title: Metabolism of pro-luteinizing hormone-releasing hormone in immortalized hypothalamic neurons.
abstract: An immortalized hypothalamic neuronal cell line was recently developed by genetically targeting the expression of the simian virus-40 large T-antigen in LHRH neurons. These GT1 cells were subcloned to GT1-1, GT1-3, and GT1-7 cells, and they have been shown to express the mRNA for pro-LHRH and secrete LHRH-like immunoreactive (IR) materials into the media. The purpose of our study was to biochemically and immunologically characterize the IR materials within and secreted from these cells. Both LHRH- and GnRH-associated peptide (GAP)-like IR materials were present and were secreted from these four cell lines. Up to 3% of the total cellular protein was composed of LHRH and GAP materials. When materials from the cell lysate and media were separated according to mol wt (Mr), at least three different pro-LHRH species were detected. These precursors contained both LHRH- and GAP-like IR determinants, and they eluted in the void volume and at approximately 10,000-12,000 and 8,400-8,500 Mr. A material that contained GAP-like IR eluted at approximately 6,500-6,800 Mr. This species is probably mouse GAP-(1-56) because it eluted on a reverse phase column in the approximate position of rat GAP-(1-56). Cell lysates contained a single LHRH-like IR form which coeluted on a size-exclusion column with synthetic LHRH. This material stimulated secretion of LH from anterior pituitary cells in a dose-response manner. By comparison, two different molecular forms of LHRH were detected in media at approximately 1,500 and 540 Mr. HPLC analyses revealed these peaks to be heterogeneous and to contain at least (Gln1)LHRH-(Gly11,Lys12,Arg13), (Gln1)LHRH-(Gly1,Lys12), LHRH-(Gly11), and LHRH. These experiments demonstrate that the cells contain and secrete multiple molecular forms of the pro-LHRH and that processing of the prohormone must involve 1) cleavage by an endopeptidase to give GAP-(1-56) and a C-terminally extended LHRH, 2) removal of C-terminal basic amino acids by a carboxypeptidase, 3) amidation of LHRH-(Gly11) to LHRH, and 4) cyclization of glutamine to pyroglutamate at the N-terminal of LHRH. These results provide the first evidence for intermediates in the metabolic pathway of pro-LHRH to LHRH.
authors.affiliation: Reproductive Neuroendocrinology Section, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
authors.forename: W C
authors.initials: WC
authors.lastname: Wetsel
authors.forename: P L
authors.initials: PL
authors.lastname: Mellon
authors.forename: R I
authors.initials: RI
authors.lastname: Weiner
authors.forename: A
authors.initials: A
authors.lastname: Negro-Vilar
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D002460:Cell Line; D002999:Clone Cells; D000939:Epitopes / Q000235:genetics; D015870:Gene Expression; D007987:Gonadotropin-Releasing Hormone / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007031:Hypothalamus / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism*; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D012689:Sequence Homology, Nucleic Acid; D013539:Simian virus 40 / Q000235:genetics / Q000276:immunology; D014162:Transfection
chemical_list: D000952:Antigens, Polyomavirus Transforming; D000939:Epitopes; D011498:Protein Precursors; D012333:RNA, Messenger; C050866:progonadoliberin I; D007987:Gonadotropin-Releasing Hormone