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, 21 (2), 111-120

Internalization of Rat FSH and LH/CG Receptors by rec-eCG in CHO-K1 Cells

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Internalization of Rat FSH and LH/CG Receptors by rec-eCG in CHO-K1 Cells

Jong-Ju Park et al. Dev Reprod.

Abstract

Equine chorionic gonadotropin (eCG) is a unique molecule that elicits the response characteristics of both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in other species. Previous studies from this laboratory had demonstrated that recombinant eCG (rec-eCG) from Chinese hamster ovary (CHO-K1) cells exhibited both FSH- and LH-like activity in rat granulosa and Leydig cells. In this study, we analyzed receptor internalization through rec-eCGs, wild type eCG (eCGβ/α) and mutant eCG (eCGβ/αΔ56) with an N-linked oligosaccharide at Asn56 of the α-subunit. Both the rec-eCGs were obtained from CHO-K1 cells. The agonist activation of receptors was analyzed by measuring stimulation time and concentrations of rec-eCGs. Internalization values in the stably selected rat follicle-stimulating hormone receptor (rFSHR) and rat luteinizing/chorionic gonadotropin receptor (rLH/CGR) were highest at 50 min after stimulation with 10 ng of rec-eCGβ/α. The dose-dependent response was highest when 10 ng of rec-eCGβ/α was used. The deglycosylated eCGβ/αΔ56 mutant did not enhance the agonist-stimulated internalization. We concluded that the state of activation of rFSHR and rLH/CGR could be modulated through agonist-stimulated internalization. Our results suggested that the eLH/CGRs are mostly internalized within 60 min by agonist-stimulation by rec-eCG. We also suggested that the lack of responsiveness of the deglycosylated eCGβ/ αΔ56 was likely because the site of glycosylation played a pivotal role in agonist-stimulated internalization in cells expressing rFSHR and rLH/CGR.

Keywords: Deglycosylated mutants; Internalization; rFSHR; rLH/CGR; rec-eCGβ/α.

Figures

Fig. 1
Fig. 1. Fig. 1. Schematic diagram of rec-eCGβ/α and receCGβ/ αΔ56.
The eCG cDNA was ligated into the pcDNA3 mammalian expression vector. Wild type (eCGβ/α) and eCGβ/αΔ56 (deglycosylated at Asn56 in the α-subunit replaced with Gln) were constructed.
Fig. 2
Fig. 2. Selection of cells expressing rFSHR and rLH/ CGR from stably transfected CHO-K1 cells.
Cloned cells were cultured at concentrations of 105-106 cells/mL. Cholera toxin (100 ng/mL) was added, incubated, and cyclic AMP was analyzed using a cAMP kit.
Fig. 3
Fig. 3. Internalization results for cells expressing rFSHR and rLH/CGR.
(A) Internalization results for time-dependent (0–70 min) at a fixed rec-eCGβ/α concentration (10 ng/mL) in rFSHR and rLH/CGR. (B) Internalization results for agonist-dependent (1–20 ng/mL). The rLH/CGR cells (wt-9) and FSHR cells (wt-7) cells were seeded at a density of 2×103 cells. The results were visualized using the In Cell Analyzer 3,000 device and the granularity of the receptors internalized into the endosomes was calculated.
Fig. 4
Fig. 4. Visualization of internalization results with In Cell Analyzer 3000 device.
The cells were labeled with CypHer5 antibody and the agonist (rec-eCGβ/α) was added, as shown in Fig. 3. The Cy5-labeled receptors and the Hoechst 33342 were visualized using the In Cell Analyzer 3,000 device.
Fig. 5
Fig. 5. Internalization results of rec-eCGβ/α and rec-eCGβ/αΔ56 using cells expressing rFSHR and rLH/CGR.
(A) rFSHR cells. (B) rLH/CGR cells. Both rec-eCGβ/α and rec-eCGβ/αΔ56 were induced by stably transfected rFSHR and rLH/CGR cells. Internalization assays were analyzed in a rec-eCGβ/ α-dose-dependent manner, as described in Fig. 3.
Fig. 6
Fig. 6. Pathways of internalization, recycling, degradation and endocytosis of G protein-coupled receptors.
When receptor cells are exposed to a agonist, the responsiveness wanes with time, in spite of the continuous presence of the agonist. It called to as desensitization that occur at the level of the hormone receptor, post-receptor steps. There are two categories of regulatory events. Uncoupling is defined as a change in the functional properties of a constant number of receptors. The other one is down-regulation defining as a reduction in the density of cell surface receptors (Hipkin et al., 1993).

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