Figure 5 In vitro characterization of the Y290F and T269M GNRHR mutations. A: Competitive binding curves for the Y290F and T269M GNRHR mutants compared.

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Figure 5 In vitro characterization of the Y290F and T269M GNRHR mutations. A: Competitive binding curves for the Y290F and T269M GNRHR mutants compared to the wildtype (WT) receptor in transiently transfected COS-7 cells (fibroblast-like cell line derived from monkey kidney). Cells were incubated with a <sup>125</sup>I-labeled GnRH agonist in the presence of graded concentrations of unlabeled GnRH. Binding was reduced with the Y290F mutant and completely abolished with the T269M mutant. Data are means and range of a single representative experiment performed at least three times in duplicate. B: Inositol phosphate (IP) accumulation in transiently transfected COS-7 cells, showing impaired or abolished response with the Y290F and T269M GnRHR mutants. Data are means and range of a single representative experiment performed at least three times in duplicate. C: Serum responsive element (SRE)-coupled luciferase concentration-response curves in transiently transfected HEK293T cells (derived from human embryonic kidney). Luciferase activity (arbitrary units by luminometry; a.l.u.) are shown as a ratio of beta-galactosidase activity (optical density) used as internal control for transfection efficiency. SRE activity was reduced with the Y290F mutant and absent with the T269M mutant. This experiment was performed four times in triplicate. D: ERK1/2 phosphorylation by western blot in transiently transfected HEK293T cells. After transfection, cells were treated with 10 nM GnRH, 10% fetal bovine serum (FBS) or vehicle, for 10 min. ERK phosphorylation was reduced and absent with the Y290F and T269M mutants, respectively. Data are representative of three experiments. For more details see Materials and Methods and Results and Supplementary Data. From: Similarities and differences in the reproductive phenotypes of women with congenital hypogonadotrophic hypogonadism caused by GNRHR mutations and women with polycystic ovary syndrome Hum Reprod. Published online November 24, 2018. doi:10.1093/humrep/dey339 Hum Reprod | © The Author(s) 2018. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.comThis article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) 1