Characterization of [hydroxyproline9]luteinizing hormone-releasing hormone and its smallest precursor forms in immortalized luteinizing hormone-releasing hormone-secreting neurons (GT1-7), and evaluation of their mode of action on pituitary cells.
[Hydroxyproline9]luteinizing hormone-releasing hormone ([Hyp9]LHRH), an endogenous hydroxylated post-translational product of the LHRH sequence, has been isolated from mammalian hypothalamus. Using the LHRH-hypothalamic cell line (GT1-7) of fetal origin, we attempted to define the substrates available for the hydroxylation process during LHRH synthesis and to characterize immunologically the [Hyp9]LHRH and pro-[Hyp9]LHRH forms with anti-LHRH antibodies of different specificities after separation by HPLC. Their biological activity and mode of action were evaluated and compared to that of LHRH and LHRH intermediate precursors in normal pituitary cells and in a gonanodotrope cell line alpha T3-1. immunoreactivity was progressively increased in cells and media during cell culture. [Hyp9]LHRH and its two smallest precursor forms ([Hyp9]LHRH-(Gly11) and -(11-13)) were detected in cells and in media. They were simultaneously detected with the homologous LHRH molecular forms indicating that the hydroxylation occurs early in the processing of pro-LHRH. [Hyp9]LHRH-like molecules were more abundant than LHRH forms in media. This predominant release may thus represent a physiological process occurring during fetal life. Free acid forms of both decapeptides were detected only in cells. Furthermore, the results obtained suggest that conversion of Gln1 in pyroGlu1 occurs before or during processing into the hydroxylated or non-hydroxylated LHRH intermediate (11-13)-precursors. The biosynthetic pathway is thus common for both decapeptides and it is not altered by the hydroxylation process. LHRH and [Hyp9]LHRH shared the same receptor for their biological activity, as assessed by measuring luteinizing hormone release and activation of phospholipase C and A2. [Hyp9]LHRH was, however, less potent than LHRH.
Gautron
J P
JP
Unité de Dynamique des Systèmes Neuroendocriniens (U159) de l'INSERM Centre Paul Broca, Paris, France.
Poulin
B
B
Kordon
C
C
Drouva
S V
SV
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D002850:Chromatography, Gel; D000242:Cyclic AMP / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D007987:Gonadotropin-Releasing Hormone / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism* / Q000494:pharmacology; D007295:Inositol Phosphates / Q000378:metabolism; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism; D010902:Pituitary Gland / Q000187:drug effects*; D011498:Protein Precursors / Q000032:analysis / Q000378:metabolism* / Q000494:pharmacology; D051381:Rats; D010738:Type C Phospholipases / Q000378:metabolism
D007295:Inositol Phosphates; D011498:Protein Precursors; D007987:Gonadotropin-Releasing Hormone; C074293:LHRH, hydroxyprolyl(9)-; D000242:Cyclic AMP; D010741:Phospholipases A; D010738:Type C Phospholipases