Abstract
Placental hCG and pitutary LH transduce signals in target tissues through a common receptor (LHCGR). We demonstrate that recombinant LHCGR proteins which include the hormone-binding domain are secreted from transfected cells and that natural LHCGR is also secreted from human placental explants. LHCGR recombinant proteins representing varying lengths of the N-terminal extracellular domain were expressed in Chinese Hamster Ovary cells in suspension culture. Secretion was minimal up to 72h but by 96h 24-37% of the LHCGR had been released into the culture medium. The secreted proteins were folded and sensitive to glycosidases suggesting N-linked glycosylation. Secretion was independent of recombinant size and was mediated via structurally defined membrane vesicles (50-150nm). Similarly cultured human early pregnancy placental explants also released LHCGR via microvesicles. These studies provide the first experimental evidence of the possible mechanistic basis of the secretion of LHCGR.
Highlights
Reproductive hormones]) are collectively known as gonadotropins because they stimulate gonads
Expression of LHCGR recombinant proteins in CHO cell suspension culture Chinese hamster ovary suspension (CHO-S) cells grown in serum-free medium at a cell density of 106 cells per ml were transfected with mock or with one of three LHCGR recombinants; LHCGR-229, LHCGR-291, LHCGR-318 (Figure 1a) and the cell density and cell viability were recorded for up to seven days post-transfection
Only those microvesicles derived from transfected cells reacted with anti-FLAG antibody. These experiments confirm that the LHCGR protein is integral to the microvesicles released into the culture supernatant for both recombinant transfectants and cultured placental explants and further show that only a fraction of the microvesicles released from the placenta contain LHCGR. In this manuscript, we demonstrate that the secretion of soluble LHCGR from transfected cells as well as from placental tissues is independent of the size of the receptor and is mediated via microvesicles
Summary
Reproductive hormones (luteinizing hormone [LH], follicle stimulating hormone [FSH] and human chorionic gonadotropin [hCG)]) are collectively known as gonadotropins because they stimulate gonads (testes in male and ovaries in female). Tsai-Morris et al [11] first reported the production and secretion of soluble LH receptor following transient transfection of a naturally truncated variant of the receptor in COS cells. These data were independently substantiated using different combinations of in vitro expression systems [12,13], In some studies, the lack of detection of an actively secreted soluble form of expressed LH receptor was attributed to misfolding and intra-cellular retention of the expressed protein or proteolytic degradation of the protein released into the culture media [13,14]. The lack of a secreted form of the receptor was overcome by co-expression of hCG with the extra-cellular domain of porcine LH receptor leading to the secretion of a mixture of free as well as hCG-receptor complex [13]
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