Fig. 2

Results of in vitro analyses of MC4R variant p.Met215Ile - a) cell surface expression, b) cAMP accumulation, C) MAPK/ERK assay HEK293 cells for cAMP accumulation and MAP kinase determination (b, c) and COS-7 cells for cell surface ELISA (a) were transfected as indicated in the Methods section. a Cell surface ELISA with N-terminally HA tagged receptors show a slight reduction in cell surface expression compared to the wild-type. The result of five independent experiments performed in sextuplicate is shown. Data represents mean ± SEM. A test with Welsh correction was performed for statistical analysis comparing wild-type to p.215I. b cAMP accumulation after stimulation with increasing amounts of NDP-α-MSH and α-MSH stimulation indicate a loss of maximal stimulation of p.M215I. EC50 values for alpha-MSH induced signaling for wt-MC4R and MC4R-M215I are 30 nM and 33 nM, respectively and for NDP-α-MSH induced signaling 1.6 nM and 0.7 nM. The result of four independent experiments performed in triplicate is shown. Data were calculated as fold level over basal stimulation and are indicated as mean ± SEM. The statistical difference in maximal stimulation was calculated by a t-test with Welsh correction. c: MAP kinase signalling was determined using an SRE-luciferase reporter gene assay after stimulation with increasing amounts of NDP-α-MSH and α-MSH stimulation. M215 resulted in a complete loss-of-function for stimulation with NDP-α-MSH and α-MSH α-MSH so that calculation of EC50 values is impossible. EC50 value for wt-MC4R after α-MSH or NDP-α-MSH challenge are 229 nM and 4.2 nM, respectively. The result of four independent experiments performed in triplicate is shown. Data were calculated as fold level over basal stimulation and are indicated as mean ± SEM. The statistical difference in maximal stimulation was calculated by a t-test with Welsh correction