Molecular cloning and expression of cDNA encoding the murine gonadotropin-releasing hormone receptor.
The primary structure of the gonadotropin-releasing hormone (GnRH) receptor was determined by sequencing a functional receptor cDNA isolated by expression cloning from an immortalized murine gonadotroph (alpha T3) cell line. Positive clone pools from a cDNA library were detected by screening expressed RNA in aequorin-injected Xenopus laevis oocytes, in which receptor-mediated calcium responses were monitored as light emission during stimulation by GnRH. The isolated receptor cDNA encodes a 327-amino acid protein that has seven putative transmembrane regions and is unique among G protein-coupled receptors in that the predicted sequence lacks a carboxyl-terminal cytoplasmic domain. COS-7 cells transfected with the receptor cDNA expressed high affinity binding sites for GnRH and its agonist and antagonist analogs and exhibited calcium responses to GnRH stimulation. These, and the prominent calcium responses of Xenopus oocytes injected with receptor RNA, were inhibited by GnRH antagonists. Northern blot analysis revealed two mRNAs (1.6 and 3.5 kilobases) in alpha T3 cells and in the mouse pituitary gland, and both transcripts were shown to encode functional GnRH receptors when expressed in Xenopus oocytes. In contrast, a single 4.6-kilobase receptor mRNA was present in rat anterior pituitary gland, ovary, and Leydig cells. The absence of a carboxyl-terminal cytoplasmic domain indicates the importance of other regions of the GnRH receptor in agonist-induced signal transduction, and possibly in receptor desensitization and sequestration.
Reinhart
J
J
Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
Mertz
L M
LM
Catt
K J
KJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA; D015870:Gene Expression; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D011966:Receptors, LHRH / Q000235:genetics*; D014982:Xenopus laevis
D012333:RNA, Messenger; D011966:Receptors, LHRH; D004247:DNA