Figure 3.

Determination of the Gα_i/o isoforms utilise by RXFP4 receptors using PTX‐resistant Gα mutants. cAMP inhibition and ERK1/2 activation following stimulation by mINSL5 in CHO‐RXFP4 cells. RXFP4 receptors inhibit forskolin‐stimulated cAMP accumulation, indicating an interaction with Gα_i/o proteins. CHO‐RXFP4 cells were transiently transfected with pcDNA3 (mock transfection) in (A, G) or G_i/o subunit constructs carrying the C351I mutation in (B, H) mGα_oA, (C, I) mGα_oB, (D, J) mGα_i1, (E, K) mGα_i2 or (F, L) mGα_i3. In the upper panels, cells were incubated with PTX (100 ng•mL⁻¹) for 16 h, and cAMP was production stimulated by forskolin (3 μM) followed by treatment with mINSL5 (100 nM). Results are normalized to the cAMP response stimulated by forskolin. In the lower panels, the ERK1/2 response was measured in cells incubated with PTX (100 ng•mL⁻¹) for 16 h followed by treatment with mINSL5 (100 nM). Results are expressed as percentage of response elicited by 10% FBS. Bars represent mean ± SEM of independent experiments [cAMP: n = 6 (mGα_oA, mGα_i3), n = 7 (pcDNA3, mGα_oB, mGα_i1, mGα_i2); ERK1/2: n = 5]. *P < 0.05; significantly different from the forskolin response (cAMP inhibition) or the baseline response (ERK1/2 activation) in PTX‐treated cells; repeated‐measures two‐way ANOVA followed by Dunnett's multiple comparisons test. ns, non‐significant.