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The pharmacological effects of the thermostabilising (m23) mutations and intra and extracellular (β36) deletions essential for crystallisation of the turkey β-adrenoceptor.

Baker JG, Proudman RG, Tate CG - Naunyn Schmiedebergs Arch. Pharmacol. (2011)

Bottom Line: The m23 mutations reduced affinity for agonists, partial agonists and neutral antagonists by about tenfold whilst the β36 deletions alone had no effect on ligand affinity.Both sets of changes appeared to reduce the agonist activation of the receptor.Although the combination of mutations severely reduced the activation ability, the final crystallised receptor (β36-m23) was still a fully functional receptor capable of binding agonist and antagonist ligands and activating intracellular agonist responses.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cell Signalling, C Floor Medical School, University of Nottingham, Queen's Medical Centre, UK. jillian.baker@nottingham.ac.uk

ABSTRACT
The X-ray crystal structure of the turkey β-adrenoceptor has recently been determined. However, mutations were introduced into the native receptor that was essential for structure determination. These may cause alterations to the receptor pharmacology. It is therefore essential to understand the effects of these mutations on the pharmacological characteristics of the receptor. This study examined the pharmacological effects of both the m23 mutations and the β36 deletions, both alone and then in combination in the β36-m23 mutant used in the crystallisation and structure determination of the turkey β-adrenoceptor. Stable CHO-K1 cell lines were made of each of the receptor mutants and the affinity and efficacy of ligands assessed by (3)H-CGP 12177 whole cell ligand binding, (3)H-cAMP accumulation, and CRE-SPAP gene transcription assays. The m23 mutations reduced affinity for agonists, partial agonists and neutral antagonists by about tenfold whilst the β36 deletions alone had no effect on ligand affinity. Both sets of changes appeared to reduce the agonist activation of the receptor. Both the m23 and the β36 receptors retained two active agonist-induced receptor conformations similar to that of the original tβtrunc receptor. The combined β36-m23 receptor bound ligands with similar affinity to the m23 receptor; however, agonist activation was only observed with a few agonists including the catecholamines. Although the combination of mutations severely reduced the activation ability, the final crystallised receptor (β36-m23) was still a fully functional receptor capable of binding agonist and antagonist ligands and activating intracellular agonist responses.

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CRE-SPAP production in response to isoprenaline in β36–m23 cells in the absence and presence of a propranolol and b CGP12177. Bars represent basal CRE-SPAP production, that in response to 10 μM isoprenaline and that in response 10, 100 1,000 nM propranolol or 1, 10 or 100 nM CGP12177 alone. Data points are mean ± SEM of triplicate determinations. Each experiment is representative of a seven and b seven separate experiments. The Schild slopes for each experiment are a 1.00 and b 1.05
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Fig8: CRE-SPAP production in response to isoprenaline in β36–m23 cells in the absence and presence of a propranolol and b CGP12177. Bars represent basal CRE-SPAP production, that in response to 10 μM isoprenaline and that in response 10, 100 1,000 nM propranolol or 1, 10 or 100 nM CGP12177 alone. Data points are mean ± SEM of triplicate determinations. Each experiment is representative of a seven and b seven separate experiments. The Schild slopes for each experiment are a 1.00 and b 1.05

Mentions: When these mutations were combined in the β36–m23 mutant receptor, many agonists did not stimulate any response (Fig. 5d, Tables 7 and 8). In addition, CGP12177 itself did not stimulate any agonist response at the β36–m23 receptor making it difficult to determine whether this mutant does indeed have two agonist-induced conformations. However, responses were seen to a few agonists and these responses were inhibited by classical antagonists in a competitive manner (propranolol, ICI118551 and CGP20712A, Fig. 8a, Table 7). In addition, CGP12177 also inhibited these agonist responses with high affinity in a similar manner to the original tβtrunc receptor (Fig. 8b, Table 7).Fig. 8


The pharmacological effects of the thermostabilising (m23) mutations and intra and extracellular (β36) deletions essential for crystallisation of the turkey β-adrenoceptor.

Baker JG, Proudman RG, Tate CG - Naunyn Schmiedebergs Arch. Pharmacol. (2011)

CRE-SPAP production in response to isoprenaline in β36–m23 cells in the absence and presence of a propranolol and b CGP12177. Bars represent basal CRE-SPAP production, that in response to 10 μM isoprenaline and that in response 10, 100 1,000 nM propranolol or 1, 10 or 100 nM CGP12177 alone. Data points are mean ± SEM of triplicate determinations. Each experiment is representative of a seven and b seven separate experiments. The Schild slopes for each experiment are a 1.00 and b 1.05
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3116118&req=5

Fig8: CRE-SPAP production in response to isoprenaline in β36–m23 cells in the absence and presence of a propranolol and b CGP12177. Bars represent basal CRE-SPAP production, that in response to 10 μM isoprenaline and that in response 10, 100 1,000 nM propranolol or 1, 10 or 100 nM CGP12177 alone. Data points are mean ± SEM of triplicate determinations. Each experiment is representative of a seven and b seven separate experiments. The Schild slopes for each experiment are a 1.00 and b 1.05
Mentions: When these mutations were combined in the β36–m23 mutant receptor, many agonists did not stimulate any response (Fig. 5d, Tables 7 and 8). In addition, CGP12177 itself did not stimulate any agonist response at the β36–m23 receptor making it difficult to determine whether this mutant does indeed have two agonist-induced conformations. However, responses were seen to a few agonists and these responses were inhibited by classical antagonists in a competitive manner (propranolol, ICI118551 and CGP20712A, Fig. 8a, Table 7). In addition, CGP12177 also inhibited these agonist responses with high affinity in a similar manner to the original tβtrunc receptor (Fig. 8b, Table 7).Fig. 8

Bottom Line: The m23 mutations reduced affinity for agonists, partial agonists and neutral antagonists by about tenfold whilst the β36 deletions alone had no effect on ligand affinity.Both sets of changes appeared to reduce the agonist activation of the receptor.Although the combination of mutations severely reduced the activation ability, the final crystallised receptor (β36-m23) was still a fully functional receptor capable of binding agonist and antagonist ligands and activating intracellular agonist responses.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cell Signalling, C Floor Medical School, University of Nottingham, Queen's Medical Centre, UK. jillian.baker@nottingham.ac.uk

ABSTRACT
The X-ray crystal structure of the turkey β-adrenoceptor has recently been determined. However, mutations were introduced into the native receptor that was essential for structure determination. These may cause alterations to the receptor pharmacology. It is therefore essential to understand the effects of these mutations on the pharmacological characteristics of the receptor. This study examined the pharmacological effects of both the m23 mutations and the β36 deletions, both alone and then in combination in the β36-m23 mutant used in the crystallisation and structure determination of the turkey β-adrenoceptor. Stable CHO-K1 cell lines were made of each of the receptor mutants and the affinity and efficacy of ligands assessed by (3)H-CGP 12177 whole cell ligand binding, (3)H-cAMP accumulation, and CRE-SPAP gene transcription assays. The m23 mutations reduced affinity for agonists, partial agonists and neutral antagonists by about tenfold whilst the β36 deletions alone had no effect on ligand affinity. Both sets of changes appeared to reduce the agonist activation of the receptor. Both the m23 and the β36 receptors retained two active agonist-induced receptor conformations similar to that of the original tβtrunc receptor. The combined β36-m23 receptor bound ligands with similar affinity to the m23 receptor; however, agonist activation was only observed with a few agonists including the catecholamines. Although the combination of mutations severely reduced the activation ability, the final crystallised receptor (β36-m23) was still a fully functional receptor capable of binding agonist and antagonist ligands and activating intracellular agonist responses.

Show MeSH
Related in: MedlinePlus