A monoclonal antibody raised against a thermo-stabilised β1-adrenoceptor interacts with extracellular loop 2 and acts as a negative allosteric modulator of a sub-set of β1-adrenoceptors expressed in stable cell lines

Abstract

Recent interest has focused on antibodies that can discriminate between different receptor conformations. Here we have characterised the effect of a monoclonal antibody (mAb3), raised against a purified thermo-stabilised turkey β₁-adrenoceptor (β₁AR-m23 StaR), on β₁-ARs expressed in CHO-K1 or HEK 293 cells. Immunohistochemical and radioligand-binding studies demonstrated that mAb3 was able to bind to ECL2 of the tβ₁-AR, but not its human homologue. Specific binding of mAb3 to tβ₁-AR was inhibited by a peptide based on the turkey, but not the human, ECL2 sequence. Studies with [³H]-CGP 12177 demonstrated that mAb3 prevented the binding of orthosteric ligands to a subset (circa 40%) of turkey β₁-receptors expressed in both CHO K1 and HEK 293 cells. MAb3 significantly reduced the maximum specific binding capacity of [³H]-CGP-12177 without influencing its binding affinity. Substitution of ECL2 of tβ₁-AR with its human equivalent, or mutation of residues D186S, P187D, Q188E prevented the inhibition of [³H]-CGP 12177 binding by mAb3. MAb3 also elicited a negative allosteric effect on agonist-stimulated cAMP responses. The identity of the subset of turkey β₁-adrenoceptors influenced by mAb3 remains to be established but mAb3 should become an important tool to investigate the nature of β₁-AR conformational states and oligomeric complexes.

Keywords: Allosterism; Alprenolol (Pubchem CID: 2119); CGP 12177 (Pubchem CID: 2687); CGP 20712A (Pubchem CID: 2685); Cimaterol (Pubchem CID: 2755); Extracellular loop 2; Furimazine (Pubchem CID: 219083); GPCR; Hoechst 33342 (Pubchem CID: 1464); IBMX (Pubchem CID: 3758); ICI 118551 (Pubchem CID: 5484725); Isoprenaline (Pubchem CID: 5807); Monoclonal antibody; Propranolol (Pubchem CID: 4946).