Identification of Key Residues in Transmembrane 4 Responsible for the Secondary, Low-Affinity Conformation of the Humanβ₁-Adrenoceptor

Abstract

The b 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 b 1 -adrenoceptor antagonists, and 2) a low-affinity secondary conformation where agonist responses, particularly CGP12177 [(2)-4-(3-tert-butylamino-2-hydroxypropoxy)-benzimidazol-2-one] are relatively resistant to inhibition by b 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 b 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 3 H-cAMP, we examined the pharmacological consequences of swapping transmembrane (TM) regions of the human b 1 -and b 2 -adrenoceptors, followed by single point mutations, to determine the key residues involved in the b 1 -adrenoceptor secondary conformation. We found that TM4 (particularly amino acids L195 and W199) had a major role in the generation of the secondary b 1 -adrenoceptor conformation. Thus, unlike at the human b 1 -wild-type adrenoceptor, at b 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 EC 50 values in the b 1 -TM4 but not b 1 -wild-type receptors. Furthermore pindolol switched from a biphasic concentration response at human b 1 -wild-type adrenoceptors to a monophasic concentration response in the b 1 -TM4 mutant receptors. Mutation of these amino acids to those found in the b 2 -adrenoceptor (L195Q and W199Y), or mutation of a single residue (W199D) in the human b 1 -adrenoceptor thus abolished this secondary conformation and created a b 1 -adrenoceptor with only one high-affinity agonist conformation.

Use of a new proximity assay (NanoBRET) to investigate the ligand‐binding characteristics of three fluorescent ligands to the human β1‐adrenoceptor expressed in HEK‐293 cells

“…Site‐directed mutagenesis studies have attempted to isolate the regions of the β 1 ‐adrenoceptor responsible for this second site pharmacology in CHO‐K1 cells (Baker et al. , ; Joseph et al. 2004b).…”

“…Baker et al. () have indicated that residues within the dimer interface region in transmembrane domain (TM) 4 of the β 1 ‐adrenoceptor may be responsible for the secondary β 1 ‐adrenoceptor conformation (Gherbi et al. ).…”

Salmeterol’s Extremeβ2 Selectivity Is Due to Residues in Both Extracellular Loops and Transmembrane Domains

“…This cDNA was also subcloned as a HindIII/XbaI fragment into pcDNA3.1, and the sequence was confirmed by DNA sequencing. The mutations described in Table 1 were generated using QuikChange mutagenesis and BioLine PolyMate Additive for GC-rich templates (Baker et al, 2014). After subcloning in Top 10F competent cells, each mutant b2-adrenoceptor cDNA was excised on HindIII/XbaI and subcloned into native pcDNA3.1 containing a neomycin selection marker.…”

“…Following mutagenesis, each receptor DNA was digested with VspI, the fragments were purified, and the alternative N terminal sequences were ligated to give a b1-adrenoceptor with the full b2-adrenoceptor N terminus (b1-N) or a b2-adrenoceptor with the full b1-adrenoceptor N terminus (b2-N). These constructs were expressed in Chinese hamster ovary (CHO)-K1 cells, and stable cell lines were generated (see below and Baker et al, 2014). Once important EL or TM regions for the interaction of salmeterol were identified, to determine which individual amino acids were involved, several single-point mutations and chimeric receptors were made (Table 1), and these constructs were expressed transiently in CHO-K1 cells.…”

“…As the selectivity of salmeterol for the human b2-adrenoceptor is 1000-to 3000-fold greater than that for the human b1-adrenoceptor (and this is due to selective affinity, not selective efficacy; Baker, 2010), a chimeric b2/b1 mutagenesis study was undertaken in which single TM or EL regions were examined in turn before progressing to single-point mutagenesis studies (Baker et al, 2014).…”