Fig. 4.

Gain-of-function of the DRD3 Gly-9 variant. (A) Dopamine inhibited surface DRD3 binding in a biphasic manner, as measured on live HEK293 cells stably transfected; eight and nine independent clones for the Ser-9 and Gly-9 variants, respectively, were compared. Results are mean ± SEM of data obtained in three independent experiments, expressed as percentage of total specific binding. (B) The maximal inhibition by dopamine of forskolin (FSK)-stimulated cAMP accumulation in HEK293 cells stably transfected with either variant (two independent clones for each variant). Results are mean ± SEM of data obtained in four independent experiments, expressed as percentage of control cAMP accumulation measured in the absence of dopamine. ∗, P < 0.01 by the two-tailed t test. (C) Dopamine-induced activation of the MAPK (p42 and p44) was assessed in HEK293 cells transiently transfected with either variant. Levels of activated MAPK were detected by a monoclonal antibody raised against the phosphorylated forms of MAPK. Membranes were stripped and then reprobed with an anti-MAPK antibody to detect total MAPK loaded in gel. Serum-starved cells were stimulated by the indicated doses of dopamine for 5 min. (D) Serum-starved cells were stimulated by 1 μM dopamine for the indicated times. Blots are representative of four independent experiments, and quantifications were performed by densitometric analysis. ∗, P < 0.05 by the two-tailed t test.