Generation of adenosine A3 receptor functionally humanized mice for the evaluation of the human antagonists

Although the adenosine A(3) receptor (A3AR), which is a G(i/o) protein-coupled receptor, has attracted considerable interest as a potential target for drugs against asthma or inflammation, the in vivo evaluation of the antagonists using rodents in the first step of drug development has been hampered by the lack of highly potent antagonists for the rodent A3AR. To evaluate the pharmacological effects of human A3AR antagonists in mice, we previously generated A3AR-humanized mice, in which the mouse A3AR gene was replaced by its human counterpart. However, the human A3AR did not lead to the phosphoinositide 3-kinase (PI3K) gamma-signaling pathway such as IgE/antigen-dependent mast cell degranulation, probably due to the uncoupling of the mouse G(i/o) protein(s). To overcome the uncoupling, we here generated A3AR functionally humanized mice by replacing the mouse A3AR gene with a human/mouse chimeric A3AR sequence in which whole intracellular regions of the human A3AR were substituted for the corresponding regions of the mouse A3AR. The chimeric A3AR led to intracellular Ca(2+) elevation and activation of the PI3Kgamma-signaling pathway, which are equivalent to the actions induced by A3AR in wild-type mice. The human A3AR antagonist had the same binding affinities for the chimeric A3AR as the human A3AR and completely antagonized this potentiation. This is the first direct evidence that the uncoupling of mouse G protein(s) to the human A3AR is due to a sequence difference in the intracellular regions of A3AR. The A3AR functionally humanized mice can be widely employed for pharmacological evaluations of the human A3AR antagonists.