Mark Soave
A monoclonal antibody raised against a thermo-stabilised ?1-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.
Authors
Gabriella Cseke
Catherine J. Hutchings
Alastair J.H. Brown
JEANETTE WOOLARD Jeanette.Woolard@nottingham.ac.uk
Professor of Cardiovascular Physiology and Pharmacology
STEPHEN HILL STEVE.HILL@NOTTINGHAM.AC.UK
Professor of Molecular Pharmacology
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 ?1-adrenoceptor (?1AR-m23 StaR), on ?1-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 t?1-AR, but not its human homologue. Specific binding of mAb3 to t?1-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 t?1-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 ?1-adrenoceptors influenced by mAb3 remains to be established but mAb3 should become an important tool to investigate the nature of ?1-AR conformational states and oligomeric complexes.
Citation
Soave, M., Cseke, G., Hutchings, C. J., Brown, A. J., Woolard, J., & Hill, S. J. (2018). A monoclonal antibody raised against a thermo-stabilised ?1-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, https://doi.org/10.1016/j.bcp.2017.10.015
Journal Article Type | Article |
---|---|
Acceptance Date | Oct 31, 2017 |
Online Publication Date | Nov 2, 2017 |
Publication Date | Jan 31, 2018 |
Deposit Date | Nov 3, 2017 |
Publicly Available Date | Nov 3, 2017 |
Journal | Biochemical Pharmacology |
Print ISSN | 0006-2952 |
Electronic ISSN | 1873-2968 |
Publisher | Elsevier |
Peer Reviewed | Peer Reviewed |
Volume | 147 |
DOI | https://doi.org/10.1016/j.bcp.2017.10.015 |
Keywords | GPCR; Allosterism; Monoclonal antibody; Extracellular loop 2 |
Public URL | https://nottingham-repository.worktribe.com/output/907985 |
Publisher URL | http://www.sciencedirect.com/science/article/pii/S0006295217306469 |
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Copyright Statement
Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0
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