation of observed affinities (Kobs) 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 (εi) states and analogous parameters for the fractions of the free receptor population in the active (εsys) and inactive (εi-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 (Kobs and τ) as well as a parameter proportional to the activity of the free receptor complex, τsys. Two related parameters are proportional to the fraction of the free (τi-sys) and occupied (τi) receptor populations in the inactive state. We show that the estimates of the affinity constants of orthosteric ligands for the active (Kb) and inactive (Ka) states of the receptor are equivalent to τKobs/τsys and τiKobs/τi-sys, respectively. We verify our method with computer simulation techniques and apply it to the analysis of M2 and M3 muscarinic receptors. Our method is applicable in the analysis of ligand bias in drug discovery programs

Analysis of Functional Responses at G Protein-Coupled Receptors: Estimation of Relative Affinity Constants for the Inactive Receptor Sstate

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 (Kobs) 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, εi). 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 Kobs and the parameter τ, which is proportional to ε. We introduce the parameter τi, which is proportional to εi. We have previously shown that the product, Kobsτ, of one agonist, expressed relative to that of another (intrinsic relative activity, RAi), is a relative measure of the affinity constant for the active state of the receptor. In this report, we show that the product, Kobsτi, of one agonist, expressed relative to that of another (intrinsic relative inactivity, RIi), 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 M3 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 (RAi/RIi)

徳島県における小児在宅医療の今後の展望と医学教育

Thanks to advanced neonatal medical technologies, many children as very-low-birth-weight infants became to be survived in Japan. But as a result, the children with severe disabilities who need constant medical care were increased, and the same situation has also occurred in local communities in Tokushima. They used to be cared only in hospitals, therefore enough supports are not provided from welfare system as well as its system for mature patients. But majority of the health care worker doesn’t grasp the current state of this matter sufficiently. We had an opportunity to discuss about this issue with young medical students of Tokushima University Pediatric Seminar（TUPS）. They are very interested in pediatrics and working at activities in and out of medical school with enthusiasm. We believe that having more chances of hearing the real voices of children with disabilities and their families before graduate will become the good triggers for increasing young medical students who aspire to become pediatricians or home doctors

〔研究ノート〕 ムスカリン性アセチルコリン受容体の細胞膜への トラフィッキングの観察

　　G protein-coupled receptors are cell-surface receptors, many of which have conserved motif F（x）6LL in their C-terminal intracellular region. The motif is known to function when the G protein-coupled receptors are exported from the endoplasmic reticulum to the cell surface. We reported previously that the amino acid mutations of the conserved leucines of M1 muscarinic acetylcholine receptor caused significant decrease in the cell-surface expression and significant increase in the endoplasmic reticulum expression of the mutant receptor, and that in the presence of antagonist atropine, the mutant receptor showed cell-surface expression, similar to the expression of the wild-type receptor. In this study, we investigated the export trafficking of the mutant receptor by the measurements of membrane-impermeable antagonist ［3H］N-methylscopolamine binding to the cell surface, and indicated that the cell-surface expression of the mutant receptor in the presence of atropine decreased to 20％ by the depletion of atropine for 3 days, and recovered to the same amount as before by the second addition of atropine. We also investigated the export trafficking of the mutant receptor using the total internal reflection fluorescence microscope, and indicated that the mutant receptor expressed time-dependently to the cell surface by the second addition of atropine

味覚受容体の検索(第2報) : ヒトの舌より分離された THTR5 受容体の呈味物質の応答性

Gタンパク質共役受容体THTR5をCHO細胞に導入し,呈味物質への応答性を検討した。THTR5クローンにおいてはGlucose, Fructose, Sorbitol, Arginine, Denatonium benzoate, Guanosin 5monophosphate disodium salt, Inosin 5\u27monophosphate sodium saltでの刺激により培養液上清中のPGE_2濃度が上昇した。さらに,Glucose, Fructose, Sorbitolでの刺激では培養液上清中のPGE_2濃度は呈味物質の濃度に依存的に変動した。以上のようにTHTR5はGlucose, Fructose, Sorbitolに濃度依存的な応答を示したことから糖質系甘味受容体であると推察した。We introduced the THTR5 of the G-protein-coupled receptor into the CHO cell and examined its response to some tastants. In the case of the THTR5 clone, when stimulated by Glucose, Fructose, Sorbitol, Arginine, Guanosin 5-monophosphate disodium salt, Inosin 5\u27- monophosphate sodium salt and Denatonium benzoate, the concentration of the prostaglandin E_2 (PGE_2) in the culture solution rose. Due to the stimulus of Glucose, Fructose and Sorbitol, the PGE_2 concentration in the culture solution rose in accordance with the concentration of various tastants init. Thus, since the clone showed the concentration dependence-response to the sweet taste of saccharide, we guessed that this clone was a sweet taste receptor in connection with saccharide

健常者と味覚異常者における味覚受容体発現

RT-PCR法を用いて,葉状乳頭における味覚受容体の発現を検討した。すなわち葉状乳頭部から擦過法により取得した組織を使用し,味覚受容体候補であるTHTRファミリー(THTRs)およびT2Rファミリー(T2Rs)の発現をRT-PCR後,マイクロキャピラリー電気泳動により測定した。40歳以上の健常者ではTHTRs,T2Rsの受容体において,多数を発現していることが認められた。これに対し30歳以下の被験者ではいくつかのTHTRsおよびT2Rsで発現を認めたが,典型的な発現パターンはなかった。さらに薬服用者や高齢の味覚異常者においては,健常者に比べてTHTRs, T2Rsともに発現していない受容体が多かった。これらのことから,まず青年期では味覚受容体が成長段階にあり,20歳の成人になっても味覚の成長は十分ではないことが考えられた。壮年期になると味覚受容体が十分発達し,高齢者になってもその味覚受容体の発現数は減少しないことが示唆された。また薬剤の服用者や味覚異常者も,味覚受容体の欠落の可能性が示唆された。Tongue tissue from foliate papillae of 22 subjects was scraped and used. The expression of THTR family (THTRs) and T2R family (T2Rs) that are the candidate taste receptors were measured by RT-PCR and micro-capillary electrophoresis. The expression of taste receptors in the foliate papillae was measured using this scraping RT-PCR method. Normal persons over 40 years old expressed many THTRs and T2Rs. In contrast, for people under 30 years old there were some THTRs and T2Rs expressions without typical patterns. The taste disordered persons expressed few kinds of THTRs and T2Rs, and there were no or almost no expressions. These results suggested that in young adults of about 20 years old the taste receptors are not yet developed. We suggest that expression of taste receptors don\u27t decrease in a person of advanced years because taste receptors development peaks in late middle age. With further assay and analyzation of expression of THTRs and T2Rs, it maybe possible to clear up factors of disturbance in taste

Effects of Asparagine Mutagenesis of Conserved Aspartic Acids in Helix 2 (D2.50) and 3 (D3.32) of M₁–M₄ Muscarinic Receptors on the Irreversible Binding of Nitrogen Mustard Analogs of Acetylcholine and McN-A-343

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<i> N</i>-(3-chlorophenyl)­carbamate), a nitrogen mustard derivative of McN-A-343 ([4-[[<i>N</i>-(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

Quantifying Agonist Activity at G Protein-coupled Receptors

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell or tissue. This process can be analyzed at the level of a single receptor, a population of receptors, or a downstream response. Here we describe how to analyze the downstream response to obtain an estimate of the agonist affinity constant for the active state of single receptors. Receptors behave as quantal switches that alternate between active and inactive states (Figure 1). The active state interacts with specific G proteins or other signaling partners. In the absence of ligands, the inactive state predominates. The binding of agonist increases the probability that the receptor will switch into the active state because its affinity constant for the active state (Kb) is much greater than that for the inactive state (Ka). The summation of the random outputs of all of the receptors in the population yields a constant level of receptor activation in time. The reciprocal of the concentration of agonist eliciting half-maximal receptor activation is equivalent to the observed affinity constant (Kobs), and the fraction of agonist-receptor complexes in the active state is defined as efficacy (ε) (Figure 2). Methods for analyzing the downstream responses of GPCRs have been developed that enable the estimation of the Kobs and relative efficacy of an agonist 1,2. In this report, we show how to modify this analysis to estimate the agonist Kb value relative to that of another agonist. For assays that exhibit constitutive activity, we show how to estimate Kb in absolute units of M-1. Our method of analyzing agonist concentration-response curves 3,4 consists of global nonlinear regression using the operational model 5. We describe a procedure using the software application, Prism (GraphPad Software, Inc., San Diego, CA). The analysis yields an estimate of the product of Kobs and a parameter proportional to efficacy (τ). The estimate of τKobs of one agonist, divided by that of another, is a relative measure of Kb (RAi) 6. For any receptor exhibiting constitutive activity, it is possible to estimate a parameter proportional to the efficacy of the free receptor complex (τsys). In this case, the Kb value of an agonist is equivalent to τKobs/τsys 3. Our method is useful for determining the selectivity of an agonist for receptor subtypes and for quantifying agonist-receptor signaling through different G proteins

Mutagenesis of Nucleophilic Residues near the Orthosteric Binding Pocket of M1 and M2 Muscarinic receptors: Effect on the Binding of Nitrogen Mustard Analogs of Acetylcholine and McN-A-343

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 M1 and M2 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 M1 (Asp105) and M2 (Asp103) receptors to asparagine largely prevented receptor alkylation by acetylcholine mustard, although modest alkylation still occurred at M2 D103N at high concentrations of the mustard. Receptor alkylation by BR384 was also greatly inhibited in the M1 D105N mutant, but some alkylation still occurred at high concentrations of the compound. In contrast, BR384 rapidly alkylated the M2 D103N mutant. Its affinity was reduced to one tenth, however. The alkylation of M2 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 M2 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 M1 and M2 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