1. A monoclonal antibody raised against a thermo-stabilised β
- Author
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Mark, Soave, Gabriella, Cseke, Catherine J, Hutchings, Alastair J H, Brown, Jeanette, Woolard, and Stephen J, Hill
- Subjects
Monoclonal antibody ,Turkey ,Propranolol (Pubchem CID: 4946) ,IBMX (Pubchem CID: 3758) ,ICI 118551 (Pubchem CID: 5484725) ,Isoprenaline (Pubchem CID: 5807) ,CHO Cells ,CGP 20712A (Pubchem CID: 2685) ,ECL, extracellular loop ,CHO, Chinese hamster ovary ,Extracellular loop 2 ,Protein Structure, Secondary ,Article ,Propanolamines ,HEK, human embryonic kidney ,Cricetulus ,GPCR ,Allosteric Regulation ,Cricetinae ,Allosterism ,SPAP, secreted placental alkaline phosphatase ,Animals ,Humans ,Amino Acid Sequence ,mAb, monoclonal antibody ,Alprenolol (Pubchem CID: 2119) ,ComputingMethodologies_COMPUTERGRAPHICS ,Protein Stability ,Antibodies, Monoclonal ,ATCM, allosteric ternary complex model ,Adrenergic beta-Agonists ,Furimazine (Pubchem CID: 219083) ,Hoechst 33342 (Pubchem CID: 1464) ,Cimaterol (Pubchem CID: 2755) ,CGP 12177 (Pubchem CID: 2687) ,HEK293 Cells ,CRE, cAMP response element ,Receptors, Adrenergic, beta-1 ,Protein Binding ,StaR, stabilised receptor - Abstract
Graphical 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.
- Published
- 2017