Figure 7. The non-functional MT2-P95L mutant interferes with MT1/MT2 heteromer formation and blocks the effects of melatonin injection on the dark-adapted ERGs in the mouse photoreceptors.
(A, B) Coimmunoprecipitation of MT2-P95L-YFP with Flag-MT1 (A) or MT2-Rluc8 (B) receptors in HEK293T cells (upper right panel). (C) BRET donor saturation curves were performed by coexpressing equivalent amounts of MT1-Rluc8, MT2-Rluc8 or MT2-P95L-Rluc8 together with increasing quantities of MT2-P95L-YFP in HEK 293T cells. (D) Competition of the BRET between MT1-Rluc8 and MT2-P95L-YFP by increasing concentration of coexpressed Myc-MT1. Expression of the Myc-MT2-P95L mutant was determined by western blot. (E) MT2-P95L-YFP competes for MT1/MT2 heteromer formation as monitored by coimmunoprecipitation (upper middle panel). (F) Quantification of the dark-adapted response of the a- (upper graph) and b-wave (lower graph) to flashes of light recorded in MT2-P95L mutant mice after 1 h of dark adaptation and i.p. injection of melatonin (1 mg/kg) or vehicle in the middle of the day. Data are presented as mean ± SEM; n = 5–8 for each group. Melatonin injection induced no significant changes in the amplitude of the a- and b-wave (two-way ANOVA, P > 0.1). Coimmunoprecipitation data are representative of two further experiments. BRET data are obtained from 3–5 independent experiments, * P < 0.05; ** P < 0.01. Ab, antibody cross-reactivity. Anti-GFP, anti-Rluc, anti-Flag, antibodies recognizing GFP, Rluc or the Flag epitope, respectively. Luminance is expressed in candela-seconds per meter squared (cd*s/m2). ERGs in this experiment were performed at ZT 6.