A204E GHSR1a agonist-independent signal transduction via the CRE pathway in transiently transfected HEK293 cells. (A) Constitutive activity of GHSR1a as a function of its own cell-surface expression. The constitutive activity associated with increasing concentrations of WT or A204E GHSR1a vectors was assessed in cotransfection experiments with an HA-tagged GHSR1a plasmid (HA-GHSR-WT or HA-GHSR-A204E) and a CRE-containing reporter plasmid (pPOU1F1-Luc). The corresponding luminometric (RLU) signal was normalized to protein concentration (RLU/μg proteins). The HA-GHSR1a cell-surface expression detected by means of an anti-HA epitope antibody (ELISA) is expressed in OD units. Signals associated with cells transfected with the mock plasmid have been subtracted, so that plotted signals represent specific RLU. Values are the mean ± SD of 1 representative experiment performed in 4 replicates among 3 independent experiments. (B) Evaluation of a putative dominant-negative effect of the A204E mutant on the WT GHSR1a. Constitutive activity in cells expressing the GHSR1a WT, the GHSR1a A204E mutant, or both isoforms is expressed as a percentage of the basal activity of the WT GHSR1a receptor. Values are the mean ± SD of 3 independent experiments, each performed in triplicate.