Figure 2.

Study of the possible A_2AR-H₃R dimerization by functional complementation assay. A. Basis of the assay. The G protein-coupled receptor (GPCR; grey cylinders) is truncated at its carboxyl terminus to generate a nonfunctional GPCR, which is then fused to the chimeric G protein (green hexagons). The chimeric G protein is formed by a Gα_q protein in which 9 or 10 residues of the C-terminus were substituted by the corresponding sequence of a Gα_s4 or Gα_i4 protein (represented by X). Under these conditions, Ca²⁺ mobilization can only be elicited when the complex GPCR-Gα_qx is in close proximity to a wild type (WT) GPCR, either the same (homodimerization) or a different receptor (heterodimerization). B. Activation of the H₃R with its agonist RAMH resulted in Ca²⁺ mobilization only when it was co-expressed with the chimeric Gα_qi4 but not when transfected alone. C. When activated with its agonist CGS-21680, the A_2AR was uncapable to induce Ca²⁺ mobilization either alone or when co-transfected with the chimeric Gα_qs4 protein. D. Functional complementation by homodimerization of the truncated A_2AR₃₀₂, either bound to Gα_qs4 (A_2AR₃₀₂-Gα_qs4) or Gα_qi4 (A_2AR₃₀₂-Gα_qi4), with the native A_2AR was not observed. E. Activation of the truncated H₃R₄₂₇ bound to Gα_qi4 (H₃R₄₂₇-Gα_qi4) induced Ca²⁺ mobilization on its own and homodimerization with the WT-H₃R did not modify the response. No signal was observed with activation of the H₃R₄₂₇-Gα_qs4 construct when it was co-expressed with the WT-H₃R. In all graphs data are means ± SEM from 3 replicates from a representative experiment. Where SEM bars are not visible, they are smaller than the symbol size. The quantitative analysis is shown in Tables 1 and Supplementary Table 2.