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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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Related in: MedlinePlus

CRE-SPAP production in response to CGP12177 in a β6–m23 and b β36 cells in the absence and presence of propranolol. Bars represent basal CRE-SPAP production, that in response to 10 μM isoprenaline and that in response 1, 10 or 100 μM propranolol alone. Data points are mean ± SEM of triplicate determinations. Each experiment is representative of a five and b five separate experiments
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Fig7: CRE-SPAP production in response to CGP12177 in a β6–m23 and b β36 cells in the absence and presence of propranolol. Bars represent basal CRE-SPAP production, that in response to 10 μM isoprenaline and that in response 1, 10 or 100 μM propranolol alone. Data points are mean ± SEM of triplicate determinations. Each experiment is representative of a five and b five separate experiments

Mentions: Next, the β36 deletions and the m23 mutations were each examined separately to determine the effects of these mutations on the two agonist-induced conformations of the tβtrunc receptor. Several agonists stimulated CRE-SPAP responses that were inhibited by propranolol, CGP20712A and ICI118551 (Fig. 5, Tables 4, 5, 6 and 7) in a competitive manner. CGP12177 also acted as an antagonist, inhibiting both of these mutant receptor responses with high affinity (Fig. 6, Tables 4, 5, 6 and 7). Also, CGP12177 itself stimulated a partial agonist response at both of these mutants and in each case the log EC50 value was significantly higher than the KD value for CGP12177 at the mutant receptor (Fig. 7, Tables 4, 5, 6 and 7). In addition, these CGP12177 agonist responses required significantly higher concentrations of propranolol, CGP20712A and ICI118551 to be inhibited than the other agonists (Fig. 7, Tables 4, 5, 6 and 7). This is in keeping with two agonist-induced conformations (a catecholamine site conformation and a secondary CGP12177 site conformation) on each of the β36 and β6–m23 receptor mutants.Fig. 5


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 CGP12177 in a β6–m23 and b β36 cells in the absence and presence of propranolol. Bars represent basal CRE-SPAP production, that in response to 10 μM isoprenaline and that in response 1, 10 or 100 μM propranolol alone. Data points are mean ± SEM of triplicate determinations. Each experiment is representative of a five and b five separate experiments
© Copyright Policy
Related In: Results  -  Collection

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

Fig7: CRE-SPAP production in response to CGP12177 in a β6–m23 and b β36 cells in the absence and presence of propranolol. Bars represent basal CRE-SPAP production, that in response to 10 μM isoprenaline and that in response 1, 10 or 100 μM propranolol alone. Data points are mean ± SEM of triplicate determinations. Each experiment is representative of a five and b five separate experiments
Mentions: Next, the β36 deletions and the m23 mutations were each examined separately to determine the effects of these mutations on the two agonist-induced conformations of the tβtrunc receptor. Several agonists stimulated CRE-SPAP responses that were inhibited by propranolol, CGP20712A and ICI118551 (Fig. 5, Tables 4, 5, 6 and 7) in a competitive manner. CGP12177 also acted as an antagonist, inhibiting both of these mutant receptor responses with high affinity (Fig. 6, Tables 4, 5, 6 and 7). Also, CGP12177 itself stimulated a partial agonist response at both of these mutants and in each case the log EC50 value was significantly higher than the KD value for CGP12177 at the mutant receptor (Fig. 7, Tables 4, 5, 6 and 7). In addition, these CGP12177 agonist responses required significantly higher concentrations of propranolol, CGP20712A and ICI118551 to be inhibited than the other agonists (Fig. 7, Tables 4, 5, 6 and 7). This is in keeping with two agonist-induced conformations (a catecholamine site conformation and a secondary CGP12177 site conformation) on each of the β36 and β6–m23 receptor mutants.Fig. 5

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