Abstract
BACKGROUND AND PURPOSEThe 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 APPROACHResidue 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 RESULTSNECA 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 IMPLICATIONSInvestigation 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.
Highlights
GPCR are composed of seven transmembrane (TM) spanning α-helices and are responsible for translating signals from the extracellular milieu to intracellular responses
Data are normalized to basal and maximal internalization mediated by 10 μM NECA and each data point represent the mean ± SEM of three experiments performed in triplicate
The binding of an agonist to a GPCR leads to structural changes within the TM regions allowing activation of a
Summary
GPCR are composed of seven transmembrane (TM) spanning α-helices and are responsible for translating signals from the extracellular milieu to intracellular responses. For the angiotensin II receptor, mutation of residues within the DRY motif at the bottom of TM helix 3 can result in the receptor no longer being able to activate G-protein signalling, but which is still internalized effectively in an agonist-dependent manner (Gaborik et al, 2003; Wei et al, 2003). Analysis of the purified thermostabilized turkey β1-adrenoceptor has indicated that a bent conformation of TM6 is required for the formation of the inactivating ‘ionic lock’ (Moukhametzianov et al, 2011) Movement around this kink is believed to be the basis for forming an active conformation of the GPCR that can stimulate intracellular signalling pathways. Further experiments were carried out to investigate agonist-mediated ERK1/2 phosphorylation, inhibition of [3H]-cAMP accumulation and β-arrestin binding
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