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Multiple GPCR conformations and signalling pathways: implications for antagonist affinity estimates.

Baker JG, Hill SJ - Trends Pharmacol. Sci. (2007)

Bottom Line: As a consequence, changes in antagonist affinity values have been taken as initial evidence for the presence of novel receptor subtypes.Emerging evidence suggests, however, that receptors can possess multiple binding sites and the same receptor can show different antagonist affinity measurements under distinct experimental conditions.Here, we discuss several mechanisms by which antagonists have different affinities for the same receptor as a consequence of allosterism, coupling to different G proteins, multiple (but non-interacting) receptor sites, and signal-pathway-dependent pharmacology (where the pharmacology observed varies depending on the signalling pathway measured).

View Article: PubMed Central - PubMed

Affiliation: Institute of Cell Signalling, Medical School, Nottingham, NG7 2UH, UK.

ABSTRACT
Antagonist affinity measurements have traditionally been considered important in characterizing the cell-surface receptors present in a particular cell or tissue. A central assumption has been that antagonist affinity is constant for a given receptor-antagonist interaction, regardless of the agonist used to stimulate that receptor or the downstream response that is measured. As a consequence, changes in antagonist affinity values have been taken as initial evidence for the presence of novel receptor subtypes. Emerging evidence suggests, however, that receptors can possess multiple binding sites and the same receptor can show different antagonist affinity measurements under distinct experimental conditions. Here, we discuss several mechanisms by which antagonists have different affinities for the same receptor as a consequence of allosterism, coupling to different G proteins, multiple (but non-interacting) receptor sites, and signal-pathway-dependent pharmacology (where the pharmacology observed varies depending on the signalling pathway measured).

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Antagonism of histamine H2 receptor responses. (a) Antagonism of histamine-stimulated CRE (cAMP response element) gene transcription, mediated through the H2 receptor, by increasing concentrations of the H2 antagonist famotidine in CHO cells expressing the human H2 receptor. Gene transcription was measured by using a secreted placental alkaline phosphatase (SPAP) reporter gene. (b) Schild plots of the famotidine antagonism of H2 responses stimulated by histamine, Nα-methylhistamine and amthamine. The x-axis intercept gives the −log Kb value. Data are from Ref. [16] and J.G.B. (unpublished observations).
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fig1: Antagonism of histamine H2 receptor responses. (a) Antagonism of histamine-stimulated CRE (cAMP response element) gene transcription, mediated through the H2 receptor, by increasing concentrations of the H2 antagonist famotidine in CHO cells expressing the human H2 receptor. Gene transcription was measured by using a secreted placental alkaline phosphatase (SPAP) reporter gene. (b) Schild plots of the famotidine antagonism of H2 responses stimulated by histamine, Nα-methylhistamine and amthamine. The x-axis intercept gives the −log Kb value. Data are from Ref. [16] and J.G.B. (unpublished observations).

Mentions: Analysis of logarithmic concentration–response curves has long been used to evaluate the nature of the competitive interactions between agonists and antagonists from functional measurements and particularly so in the case of G-protein-coupled receptors (GPCRs) (Figure 1). The standard approach is the construction of full agonist concentration–response curves in the absence and presence of fixed concentrations of antagonist. The extent of the parallel rightward shift in the position of the agonist concentration–response curve is then used to calculate the antagonist affinity directly (assuming competitive antagonism) or (if various antagonist concentrations have been used) to construct a Schild plot, which will have a slope of one if the interaction is competitive (Figure 1).


Multiple GPCR conformations and signalling pathways: implications for antagonist affinity estimates.

Baker JG, Hill SJ - Trends Pharmacol. Sci. (2007)

Antagonism of histamine H2 receptor responses. (a) Antagonism of histamine-stimulated CRE (cAMP response element) gene transcription, mediated through the H2 receptor, by increasing concentrations of the H2 antagonist famotidine in CHO cells expressing the human H2 receptor. Gene transcription was measured by using a secreted placental alkaline phosphatase (SPAP) reporter gene. (b) Schild plots of the famotidine antagonism of H2 responses stimulated by histamine, Nα-methylhistamine and amthamine. The x-axis intercept gives the −log Kb value. Data are from Ref. [16] and J.G.B. (unpublished observations).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169386&req=5

fig1: Antagonism of histamine H2 receptor responses. (a) Antagonism of histamine-stimulated CRE (cAMP response element) gene transcription, mediated through the H2 receptor, by increasing concentrations of the H2 antagonist famotidine in CHO cells expressing the human H2 receptor. Gene transcription was measured by using a secreted placental alkaline phosphatase (SPAP) reporter gene. (b) Schild plots of the famotidine antagonism of H2 responses stimulated by histamine, Nα-methylhistamine and amthamine. The x-axis intercept gives the −log Kb value. Data are from Ref. [16] and J.G.B. (unpublished observations).
Mentions: Analysis of logarithmic concentration–response curves has long been used to evaluate the nature of the competitive interactions between agonists and antagonists from functional measurements and particularly so in the case of G-protein-coupled receptors (GPCRs) (Figure 1). The standard approach is the construction of full agonist concentration–response curves in the absence and presence of fixed concentrations of antagonist. The extent of the parallel rightward shift in the position of the agonist concentration–response curve is then used to calculate the antagonist affinity directly (assuming competitive antagonism) or (if various antagonist concentrations have been used) to construct a Schild plot, which will have a slope of one if the interaction is competitive (Figure 1).

Bottom Line: As a consequence, changes in antagonist affinity values have been taken as initial evidence for the presence of novel receptor subtypes.Emerging evidence suggests, however, that receptors can possess multiple binding sites and the same receptor can show different antagonist affinity measurements under distinct experimental conditions.Here, we discuss several mechanisms by which antagonists have different affinities for the same receptor as a consequence of allosterism, coupling to different G proteins, multiple (but non-interacting) receptor sites, and signal-pathway-dependent pharmacology (where the pharmacology observed varies depending on the signalling pathway measured).

View Article: PubMed Central - PubMed

Affiliation: Institute of Cell Signalling, Medical School, Nottingham, NG7 2UH, UK.

ABSTRACT
Antagonist affinity measurements have traditionally been considered important in characterizing the cell-surface receptors present in a particular cell or tissue. A central assumption has been that antagonist affinity is constant for a given receptor-antagonist interaction, regardless of the agonist used to stimulate that receptor or the downstream response that is measured. As a consequence, changes in antagonist affinity values have been taken as initial evidence for the presence of novel receptor subtypes. Emerging evidence suggests, however, that receptors can possess multiple binding sites and the same receptor can show different antagonist affinity measurements under distinct experimental conditions. Here, we discuss several mechanisms by which antagonists have different affinities for the same receptor as a consequence of allosterism, coupling to different G proteins, multiple (but non-interacting) receptor sites, and signal-pathway-dependent pharmacology (where the pharmacology observed varies depending on the signalling pathway measured).

Show MeSH