[논문]Functional characterization of naturally-occurring constitutively activating/inactivating mutations in equine follicle-stimulating hormone receptor

Byambaragchaa, Munkhzaya; Ahn, Tae-Young; Choi, Seung-Hee; Kang, Myung-Hwa; Min, Kwan-Sik

doi:10.5713/ab.21.0246

Functional characterization of naturally-occurring constitutively activating/inactivating mutations in equine follicle-stimulating hormone receptor 원문보기

Animal bioscience, v.35 no.3, 2022년, pp.399 - 409

Byambaragchaa, Munkhzaya (Animal Biotechnology, Graduate School of Future Convergence Technology, Hankyong National University) , Ahn, Tae-Young (Animal Biotechnology, Graduate School of Future Convergence Technology, Hankyong National University) , Choi, Seung-Hee (Animal Biotechnology, Graduate School of Future Convergence Technology, Hankyong National University) , Kang, Myung-Hwa (Department of Food Science and Nutrition, Hoseo University) , Min, Kwan-Sik (Animal Biotechnology, Graduate School of Future Convergence Technology, Hankyong National University)

Abstract ▼ AI-Helper

Objective: Follicle-stimulating hormone (FSH) is the central hormone involved in mammalian reproduction, maturation at puberty, and gamete production that mediates its function by control of follicle growth and function. The present study investigated the mutations involved in the regulation of FSH receptor (FSHR) activation. Methods: We analyzed seven naturally-occurring mutations that were previously reported in human FSHR (hFSHR), in the context of equine FSHR (eFSHR); these include one constitutively activation variant, one allelic variant, and five inactivating variants. These mutations were introduced into wild-type eFSHR (eFSHR-wt) sequence to generate mutants that were designated as eFSHR-D566G, -A306T, -A189V, -N191I, -R572C, -A574V, and -R633H. Mutants were transfected into PathHunter EA-parental CHO-K1 cells expressing β-arrestin. The biological function of mutants was analyzed by quantitating cAMP accumulation in cells incubated with increasing concentrations of FSH. Results: Cells expressing eFSHR-D566G exhibited an 8.6-fold increase in basal cAMP response, as compared to that in eFSHR-wt. The allelic variation mutant eFSHR-A306T was not found to affect the basal cAMP response or half maximal effective concentration (EC50) levels. On the other hand, eFSHR-D566G and eFSHR-A306T displayed a 1.5- and 1.4-fold increase in the maximal response, respectively. Signal transduction was found to be completely impaired in case of the inactivating mutants eFSHR-A189V, -R572C, and -A574V. When compared with eFSHR-wt, eFSHR-N191I displayed a 5.4-fold decrease in the EC50 levels (3,910 ng/mL) and a 2.3-fold decrease in the maximal response. In contrast, cells expressing eFSHR-R633H displayed in a similar manner to that of the cells expressing the eFSHR-wt on signal transduction and maximal response. Conclusion: The activating mutant eFSHR-D566G greatly enhanced the signal transduction in response to FSH, in the absence of agonist treatment. We suggest that the state of activation of the eFSHR can modulate its basal cAMP accumulation.

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표/그림 (6)

그림 Figure 1. Schematic representation of the eFSHR structure. The location of the constitutively activating mutation (D566G), the five inactivating mutations (A189V, N191I, R572C, A574V, and R633H), and the allelic variant (A306T) are indicated in the structure of eFSHR. Red circles indicate the constitutively activating mutation and the allelic variant, while green circles indicate the inactivating mutations. Two inactivating mutations (A189V and N191I) and the allelic variant (A306T) are located in the EC domain. The activating mutation (D566G) is located in the third IC loop. The other two inactivating mutations (R572C and A574V) are located between the third IC loop region and the six TM. R633H is located in the intraplasmic tail region. eFSHR, equine follicle-stimulating hormone receptor; EC, extracellular domain; TM, transmembrane domain; IC, intracellular domain.
그림 Figure 2. Total cAMP levels stimulated by FSH in PathHunter EA-parental CHO-K1 cells transfected with constitutively active and allelic variant eFSHR mutants. PathHunter EA-parental CHO-K1 cells transiently transfected with wild-type and mutant (D566G and A306T) eFSHRs were stimulated with FSH in a medium containing 0.5 mM 3-isobutyl-1-methyl xanthine for 30 min. cAMP production was detected using homogeneous time-resolved fluorescence assay. The cAMP accumulation is represented as Delta F%. cAMP concentration was recalculated and presented using GraphPad Prism software. The results of the mock-transfected cells subtracted from each data set (see Methods). Each point represents the average±standard error of the mean of triplicate experiments. The mean data were fitted to the equation for a one-phase exponential decay curve. The blank circles were the same curves of wild-type receptor. FSH, equine follicle-stimulating hormone; eFSHR, equine follicle-stimulating hormone receptor.
표 Table 1. Bioactivity of eFSH receptors in cells expressing wild-type as well as activating and allelic mutations
그림 Figure 3. cAMP production, stimulated by FSH treatment, in PathHunter EA-parental CHO-K1 cells transfected with inactivating eFSHR mutants. PathHunter EA-parental CHO-K1 cells transiently transfected with wild-type eFSHR and eFSHR inactivating mutants (eFSHR-A189V, -N191I, -R572C, -A574V, and -R633H) were stimulated with FSH for 30 min. Total cAMP accumulation was analyzed using a homogeneous time-resolved fluorescence assay. The empty circles denote wild-type eFSHR and black circles denote the mutants. The values for the mock-transfected cells subtracted from each data point. Each point represents the average±standard error of the mean of triplicate experiments. The mean data were fitted to the equation for a one-phase exponential decay curve. FSH, equine follicle-stimulating hormone; eFSHR, equine follicle-stimulating hormone receptor.
그림 Figure 4. Determination of cAMP production in PathHunter parental CHO-K1 cells transiently expressing the wild-type and mutant eFSHRs. cAMP production in cells expressing the wild-type and mutant eFSHRs. (A) Basal accumulation in the absence of agonist treatment; (B) Rmax levels among eFSHR-wt and eFSHR mutants with activating mutation D566G and allelic variant A306T, and (C) Rmax levels among eFSHR-wt and eFSHR inactivating mutants. Data have been expressed as mean±standard error of the mean of triplicate experiments. eFSHR, equine follicle-stimulating hormone receptor. Values with different superscripts were significantly different (p<0.05).
표 Table 2. Bioactivity of eFSH receptors in cells expressing inactivating receptor mutations

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