Identification of Key Residues in Transmembrane 4 Responsible for the Secondary, Low-Affinity Conformation of the Human β1-Adrenoceptor

The beta(1)-adrenoceptor exists in two agonist conformations/states: 1) a high-affinity state where responses to catecholamines and other agonists (e.g., cimaterol) are potently inhibited by beta(1)-adrenoceptor antagonists, and 2) a low-affinity secondary conformation where agonist responses, particularly CGP12177 [(-)-4-(3-tert-butylamino-2-hydroxypropoxy)-benzimidazol-2-one] are relatively resistant to inhibition by beta(1)-adrenoceptor 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 beta(2)-adrenoceptor. Here, using site-directed mutagenesis and functional measurements of production of a cyclic AMP response element upstream of a secreted placental alkaline phosphatase reporter gene and accumulation of H-3-cAMP, we examined the pharmacological consequences of swapping transmembrane (TM) regions of the human beta(1)- and beta(2)-adrenoceptors, followed by single point mutations, to determine the key residues involved in the beta(1)-adrenoceptor secondary conformation. We found that TM4 (particularly amino acids L195 and W199) had a major role in the generation of the secondary beta(1)-adrenoceptor conformation. Thus, unlike at the human beta(1)-wild-type adrenoceptor, at beta(1)-TM4 mutant receptors, cimaterol and CGP12177 responses were both potently inhibited by antagonists. CGP12177 acted as a simple partial agonist with similar K-B and EC50 values in the beta(1)-TM4 but not beta(1)-wild-type receptors. Furthermore pindolol switched from a biphasic concentration response at human beta(1)-wild-type adrenoceptors to a monophasic concentration response in the beta(1)-TM4 mutant receptors. Mutation of these amino acids to those found in the b2-adrenoceptor (L195Q and W199Y), or mutation of a single residue (W199D) in the human beta(1)-adrenoceptor thus abolished this secondary conformation and created a beta(1)-adrenoceptor with only one high-affinity agonist conformation.