Skip to main content

Research Repository

Advanced Search

Effect of a toggle switch mutation in TM6 of the human adenosine A3 receptor on Gi protein-dependent signalling and Gi-independent receptor internalization

Stoddart, Leigh A.; Kellam, Barrie; Briddon, Stephen J.; Hill, Stephen J.

Authors

Leigh A. Stoddart

Barrie Kellam

Stephen J. Briddon

Stephen J. Hill



Abstract

Background and Purpose: The highly conserved tryptophan (W6.48) in transmembrane domain 6 of GPCRs has been shown to play a central role in forming an active conformation in response to agonist binding. We set out to characterize the effect of this mutation on the efficacy of two agonists at multiple signalling pathways downstream of the adenosine A3 receptor.
Experimental Approach: Residue W6.48 in the human adenosine A3 receptor fused to yellow fluorescent protein was mutated to phenylalanine and expressed in CHO-K1 cells containing a cAMP response element reporter gene. The effects on agonist-mediated receptor internalization were monitored by automated confocal microscopy and image analysis. Further experiments were carried out to investigate agonist-mediated ERK1/2 phosphorylation, inhibition of [3H]-cAMP accumulation and β-arrestin2 binding.
Key Results: NECA was able to stimulate agonist-mediated internalization of the W6.48F mutant receptor, while the agonist HEMADO was inactive. Investigation of other downstream signalling pathways indicated that G-protein coupling was impaired for both agonists tested. Mutation of W6.48F therefore resulted in differential effects on agonist efficacy, and introduced signalling pathway bias for HEMADO at the adenosine A3 receptor.
Conclusions and Implications: Investigation of the pharmacology of the W6.48F mutant of the adenosine A3 receptor confirms that this region is important in forming the active conformation of the receptor for stimulating a number of different signalling pathways and that mutations in this residue can lead to changes in agonist efficacy and signalling bias.

Journal Article Type Article
Publication Date Jul 25, 2014
Journal British Journal of Pharmacology
Print ISSN 0007-1188
Electronic ISSN 0007-1188
Publisher Wiley
Peer Reviewed Peer Reviewed
Volume 171
Issue 16
APA6 Citation Stoddart, L. A., Kellam, B., Briddon, S. J., & Hill, S. J. (2014). Effect of a toggle switch mutation in TM6 of the human adenosine A3 receptor on Gi protein-dependent signalling and Gi-independent receptor internalization. British Journal of Pharmacology, 171(16), doi:10.1111/bph.12739
DOI https://doi.org/10.1111/bph.12739
Publisher URL http://onlinelibrary.wiley.com/doi/10.1111/bph.12739/abstract;jsessionid=E289E50D7766D9EE65349A8C9DC19397.f02t03
Copyright Statement Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0

Files

Stoddart_et_al-2014-British_Journal_of_Pharmacology.pdf (2 Mb)
PDF

Copyright Statement
Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0





You might also like



Downloadable Citations

;