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. 2017 Aug 10;6:e26985. doi: 10.7554/eLife.26985

Figure 1. mGlu4 activates G protein in the mGlu2-4 heterodimer.

(A) Cartoons illustrating mGlu2 and mGlu4 homodimers, and mGlu2-4 (2–4 or 4–2) heterodimers with each subunit carrying the quality control C1 or C2 system as C terminal tails, and the indicated HA or Flag tag at their N terminus. (B) Quantification of cell surface expressed HA-tagged or Flag-tagged constructs by ELISA on intact cells transfected with the indicated subunits (2C1, 2C2, 4C1, 4C2) alone or together. Data are expressed as means ± SEM (n ≥ 3). **p<0.01, ***p<0.001 (unpaired t test). (C, D, E, F) Intracellular Ca2+ responses mediated by the indicated subunits upon stimulation with increasing concentrations of glutamate, in the presence of the chimeric Gqi9, with the control subunits (C), the mGlu2 homodimer with no, one or both subunits mutated (D), same with mGlu4 homodimer (E) or mGlu2-4 heterodimer (F). The red cross indicates the subunit carries the FS mutation that prevents G protein activation. Data are expressed as means ± SEM of triplicates from a typical experiment repeated at least three times.

Figure 1—source data 1. Glutamate potency at the indicated heterodimers.
Intracellular Ca2+ responses mediated by the indicated subunits upon stimulation with increasing concentrations of glutamate. 2X and 4X indicated subunits carrying the F756S (mGlu2) or F781S (mGlu4) mutation preventing G protein activation. Data represent the means ± SEM of (n) independent experiments. N.D., not determined.
elife-26985-fig1-data1.docx (63.5KB, docx)
DOI: 10.7554/eLife.26985.006

Figure 1.

Figure 1—figure supplement 1. G protein coupling of the mGlu subunits with C1 or C2 tail.

Figure 1—figure supplement 1.

Intracellular Ca2+ response mediated by indicated subunits co-expressed with the chimeric Gqi9 upon stimulation with DCG-IV (30 μM, green), L-AP4 (30 μM, red) or glutamate (1 mM, blue). Data are means ±SEM of triplicates from a typical experiment repeated at least three times.
Figure 1—figure supplement 2. Cell surface and total expression of various mGlu dimer combination.

Figure 1—figure supplement 2.

Quantification of cell surface-total expression of HA-tagged or Flag-tagged constructs (as indicated in Figure 1) by ELISA on intact cells transfected with indicated subunits. Data were obtained from a same cells used for with Figure 1D,E,F and expressed as means ± SEM of triplicates from a typical experiment repeated at least three times.
Figure 1—figure supplement 3. mGlu4 is responsible for G protein coupling in the mGlu2-4 heterodimer.

Figure 1—figure supplement 3.

(A) Intracellular Ca2+ responses mediated by indicated subunits upon stimulation with increasing concentration of glutamate, in the presence of the chimeric Gqi9. (B) Cartoons illustrating the receptor combinations used in panels C and D. (C) Intracellular Ca2+ response mediated by indicated subunits co-expressed with Gqi9 upon stimulation with DCG-IV (30 μM), L-AP4 (30 μM), or DCG-IV +L-AP4. (D) Same as in C with Gqo. Data are means ±SEM of triplicates from a typical experiment repeated at least three times.
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