Supporting Information on Line Invertebrate Neuroscience

Supporting Information on line Invertebrate Neuroscience

Characterisation of AmphiAmR4, an amphioxus α2-adrenergic-like G-protein-coupled receptor.

By

Asha Bayliss and Peter D. Evans

Supplementary Figures S1-S4

Supplementary Figure Legends Bayliss and Evans

Fig. S1 Effect of biogenic amines (a) and synthetic agonists (b) on forskolin-stimulated cAMP levels in AmphiAmR4-expressing (black bars) and wild type CHO-K1 cells (open bars). Cells were pre-incubated with 100 µM IBMX for 20 min, followed by incubation with various agonists at 1 µM, 100 µM IBMX and 10 µM forskolin for a further 20 min. Data are expressed as the mean ± SEM. (A) n ≥ 3 and (B) n > 3.

Fig. S2 Time courses for the effect of the biogenic amines on ERK1/2 phosphorylation in AmphiAmR4-expressing CHO-K1 cells. AmphiAmR4-expressing CHO-K1 cells were serum-starved for 2 h, prior to stimulation with various biogenic amines (1 µM) for varying lengths of time. Equal quantities of cell lysates were separated by SDS-PAGE and analysed for p-ERK or t-ERK by Western blotting. (a) Representative blots in the presence and absence of 1µM noradrenaline, plus a 0.01% FBS control, from three to four independent experiments. (b) Summary of the quantified time course blots for the different amines tested. Data are expressed as the mean ± SEM. n = 3-4.

Fig. S3 The relative effectiveness of the biogenic amines, synthetic agonists and synthetic antagonists on ERK1/2 activation in Wild type CHO-K1 cells. Cells were serum-starved for 2 h, prior to stimulation with various biogenic amines (a and b at 1 μM) or synthetic agonists (c and d at 100 nM) for 5 min. (e and f) Cells were serum-starved for 2 h, including incubation with 1 µM of antagonist or F-12 Hams control for the final 10 min. Cells were then stimulated with 0.01 % FBS or F-12 Hams control in the presence of antagonist for 5 min. Basal measurements in the absence of antagonist are shown as 100%. Equal quantities of cell lysates were separated by SDS-PAGE and analysed for p-ERK or t-ERK by Western blotting. (a, c, and e) Representative blots from three to four independent experiments. (b, d, and f) Summaries of the quantified blots for each of the different experiments. Data are expressed as the mean ± SEM. n = 3-4. B, F-12 Hams-treated basal; NA, noradrenaline; Adr, adrenaline; DA, dopamine; OA, octopamine; Syn, synephrine; TA, tyramine; Phenyl, phenylethylamine; HA, histamine; Nap, naphazoline; Phe, phenylephrine; UK, UK14,304; Clo, clonidine; Iso, isoproterenol; 6-C, 6-Chloro-APB; Qui, quinpirole; Phent, phentolamine; WB, WB4101; Mia, mianserin; Pra, prazosin; Pro, propranolol; But, butaclamol; Spi, spiperone; Yoh, yohimbine; Rau, rauwolscine; Flu, flupenthixol; Met, metoclopramide.

Fig. S4 Effect of signalling inhibitors on noradrenaline-induced ERK1/2 activation in AmphiAmR4-expressing and wild type CHO-K1 cells. (a – h) AmphiAmR4-expressing (black bars) and wild type (open bars) CHO-K1 cells were serum-starved for 2 h including incubation with 10 µM of the indicated inhibitors (AG1478, AG1296, PP2, Ro 31-8220 or LY294002) or F-12 Hams control for the final 20 min. Cells were then stimulated for 5 min with 10 nM noradrenaline/0.01 % FBS or F-12 Hams control, as indicated. (a, c, e and g) Representative blots from three or four independent experiments. (b, d, f and h) Summary of the quantified blots. Data are expressed as the mean ± SEM. n = 3 - 4. B, F-12 Hams-treated basal; NA, noradrenaline; Ro, Ro 31-8220; LY, LY294002.

Bayliss and Evans Figure S1

Bayliss and Evans Figure S2

Bayliss and Evans Figure S3

(a) (c)

Bayliss and Evans Figure S4