Limits...
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.

Show MeSH
The amino acid sequence of the turkey β-adrenoceptor after deletion of the C-terminal 50 amino acid residues (tβtrunc) and the three other mutants, β6–m23, β36 and β36–m23 used in this study. Thermostabilising point mutations are coloured blue, the original C116L mutation in yellow and the C358A mutation to remove the palmitoylation sites in orange
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3116118&req=5

Fig1: The amino acid sequence of the turkey β-adrenoceptor after deletion of the C-terminal 50 amino acid residues (tβtrunc) and the three other mutants, β6–m23, β36 and β36–m23 used in this study. Thermostabilising point mutations are coloured blue, the original C116L mutation in yellow and the C358A mutation to remove the palmitoylation sites in orange

Mentions: Initially, part of the very large C-terminus was deleted and a C116L mutation made to make the starting construct: tβtrunc construct (Baker 2010a; Warne et al. 2008). Deletion of the N-terminus, made the β6 construct, which significantly increased heterologous expression in the baculovirus expression systems (Warne et al. 2003). This β6 construct was then modified, firstly by introducing 6-point mutations to increase the thermostability (the m23 mutations; Serrano-Vega et al. 2008, Fig. 1b) and secondly residues in intracellular loop three, C-terminus and the palmitoylation site were removed (β36 deletions; Warne et al. 2009, Fig. 1c). Combination of the receptor truncations (β36 deletions) and the thermostabilising mutations (m23 mutations) to form the construct β36–m23 (Fig. 1d) allowed crystallisation and structure determination (Warne et al. 2008, 2009).Fig. 1


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)

The amino acid sequence of the turkey β-adrenoceptor after deletion of the C-terminal 50 amino acid residues (tβtrunc) and the three other mutants, β6–m23, β36 and β36–m23 used in this study. Thermostabilising point mutations are coloured blue, the original C116L mutation in yellow and the C358A mutation to remove the palmitoylation sites in orange
© Copyright Policy
Related In: Results  -  Collection

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

Fig1: The amino acid sequence of the turkey β-adrenoceptor after deletion of the C-terminal 50 amino acid residues (tβtrunc) and the three other mutants, β6–m23, β36 and β36–m23 used in this study. Thermostabilising point mutations are coloured blue, the original C116L mutation in yellow and the C358A mutation to remove the palmitoylation sites in orange
Mentions: Initially, part of the very large C-terminus was deleted and a C116L mutation made to make the starting construct: tβtrunc construct (Baker 2010a; Warne et al. 2008). Deletion of the N-terminus, made the β6 construct, which significantly increased heterologous expression in the baculovirus expression systems (Warne et al. 2003). This β6 construct was then modified, firstly by introducing 6-point mutations to increase the thermostability (the m23 mutations; Serrano-Vega et al. 2008, Fig. 1b) and secondly residues in intracellular loop three, C-terminus and the palmitoylation site were removed (β36 deletions; Warne et al. 2009, Fig. 1c). Combination of the receptor truncations (β36 deletions) and the thermostabilising mutations (m23 mutations) to form the construct β36–m23 (Fig. 1d) allowed crystallisation and structure determination (Warne et al. 2008, 2009).Fig. 1

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