Expression of candidate pro-GnRH processing enzymes in rat hypothalamus and an immortalized hypothalamic neuronal cell line.
Since gonadotropin-releasing hormone (GnRH, also referred to as LHRH) is a major hormone regulating mammalian reproduction, identification of the processing steps involved in the conversion of the pro-LHRH to LHRH is fundamental to our understanding of its physiology. Extracts from immortalized LHRH neurons (GT1) were used to isolate the pro-LHRH intermediate products and to identify the enzymes which may participate in these conversions. The GT1 cells contain and secrete a pro-LHRH species that elutes at approximately 10,000-12,000 molecular weight. The pro-LHRH is metabolized to various N- and C-terminally modified LHRH products and to gonadotropin-releasing hormone-associated peptide (GAP). Analyses of these intermediates suggests that, at least, four different enzymatic steps are involved in pro-LHRH processing. Northern blot analyses reveal that prohormone convertase 2 (PC2), carboxypeptidase E, glutaminyl cyclase, and peptidyl-glycine alpha-amidating monooxygenase are expressed in the GT1 cells and rat hypothalamus. PC2 immunoreactivity is localized to the perikarya and beaded axon-like processes of these cells. SDS-PAGE analyses indicate that PC2 is biosynthesized, processed and secreted by the immortalized LHRH neurons. Our results indicate that the GT1 cell line may serve as a useful model to study the regulation of pro-LHRH processing and that it may also represent an important tool for dissecting the molecular and cellular basis of mammalian reproduction.
Wetsel
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WC
Laboratory of Cellular and Molecular Pharmacology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.
Liposits
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Seidah
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Collins
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D000217:Acyltransferases / Q000235:genetics; D000595:Amino Acid Sequence; D019881:Aminoacyltransferases; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D043423:Carboxypeptidase H; D002268:Carboxypeptidases / Q000235:genetics; D002461:Cell Line, Transformed; D015870:Gene Expression; D007987:Gonadotropin-Releasing Hormone / Q000737:chemistry / Q000378:metabolism*; D007031:Hypothalamus / Q000201:enzymology*; D008297:Male; D006899:Mixed Function Oxygenases / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes; D009474:Neurons / Q000201:enzymology*; D045706:Proprotein Convertase 2; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013381:Subtilisins / Q000032:analysis / Q000235:genetics / Q000378:metabolism
D009097:Multienzyme Complexes; D011498:Protein Precursors; D012333:RNA, Messenger; C050866:progonadoliberin I; D007987:Gonadotropin-Releasing Hormone; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D000217:Acyltransferases; D019881:Aminoacyltransferases; C059280:glutaminyl-peptide cyclotransferase; D002268:Carboxypeptidases; D043423:Carboxypeptidase H; D013381:Subtilisins; D045706:Proprotein Convertase 2