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Identification of transmembrane 4 as a key site for the secondary, low affinity, conformation of the human β1-adrenoceptor. The β1-adrenoceptor (AR) exists in two agonist conformations/states: state 1 - a high affinity state where responses to catecholamines (and cimaterol) are potently inhibited by β1-antagonists and state 2 - a low affinity secondary state where agonist responses (particularly CGP 12177) are relatively resistant to inhibition by β1-antagonists (Kaumann and Molenaar 2008). The precise nature of the secondary state is unknown but does not occur in the closely related β2-AR. Using site directed mutagenesis, we made point mutations in the wildtype (WT) human β1-AR receptor such that each transmembrane (tm) region of β1-AR was in turn mutated to resemble that of the human β2-AR (Baker et al., 2008). Thus β1tm1 is the β1-AR but with the amino acid sequence of the β2-AR in transmembrane 1. CHO-CRE-SPAP cells were transfected and stable cell lines were made expressing the WT or mutant receptors. Responses to cimaterol and CGP 12177 and antagonism of these with CGP 20712A, propranolol and bisoprolol was examined using CRE-SPAP production (Baker et al., 2008).
Log EC50, % isoprenaline maximum and log KD values for ligands at the wildtype (WT) and chimeric receptors where each tm region in turn has been mutated to that of the human β 2-adrenoceptor In conclusion, swapping entire transmembrane domains of the human β1 and β2-adrenoceptors had little effect of the agonist responses to cimaterol, and inhibition of these cimaterol responses by antagonists. However, the agonist responses to CGP 12177 were far more potent, and the affinity of antagonist far greater in the β1tm4 receptor (in bold in the Table above). This strongly suggests that transmembrane 4 has a major role in the secondary conformation of the β1-adrenoceptor. Baker et al., (2008) Mol. Pharmacol. 74: 1246-1260 Kaumann and Molenaar (2008) Pharmacol. Ther. 118: 303-336.
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