Microvesicle-mediated release of soluble LH/hCG receptor (LHCGR) from transfected cells and placenta explants
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RESEARCH
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Microvesicle-mediated release of soluble LH/hCG receptor (LHCGR) from transfected cells and placenta explants Anne E Chambers1, Paul F Stanley2, Harpal Randeva3 and Subhasis Banerjee1*
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. Background 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). The hormonal activation occurs through specific ligand-receptor interactions on the surface of the target cells. The LH and hCG utilize a common receptor encoded by a single copy ~70 kb LHCGR gene, located at human chromosome 2p21 [1]. LHCGR has 11 exons and codes for multiple alternatively spliced species (at least 6) of mRNA. Transcriptional activation to generate these mRNAs is initiated at multiple sites spanning a region more than a kilobase upstream of the first exon [2]. Alternatively spliced mRNAs produce several truncated proteins which have the ligand binding capacity but are ineffective in transducing signals [3-7]. In addition to testis, ovary and placenta, various isoforms of LHCGR are expressed in uterine myometrium, vascular endothelial and smooth muscle cells, adrenals,
* Correspondence: [email protected] 1 Department of Clinical Biochemistry, Laboratory Medicine, Birmingham Heartlands Hospital, Bordesley Green East, Birmingham B9 5SS, UK Full list of author information is available at the end of the article
brain, skin, lymphocytes, human sperm, macrophages and in fetal tissues [[8], for a review]. The earliest biochemical evidence on the existence of cell-free soluble LH receptor was the purification of an hCG-binding protein, relative molecular mass of 65K (Mr, 65K) from porcine follicular fluid and was based on gel filtration followed by affinity chromatography [9]. A different experimental approach by West and Cooke [10] demonstrated the secretion of Mr 80-90K hCG-LH receptor complex into the culture media from ligand-induced rat and mouse leydig cells.
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