PT - JOURNAL ARTICLE AU - Jillian G Baker AU - Richard G W Proudman AU - Stephen J Hill TI - Identification of Key Residues in Transmembrane 4 Responsible for the Secondary Site of the Human β1-Adrenoceptor AID - 10.1124/mol.114.091587 DP - 2014 Mar 07 TA - Molecular Pharmacology PG - mol.114.091587 4099 - http://molpharm.aspetjournals.org/content/early/2014/03/07/mol.114.091587.short 4100 - http://molpharm.aspetjournals.org/content/early/2014/03/07/mol.114.091587.full AB - The β1-adrenoceptor can exist in two agonist conformations/states: (1) a high affinity state where responses to catecholamines (and other agonists such as cimaterol) are potently inhibited by β1-antagonists and (2) a low affinity secondary state where agonist responses (particularly CGP12177) are relatively resistant to inhibition by β1-antagonists. Although both states have been demonstrated in many species (including human), the precise nature of the secondary state is unknown and does not occur in the closely related β2-adrenoceptor. Here, using site directed mutagenesis and functional measurements of 3H-cAMP accumulation and CRE-SPAP production, we examined the pharmacological consequences of swapping transmembrane regions of the human β1 and β2-adrenoceptors, followed by single point mutations, in order to determine the key residues involved in the β1-adrenoceptor secondary state. We found that transmembrane 4 (particularly amino acids L195 and W199) had a major role in the generation of the secondary β1-adrenoceptor conformation. Thus, unlike at the human β1-wildtype receptor, at β1-transmembrane 4 mutant receptors cimaterol and CGP12177 responses were both potently inhibited by antagonists, CGP12177 acted as a simple partial agonist with similar KD and EC50 values and pindolol switched from biphasic to monophasic concentration response relationships. Mutation of these amino acids to those found in the β2-adrenoceptor (L195Q and W199Y), or mutation of a single residue (W199D) in the human β1-adrenoceptor, thus abolished this secondary conformation and created a β1-receptor with only one high affinity agonist conformation.