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A TM6 toggle switch mutant of the adenosine A3 receptor has reduced ability to couple to multiple signalling pathways The tryptophan residue at position 6.48 of family A GPCRs is highly conserved. It is thought to allow formation of the active receptor conformation through the release of constraints around the proline kink in TM6, (Shi et al, 2002). In the human adenosine A3 (A3-AR) receptor, mutation of this residue (tryptophan 243; W243) results in a receptor which is unable to couple to G proteins (Gao et al., 2002). To investigate the role of this residue further, we have created a point mutation in the human A3-AR-YFP fusion protein, replacing W243 with phenylalanine (A3-AR W243F-YFP). A3-AR-YFP and A3-AR W243F-YFP constructs were stably expressed in CHO cells containing a reporter gene consisting of a cAMP response element (CRE) promoter linked to secreted placental alkaline phosphatase (SPAP). Confocal imaging, SPAP assays and measurement of intracellular calcium concentrations were carried out as described previously (Middleton et al., 2007; May et al., 2007). Confocal imaging confirmed that both wild type and mutant receptors were expressed at the cell surface. Both also showed membrane binding of the fluorescent A3-AR agonist, ABEA-X-BY630 (50nM, Middleton et al., 2007) which was displaced by pre-incubation with MRS1220 (100nM, 30 min, 37°C, n = 4). Both A3-AR-YFP and A3-AR W243F-YFP underwent internalisation when exposed to 1μM NECA (30 mins, 37oC) which was insensitive to pertussis toxin (PTx) treatment (100ng/ml-1, 16h), suggesting that internalisation of the A3-AR is independent of Gi/o activation. In functional assays, NECA inhibited forskolin-stimulated (3μM) SPAP production with similar potency in A3-AR-YFP and A3-AR W243F-YFP cells (pEC50 = 7.03±0.25 and 6.82±0.29, respectively, n = 6). However, maximum inhibition was reduced from 79±7% for A3-AR-YFP cells to only 30±4% in A3-AR W243F-YFP cells (n = 6, P<0.01, unpaired Student’s t-test). In both cases, the response was abolished by PTx treatment. Similarly, NECA increased intracellular calcium in both cell lines in a concentration dependent manner (pEC50 = 6.59±0.09 and 6.46±0.11 for A3-AR-YFP and A3-AR W243F-YFP, respectively, n = 3). Intracellular calcium was increased by 4.48±0.76 fold in A3-AR-YFP cells and by 1.32±0.38 fold in A3-AR W243F-YFP cells (n = 3). Again, in both cases the response was sensitive to PTx treatment (n = 3). Taken together this indicates that, whilst the responses mediated by A3-AR W243F are reduced, it is still capable of forming an active confirmation. In addition, internalisation of the receptor, which appears PTx-insensitive, is unaffected by the mutation.
Gao et al., (2002) J Biol Chem, 277:19056-19063. We thank the MRC for financial support
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