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

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).

Figure 1

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, N^α-methylhistamine and amthamine. The x-axis intercept gives the −log K_b value. Data are from Ref. and J.G.B. (unpublished observations).

Figure 2

5′-N-ethylcarboxamidoadenosine (NECA)-induced gene transcription mediated by the human A1 adenosine receptor. (a) G_i and G_s signalling pathways from the adenosine A₁ receptor to CRE-mediated gene transcription in CHO cells expressing the human A1-receptor. Abbreviations: CREB, CRE-binding protein; PKA, protein kinase A. 6 × CRE–SPAP signifies a SPAP reporter gene containing six CRE elements. (b) Concentration–response curves for the effect of the agonist NECA on forskolin-stimulated CRE gene transcription in the presence and absence of increasing concentrations of DPCPX. Data are from Ref. .

Figure 3

Differential affinities of antagonists for the ‘catecholamine’ and ‘CGP 12177’ sites of the human β₁ adrenoceptor expressed in CHO cells. (a,b) Effect of increasing concentrations of the selective β₁ adrenoceptor antagonist CGP 20712A (CGPA) on isoprenaline-stimulated (a) and CGP-12177-stimulated (b) CRE-mediated luciferase expression. Note that the blue data points and line in (a) and (b) represent the same concentration of CGP 20712A (300 nM). (c) CRE–luciferase response to CGP 12177 in the absence and presence of fixed concentrations of cimaterol. (d) Correlation between the logarithm of the antagonist dissociation constant (log K_d) obtained for 12 antagonists with the agonist isoprenaline (x axis) and log K_d determined with the same 12 antagonists but with adrenaline, noradrenaline, cimaterol or CGP 12177 as the agonist (y axis). Data are from Refs .

Figure 4

Dual efficacy of propranolol on β2-adrenoceptor-mediated responses in CHO cells expressing the human β₂ adrenoceptor. (a) Inverse agonist effect of propranolol on [³H]cAMP accumulation. 6 × CRE–SPAP signifies a SPAP reporter gene containing six CRE elements. (b) Agonist effects of isoprenaline and propranolol on levels of phosphorylated ERK1/2 monitored by an ELISA assay. (c) Agonist effect of propranolol on CRE-mediated gene transcription. Data are from Ref. .