title: Effect of a toggle switch mutation in TM6 of the human adenosine A₃ receptor on Gi protein-dependent signalling and Gi-independent receptor internalization.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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 [(3)H]-cAMP accumulation and β-arrestin2 binding.
  
  
  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
  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.
authors.affiliation: Institute of Cell Signalling, School of Life Sciences, The University of Nottingham, Nottingham, UK.
authors.forename: Leigh A
authors.initials: LA
authors.lastname: Stoddart
authors.forename: Barrie
authors.initials: B
authors.lastname: Kellam
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Briddon
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Hill
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D019830:Adenosine-5'-(N-ethylcarboxamide) / Q000494:pharmacology; D000818:Animals; D019390:Arrestins / Q000378:metabolism; D016466:CHO Cells; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects; D009154:Mutation; D017434:Protein Structure, Tertiary; D058906:Purinergic P1 Receptor Agonists / Q000494:pharmacology*; D043684:Receptor, Adenosine A3 / Q000235:genetics* / Q000378:metabolism*; D015398:Signal Transduction; D000071557:beta-Arrestins
chemical_list: C518542:2-hexyn-1-yl-N(6)-methyladenosine; D019390:Arrestins; D058906:Purinergic P1 Receptor Agonists; D043684:Receptor, Adenosine A3; D000071557:beta-Arrestins; D019830:Adenosine-5'-(N-ethylcarboxamide); D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D000241:Adenosine
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