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

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.

Fig. 1

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

Fig. 2

Inhibition of ³H-CGP12177 specific binding to whole cells by a the antagonist timolol and b the agonist isoprenaline at each of the turkey β-adrenoceptors mutants. Nonspecific binding was determined by 10 μM propranolol. The concentration of 3H-CGP 12177 present in these experiments was 1.6 nM. Data points are mean ± SE mean of triplicate determinations. These single experiments are representative of a five and b seven separate experiments

Fig. 3

Correlation plots of the log K _D 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

Fig. 4

³H-cAMP accumulation in response to noradrenaline, adrenaline, clenbuterol and xamoterol in a tβtrunc cells, b β6–m23 cells, c β36 cells and d β36–m23 cells expressed as a percentage of the maximal response to isoprenaline in each experiment. Data points are mean ± SE mean of triplicate determinations. Each experiment is representative of a four, b four, c four and d three separate experiments

Fig. 5

CRE-SPAP production in response to clenbuterol in the absence and presence of propranolol in a tβtrunc cells, b β6–m23 cells, c β36 cells and d β36–m23 cells. Bars represent basal CRE-SPAP production, that in response to 10 μM isoprenaline and that in response 10, 100, 1,000 or 30, 300, 3,000 nM propranolol alone. Data points are mean ± SEM of triplicate determinations. The Schild slopes for each experiment are a 0.95, b 0.98 and c 1.00. Each experiment is representative of a five, b five, c five, and d three separate experiments

Fig. 6

CRE-SPAP production in response to cimaterol in a β6–m23 and b β36 cells in the absence and presence of CGP12177. Bars represent basal CRE-SPAP production that in response to 10 μM isoprenaline and that in response 1, 10, 30 or 100 nM CGP12177 alone. Data points are mean ± SEM of triplicate determinations. Each experiment is representative of a five and b five separate experiments

Fig. 7

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

Fig. 8

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

Fig. 9

³H-cAMP accumulation in response to cyanopindolol in a tβtrunc cells, b β6–m23 cells, c β36 cells and d β36–m23 cells. Bars represent basal ³H-cAMP accumulation and that in response to 10 μM isoprenaline. Data points are mean ± SE mean of triplicate determinations. Each experiment is representative of a four, b 12, c six and d three separate experiments

Fig. 10

³H-cAMP accumulation in response to CGP12177 in a tβtrunc cells, b β6–m23 cells, c β36 cells and d β36–m23 cells in the absence and presence of cimaterol. Bars represent basal ³H-cAMP accumulation, that in response to 10 μM isoprenaline and that in response to 3, 10, 30, 100, 300 or 1,000 nM cimaterol. Data points are mean ± SE mean of triplicate determinations. Each experiment is representative of a four, b six, c six and d four separate experiments