Extended Data Fig. 4: Dose response analysis and expression levels of functional receptors on the plasma membrane.
From: The structure and function of the ghrelin receptor coding for drug actions
(a) Ca2+ mobilization assay for ghrelin receptor mutants. Wild-type and alanine mutants are shown in gray and black, respectively. The change in maximum [Ca2+]i upon the addition of each concentration of anamorelin was determined in quadruplicate (n = 4 wells). Symbols and error bars represent mean and standard error. A representative result from three independent experiments with similar results was presented. (b) Cell surface expression levels of wild-type and mutant ghrelin receptors. The wild-type and mutant receptors were fluorescently labeled through an N-terminal Flag tag on each receptor. The expression levels of the mutants were quantified by calculating the ratio of green fluorescence intensity to that of the wild type. The expression levels of each mutant were 50–145% of the wild type, except for L2105.36A, S2175.43A, and F3127.42A. Statistics: results are expressed as mean ± SD (One-way ANOVA followed by Dunnett multiple comparison test, P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; n = 3 wells). Adjusted P values in comparison with the wild-type receptor are as follows: <0.0001 for E124E, R283A, S123A, S217A, F309A, F312A, and L210A; 0.0001 for D99A; 0.0003 for N305A; 0.0009 for F286A; 0.0012 for Q120A; 0.0041 for L103A; 0.0400 for R102A; 0.0608 for L181A; 0.2281 for M213A; 0.9076 for I178A; >0.9999 for V214A.
