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 1- adrenoceptors expressed in stable cell lines
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 29...
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Elsevier
2018
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| Online Access: | https://eprints.nottingham.ac.uk/47824/ |
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| author | Soave, Mark Cseke, Gabriella Hutchings, Catherine J. Brown, Alastair J.H. Woolard, Jeanette Hill, Stephen J. |
| author_facet | Soave, Mark Cseke, Gabriella Hutchings, Catherine J. Brown, Alastair J.H. Woolard, Jeanette Hill, Stephen J. |
| author_sort | Soave, Mark |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | 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 1-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. |
| first_indexed | 2025-11-14T20:07:04Z |
| format | Article |
| id | nottingham-47824 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| last_indexed | 2025-11-14T20:07:04Z |
| publishDate | 2018 |
| publisher | Elsevier |
| recordtype | eprints |
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| spelling | nottingham-478242020-05-04T19:29:17Z https://eprints.nottingham.ac.uk/47824/ 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 1- adrenoceptors expressed in stable cell lines Soave, Mark Cseke, Gabriella Hutchings, Catherine J. Brown, Alastair J.H. Woolard, Jeanette Hill, Stephen J. 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 1-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. Elsevier 2018-01-31 Article PeerReviewed Soave, Mark, Cseke, Gabriella, Hutchings, Catherine J., Brown, Alastair J.H., Woolard, Jeanette and Hill, Stephen J. (2018) 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 1- adrenoceptors expressed in stable cell lines. Biochemical Pharmacology, 147 . pp. 38-54. ISSN 1873-2968 GPCR; Allosterism; Monoclonal antibody; Extracellular loop 2 http://www.sciencedirect.com/science/article/pii/S0006295217306469 doi:10.1016/j.bcp.2017.10.015 doi:10.1016/j.bcp.2017.10.015 |
| spellingShingle | GPCR; Allosterism; Monoclonal antibody; Extracellular loop 2 Soave, Mark Cseke, Gabriella Hutchings, Catherine J. Brown, Alastair J.H. Woolard, Jeanette Hill, Stephen J. 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 1- adrenoceptors expressed in stable cell lines |
| title | 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 1- adrenoceptors expressed in stable cell lines |
| title_full | 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 1- adrenoceptors expressed in stable cell lines |
| title_fullStr | 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 1- adrenoceptors expressed in stable cell lines |
| title_full_unstemmed | 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 1- adrenoceptors expressed in stable cell lines |
| title_short | 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 1- adrenoceptors expressed in stable cell lines |
| title_sort | 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 1- adrenoceptors expressed in stable cell lines |
| topic | GPCR; Allosterism; Monoclonal antibody; Extracellular loop 2 |
| url | https://eprints.nottingham.ac.uk/47824/ https://eprints.nottingham.ac.uk/47824/ https://eprints.nottingham.ac.uk/47824/ |