Figure 3.

Ligand modulation of the A_2AR-H₃R interaction in HEK-293 cells. Activation of the WT-H₃R with its agonist RAMH led to functional complementation when co-expressed with the A_2AR₃₀₂-Gα_qi4 but not with A_2AR₃₀₂-Gα_qs4. B. Ca²⁺ mobilization was not observed when the WT-A_2AR was co-transfected with the truncated H₃R₄₂₇-Gα_qs4 or H₃R₄₂₇-Gα_qi4, in accord with the incapability of the receptor to activate the chimeric proteins. C. As shown in Figure 1D, activation of the H₃R₄₂₇-Gα_qi4 resulted in Ca²⁺ mobilization, and this response was prevented when it was co-expressed with the WT-A_2AR, suggesting a preference of the H₃R₄₂₇-Gα_qi4 to form heterodimers. D. The well-studied A_2AR-D₂R heterodimer was used as a positive control for these experiments. Activation of the D₂R with increasing concentrations of the agonist quinpirole caused marked Ca²⁺ mobilization only when co-expressed with the chimeric A_2AR₃₀₂-Gα_qi4. E. The histamine H₄ receptor (H₄R) as a negative control. No functional complementation was observed when the receptor was co-expressed with the A_2AR₃₀₂-Gα_qi4, showing the specificity of the A_2AR-H₃R interaction. F. Functional complementation between the A_2AR₃₀₂-Gα_qi4 and the WT-H₃R was not observed when the latter receptor was activated with the endogenous agonist histamine. For all graphs data are means ± SEM from 3 replicates from representative experiments. Where SEM bars are not visible, they are smaller than the symbol size. The quantitative analysis is shown in Table 1 and Supplementary Table 2.