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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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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. [9].
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fig4: 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. [9].

Mentions: Some insight into the potential for the human β2 adrenoceptor to exist in different conformations (each with its own unique pharmacology) and to couple to distinct signalling pathways has been provided by detailed studies of the agonist and inverse agonist properties of propranolol on activation of the ERK1/2 mitogen-activated protein (MAP) kinase pathway and cAMP accumulation in CHO and human embryonic kidney 293 (HEK293) cells [9,43,49] (Figure 4). In the case of cAMP accumulation in CHO or HEK293 cells expressing the human β2 adrenoceptor, propranolol shows clear inverse agonist properties [9,43,49]. When ERK1/2 activation is measured in the same cells, however, propranolol behaves as a partial agonist. It also seems that stimulation of ERK1/2 phosphorylation by propranolol is independent of Gi or Gs protein activation [9,43]. Furthermore, it has been shown in HEK293 cells that ERK1/2 stimulation involves an interaction with β-arrestins [43]. These data suggest that ligand-specific conformations of the β2 adrenoceptor do indeed exist that can differentially activate distinct signalling pathways with very different pharmacologies. A similar observation has also been made for the murine β3 adrenoceptor, for which SR59230A is an antagonist of CL316243-mediated increases in cAMP accumulation in adipocytes, but is an agonist with greater efficacy than CL316243 for extracellular acidification in the same cells [50].


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

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

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. [9].
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2169386&req=5

fig4: 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. [9].
Mentions: Some insight into the potential for the human β2 adrenoceptor to exist in different conformations (each with its own unique pharmacology) and to couple to distinct signalling pathways has been provided by detailed studies of the agonist and inverse agonist properties of propranolol on activation of the ERK1/2 mitogen-activated protein (MAP) kinase pathway and cAMP accumulation in CHO and human embryonic kidney 293 (HEK293) cells [9,43,49] (Figure 4). In the case of cAMP accumulation in CHO or HEK293 cells expressing the human β2 adrenoceptor, propranolol shows clear inverse agonist properties [9,43,49]. When ERK1/2 activation is measured in the same cells, however, propranolol behaves as a partial agonist. It also seems that stimulation of ERK1/2 phosphorylation by propranolol is independent of Gi or Gs protein activation [9,43]. Furthermore, it has been shown in HEK293 cells that ERK1/2 stimulation involves an interaction with β-arrestins [43]. These data suggest that ligand-specific conformations of the β2 adrenoceptor do indeed exist that can differentially activate distinct signalling pathways with very different pharmacologies. A similar observation has also been made for the murine β3 adrenoceptor, for which SR59230A is an antagonist of CL316243-mediated increases in cAMP accumulation in adipocytes, but is an agonist with greater efficacy than CL316243 for extracellular acidification in the same cells [50].

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