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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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Correlation plots of the log KD values from 3H-CGP12177 whole cell binding for the turkey β-adrenoceptor mutants compared with each other. Ligands are shown as either full agonists (filled circles), partial agonists (asterisks) or antagonists (open circles). Categorisation into these classes was performed from data in Tables 2 and 3, where ligands are defined as agonists if they stimulated more than 85% of the response at the human β1-adrenoceptor, as partial agonists if they stimulated 10–85% of the full response and as antagonists if they stimulated less than 10% of a full agonist response at the tβtrunc receptor
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Fig3: Correlation plots of the log KD values from 3H-CGP12177 whole cell binding for the turkey β-adrenoceptor mutants compared with each other. Ligands are shown as either full agonists (filled circles), partial agonists (asterisks) or antagonists (open circles). Categorisation into these classes was performed from data in Tables 2 and 3, where ligands are defined as agonists if they stimulated more than 85% of the response at the human β1-adrenoceptor, as partial agonists if they stimulated 10–85% of the full response and as antagonists if they stimulated less than 10% of a full agonist response at the tβtrunc receptor

Mentions: The agonist response to 24 ligands (seven-point concentration–response curves) was examined for each of the turkey β-adrenoceptor mutants (Table 2). A further 24 ligands were examined at maximum concentrations only (Table 3). This enabled ligands to be classified into full agonists (those ligands that stimulated a greater than 85% maximum response to isoprenaline in the cAMP assays at the tβtrunc receptor), partial agonists (those that stimulated between 10–85% maximum responses at the tβtrunc receptor) and antagonists (those ligands that stimulated less than 10% maximum response at the tβtrunc receptor). This allowed the study of ligand affinity to be further examined to assess whether a particular subset of ligands (e.g. agonists) were disproportionately affected by the mutations (Fig. 3).Table 2


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)

Correlation plots of the log KD values from 3H-CGP12177 whole cell binding for the turkey β-adrenoceptor mutants compared with each other. Ligands are shown as either full agonists (filled circles), partial agonists (asterisks) or antagonists (open circles). Categorisation into these classes was performed from data in Tables 2 and 3, where ligands are defined as agonists if they stimulated more than 85% of the response at the human β1-adrenoceptor, as partial agonists if they stimulated 10–85% of the full response and as antagonists if they stimulated less than 10% of a full agonist response at the tβtrunc receptor
© Copyright Policy
Related In: Results  -  Collection

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

Fig3: Correlation plots of the log KD values from 3H-CGP12177 whole cell binding for the turkey β-adrenoceptor mutants compared with each other. Ligands are shown as either full agonists (filled circles), partial agonists (asterisks) or antagonists (open circles). Categorisation into these classes was performed from data in Tables 2 and 3, where ligands are defined as agonists if they stimulated more than 85% of the response at the human β1-adrenoceptor, as partial agonists if they stimulated 10–85% of the full response and as antagonists if they stimulated less than 10% of a full agonist response at the tβtrunc receptor
Mentions: The agonist response to 24 ligands (seven-point concentration–response curves) was examined for each of the turkey β-adrenoceptor mutants (Table 2). A further 24 ligands were examined at maximum concentrations only (Table 3). This enabled ligands to be classified into full agonists (those ligands that stimulated a greater than 85% maximum response to isoprenaline in the cAMP assays at the tβtrunc receptor), partial agonists (those that stimulated between 10–85% maximum responses at the tβtrunc receptor) and antagonists (those ligands that stimulated less than 10% maximum response at the tβtrunc receptor). This allowed the study of ligand affinity to be further examined to assess whether a particular subset of ligands (e.g. agonists) were disproportionately affected by the mutations (Fig. 3).Table 2

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