Mouse β3a and β3b-adrenoceptors expressed in Chinese hamster ovary cells display identical pharmacology but utilize distinct signalling pathways

Dana S. Hutchinson, Tore Bengtsson, Bronwyn A. Evans, Roger J. Summers

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Abstract

1. This study characterizes the mouse β3a-adrenoceptor (AR) and the splice variant of the β3-AR (β3b-AR) expressed in Chinese hamster ovary cells (CHO-K1). 2. Stable clones with high (∼ 1200), medium (∼ 500) or low receptor expression (∼ 100 fmol mg protein-1) were determined by saturation binding with [125I]-(-)-cyanopindolol. Competition binding studies showed no significant differences in affinity of β-AR ligands for either receptor. 3. Several functional responses of each receptor were measured, namely extracellular acidification rate (EAR; cytosensor microphysiometer), cyclic AMP accumulation, and Erk1/2 phosphorylation. The β3-AR agonists BRL37344, CL316243, GR265162X, L755507, SB251023, the non-conventional partial β-AR agonist CGP12177 and the β-AR agonist (-)-isoprenaline caused concentration-dependent increases in EAR in cells expressing either splice variant. CL316243 caused concentration-dependent increases in cyclic AMP accumulation and Erk1/2 phosphorylation in cells expressing either receptor. 4. PTX treatment increased maximum EAR and cyclic AMP responses to CL316243 in cells expressing the β3b-AR but not in cells expressing the β3a-AR at all levels of receptor expression. 5. CL316243 increased Erk1/2 phosphorylation with pEC50 values and maximum responses that were not significantly different in cells expressing either splice variant. Erk1/2 phosphorylation was insensitive to PTX or H89 (PKA inhibitor) but was inhibited by LY294002 (PI3Kγ inhibitor), PP2 (c-Src inhibitor), genistein (tyrosine kinase inhibitor) and PD98059 (MEK inhibitor). 6. The adenylate cyclase activators forskolin or cholera toxin failed to increase Erk1/2 levels although both treatments markedly increased cyclic AMP accumulation in both β3a- or β3b-AR transfected cells. 7. These results suggest that in CHO-K1 cells, the β3b-AR, can couple to both Gs and Gi to stimulate and inhibit cyclic AMP production respectively, while the β3a, -AR, couples solely to Gs to increase cyclic AMP levels. However, the increase in Erk1/2 phosphorylation following receptor activation is not dependent upon coupling of the receptors to Gi or the generation of cyclic AMP.

Original languageEnglish
Pages (from-to)1903-1914
Number of pages12
JournalBritish Journal of Pharmacology
Volume135
Issue number8
DOIs
Publication statusPublished - 1 Jan 2002

Keywords

  • Cyclic AMP
  • Mouse
  • Splice variant cytosensor microphysiometer Erk1/2

Cite this

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title = "Mouse β3a and β3b-adrenoceptors expressed in Chinese hamster ovary cells display identical pharmacology but utilize distinct signalling pathways",
abstract = "1. This study characterizes the mouse β3a-adrenoceptor (AR) and the splice variant of the β3-AR (β3b-AR) expressed in Chinese hamster ovary cells (CHO-K1). 2. Stable clones with high (∼ 1200), medium (∼ 500) or low receptor expression (∼ 100 fmol mg protein-1) were determined by saturation binding with [125I]-(-)-cyanopindolol. Competition binding studies showed no significant differences in affinity of β-AR ligands for either receptor. 3. Several functional responses of each receptor were measured, namely extracellular acidification rate (EAR; cytosensor microphysiometer), cyclic AMP accumulation, and Erk1/2 phosphorylation. The β3-AR agonists BRL37344, CL316243, GR265162X, L755507, SB251023, the non-conventional partial β-AR agonist CGP12177 and the β-AR agonist (-)-isoprenaline caused concentration-dependent increases in EAR in cells expressing either splice variant. CL316243 caused concentration-dependent increases in cyclic AMP accumulation and Erk1/2 phosphorylation in cells expressing either receptor. 4. PTX treatment increased maximum EAR and cyclic AMP responses to CL316243 in cells expressing the β3b-AR but not in cells expressing the β3a-AR at all levels of receptor expression. 5. CL316243 increased Erk1/2 phosphorylation with pEC50 values and maximum responses that were not significantly different in cells expressing either splice variant. Erk1/2 phosphorylation was insensitive to PTX or H89 (PKA inhibitor) but was inhibited by LY294002 (PI3Kγ inhibitor), PP2 (c-Src inhibitor), genistein (tyrosine kinase inhibitor) and PD98059 (MEK inhibitor). 6. The adenylate cyclase activators forskolin or cholera toxin failed to increase Erk1/2 levels although both treatments markedly increased cyclic AMP accumulation in both β3a- or β3b-AR transfected cells. 7. These results suggest that in CHO-K1 cells, the β3b-AR, can couple to both Gs and Gi to stimulate and inhibit cyclic AMP production respectively, while the β3a, -AR, couples solely to Gs to increase cyclic AMP levels. However, the increase in Erk1/2 phosphorylation following receptor activation is not dependent upon coupling of the receptors to Gi or the generation of cyclic AMP.",
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Mouse β3a and β3b-adrenoceptors expressed in Chinese hamster ovary cells display identical pharmacology but utilize distinct signalling pathways. / Hutchinson, Dana S.; Bengtsson, Tore; Evans, Bronwyn A.; Summers, Roger J.

In: British Journal of Pharmacology, Vol. 135, No. 8, 01.01.2002, p. 1903-1914.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Mouse β3a and β3b-adrenoceptors expressed in Chinese hamster ovary cells display identical pharmacology but utilize distinct signalling pathways

AU - Hutchinson, Dana S.

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AU - Summers, Roger J.

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N2 - 1. This study characterizes the mouse β3a-adrenoceptor (AR) and the splice variant of the β3-AR (β3b-AR) expressed in Chinese hamster ovary cells (CHO-K1). 2. Stable clones with high (∼ 1200), medium (∼ 500) or low receptor expression (∼ 100 fmol mg protein-1) were determined by saturation binding with [125I]-(-)-cyanopindolol. Competition binding studies showed no significant differences in affinity of β-AR ligands for either receptor. 3. Several functional responses of each receptor were measured, namely extracellular acidification rate (EAR; cytosensor microphysiometer), cyclic AMP accumulation, and Erk1/2 phosphorylation. The β3-AR agonists BRL37344, CL316243, GR265162X, L755507, SB251023, the non-conventional partial β-AR agonist CGP12177 and the β-AR agonist (-)-isoprenaline caused concentration-dependent increases in EAR in cells expressing either splice variant. CL316243 caused concentration-dependent increases in cyclic AMP accumulation and Erk1/2 phosphorylation in cells expressing either receptor. 4. PTX treatment increased maximum EAR and cyclic AMP responses to CL316243 in cells expressing the β3b-AR but not in cells expressing the β3a-AR at all levels of receptor expression. 5. CL316243 increased Erk1/2 phosphorylation with pEC50 values and maximum responses that were not significantly different in cells expressing either splice variant. Erk1/2 phosphorylation was insensitive to PTX or H89 (PKA inhibitor) but was inhibited by LY294002 (PI3Kγ inhibitor), PP2 (c-Src inhibitor), genistein (tyrosine kinase inhibitor) and PD98059 (MEK inhibitor). 6. The adenylate cyclase activators forskolin or cholera toxin failed to increase Erk1/2 levels although both treatments markedly increased cyclic AMP accumulation in both β3a- or β3b-AR transfected cells. 7. These results suggest that in CHO-K1 cells, the β3b-AR, can couple to both Gs and Gi to stimulate and inhibit cyclic AMP production respectively, while the β3a, -AR, couples solely to Gs to increase cyclic AMP levels. However, the increase in Erk1/2 phosphorylation following receptor activation is not dependent upon coupling of the receptors to Gi or the generation of cyclic AMP.

AB - 1. This study characterizes the mouse β3a-adrenoceptor (AR) and the splice variant of the β3-AR (β3b-AR) expressed in Chinese hamster ovary cells (CHO-K1). 2. Stable clones with high (∼ 1200), medium (∼ 500) or low receptor expression (∼ 100 fmol mg protein-1) were determined by saturation binding with [125I]-(-)-cyanopindolol. Competition binding studies showed no significant differences in affinity of β-AR ligands for either receptor. 3. Several functional responses of each receptor were measured, namely extracellular acidification rate (EAR; cytosensor microphysiometer), cyclic AMP accumulation, and Erk1/2 phosphorylation. The β3-AR agonists BRL37344, CL316243, GR265162X, L755507, SB251023, the non-conventional partial β-AR agonist CGP12177 and the β-AR agonist (-)-isoprenaline caused concentration-dependent increases in EAR in cells expressing either splice variant. CL316243 caused concentration-dependent increases in cyclic AMP accumulation and Erk1/2 phosphorylation in cells expressing either receptor. 4. PTX treatment increased maximum EAR and cyclic AMP responses to CL316243 in cells expressing the β3b-AR but not in cells expressing the β3a-AR at all levels of receptor expression. 5. CL316243 increased Erk1/2 phosphorylation with pEC50 values and maximum responses that were not significantly different in cells expressing either splice variant. Erk1/2 phosphorylation was insensitive to PTX or H89 (PKA inhibitor) but was inhibited by LY294002 (PI3Kγ inhibitor), PP2 (c-Src inhibitor), genistein (tyrosine kinase inhibitor) and PD98059 (MEK inhibitor). 6. The adenylate cyclase activators forskolin or cholera toxin failed to increase Erk1/2 levels although both treatments markedly increased cyclic AMP accumulation in both β3a- or β3b-AR transfected cells. 7. These results suggest that in CHO-K1 cells, the β3b-AR, can couple to both Gs and Gi to stimulate and inhibit cyclic AMP production respectively, while the β3a, -AR, couples solely to Gs to increase cyclic AMP levels. However, the increase in Erk1/2 phosphorylation following receptor activation is not dependent upon coupling of the receptors to Gi or the generation of cyclic AMP.

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