(a) Schematic representation showing the positions of the mutations in the D2 receptor, with coloring corresponding to the symbols and lines in (b) and (c). (b) D2/D4, a D2 mutant with 4 amino acids substituted from the D4 receptor (V91^2.61F/F110^3.29L/V111^3.28M/Y408^7.35V) making it 1000-times more sensitive to a D4-selective inhibitor (Supplementary Fig. 4 online), is activated by quinpirole, albeit with a lower potency and efficacy when compared with WT D2R. (c) All the other mutants, which are described in the text, were non-functional. Activation data were normalized as in Fig. 1. The mean±SEM of at least 3 experiments, each conducted in triplicate, are shown.

Shown are effects on signaling with the D2/D4 construct expressed either as protomer A (a), or as protomer B (D2/D4-Gqi5) (b). (a) The D4-selective antagonist L745,870 (1 µM) totally blocked signaling of the D2/D4 construct expressed as protomer A with WT-Gqi5. (b) In contrast, L745,870 increased maximal activation for WT D2R coexpressed with D2/D4-Gqi5 to156.7±7.3% (n=9) (p<0.01*** by Student’s t-test) of that observed for D2R coexpressed with WT D2R-Gqi5 (Fig. 3a). (c) Coexpression of a constitutively active mutant (Supplementary Fig. 8 online) that was unable to bind ligand (D114^3.32A/CAM-Gqi5), to enhance the fraction of protomer B in an active conformation, led to 49.6±8.4% (n=9) (p<0.01*** by Student’s t-test) of maximal activity (♦) when compared to WT D2R coexpressed with D114A-Gqi5 (◊). Activation data were normalized to surface expression as described in Fig. 3. The mean±SEM of at least 3 experiments, each conducted in triplicate, are shown.

Bound agonist is represented by a black square. Activation is represented by a trapezoid with a bold base. Extent of activation is indicated by increasingly bold trapezoid boundaries. The inverse agonist bound state is represented by an inverted trapezoid. (1) Neither protomer is activated. (2) Protomer A binds agonist and protomer B is constitutively active (or in the case of a heterodimer, is occupied by protomer B’s agonist). (3) Protomer A binds agonist, whereas protomer B cannot bind (or in a heterodimer, is not agonist-bound). Note that although protomer B is not activated by ligand, it can isomerize to the active state, which would result in configuration (2). (4) Protomer A binds agonist, whereas protomer B is stabilized in the inactive state by inverse agonist. Experimentally determined maximal activation representing these idealized conformations: (1) no ligand, (2) WT D2R coexpressed with D114A/CAM-Gqi5, (3) WT D2R coexpressed with D114A-Gqi5, (5) WT D2R coexpressed with D2/D4-Gqi5 in the presence of the selective D4 antagonist, L745,870. Activation data are normalized to that of WT D2R coexpressed with WT D2R-Gqi5, which is indicated by a dotted line to indicate the potential range of enhanced and reduced signaling achievable by modulation of the “heterodimer” partner.

We used an aequorin assay that couples Gq (or Gqi5) activation to a luminescence readout. (a) The agonist quinpirole did not lead to D2R-induced Gq activation. (b) D2R when coexpressed with free Gqi5 or (c) D2R fused with Gqi5 via a linker (D2-linker-Gqi5) led to quinpirole-induced luminescence. (c) A nonfunctional Gα deficient fusion construct, D2-linker-Gqi5_G208A failed to produce luminescence. (d) Free Gqi5 rescued the function of D2-linker-Gqi5_G208A. (e) Free Gqi5 failed to rescue the function of non-linker D2R-Gqi5, which unlike D2R-linker-Gqi5, did not signal when expressed alone. (f) Coexpressing D2R with D2R-Gqi5 (12 hour tetracycline induction) restored signaling, despite the inability of either construct to signal in this assay when expressed alone. Activation data represent luminescence relative to that seen with 0.1% triton treatment. The mean±SEM of at least 3 experiments, each conducted in triplicate, are shown. The symbols used in (Fig. 1–Fig. 4 and Fig. 6) are explained in detail in (Supplementary Fig. 2 online).

(a,b) When all mutants (as protomer A) were coexpressed with WT D2R-Gqi5 (as protomer B), only (a) WT and D2/D4 were able to signal. (b) None of the other mutants were able to restore signaling when coexpressed with WT D2R-Gqi5. (c,d,e) The results differed when WT D2R (as protomer A) was coexpressed with the various mutant-Gqi5 constructs (as protomer B). (c) D2/D4-Gqi5 (), (d) D114^3.32A-Gqi5 (), deletion 213–219-Gqi5 (), and D80^2.50A-Gqi5 () restored the ability of unfused WT D2R to signal. (e) Coexpressing R132^3.50A -Gqi5 (), V136^3.54D/M140^3.58E-Gqi5 (), or N393^7.49A-Gqi5 () with WT D2R failed to rescue signaling (d). Note that D114A-Gqi5 () and D2/D4-Gqi5 () showed a higher maximal activation than WT. Activation data represent relative luminescence when compared to WT D2R coexpressed with WT D2R-Gqi5 after normalizing for surface expression of the Gqi5 fusion construct (see Methods). The mean±SEM of at least 3 experiments, each conducted in triplicate, are shown.

(a) Structural representation of the nonameric oligomer array; the dashed box identifies the TM4 dimer contained in Model 2. (b) Structural representation of the complex formed between transducin and the nonameric oligomer array. The optimal representative structure (defined in Methods) is shown for Model 2. (c) Closeup view of the interaction between specific residues of Gα (rendered in red, CPK representation) and the IL3 (cyan) and IL2 loops of protomer A and B (magenta and blue, respectively). (d) Side view of the complex showing Gtα (red), Gtβ (wheat), Gtγ (orange), protomer A (green), protomer B (light blue), IL2 of protomer A (magenta), IL2 of protomer B (blue), and IL3 of protomer B (cyan). Other views of the model complex are shown in (Supplementary Fig. 10 online).