Isolation of the gene and hypothalamic cDNA for the common precursor of gonadotropin-releasing hormone and prolactin release-inhibiting factor in human and rat.

Abstract

Cloned cDNAs encoding the precursor protein for gonadotropin-releasing hormone (Gn-RH) and prolactin release-inhibiting factor (PIF) were isolated from libraries derived from human and rat hypothalamic mRNA. Nucleotide sequence analyses predict precursor proteins of 92 amino acids for both species and show identity between the human placental and human hypothalamic precursor proteins. Whereas the Gn-RH peptide structure is completely conserved in human and rat, the PIF domain of the precursor displays 70% interspecies homology. Genomic analyses revealed the presence of a single Gn-RH-PIF gene in human and rat containing sequences corresponding to the cDNA distributed across four exons.The decapeptide gonadotropin-releasing hormone (Gn-RH), also termed luteinizing hormone-releasing hormone (LH-RH), is a key molecule in the control of mammalian reproduction (1, 2). The peptide is secreted in a pulsatile manner from hypothalamic neurons into the capillary plexus of the median eminence to elicit the episodic release of gonadotropins from the anterior pituitary. In addition to the role of Gn-RH on pituitary gonadotrophs, Gn-RH or Gn-RH-like immunoreactivity has been found in extrahypothalamic regions of the central nervous system (3), gonads (4), mammary glands (5), and placenta (6), indicating that the decapeptide serves a variety of other functions essential for reproduction.In an attempt to study the biosynthesis of Gn-RH we recently reported the use of oligonucleotide probes to isolate a recombinant X phage carrying human genomic DNA that contained coding sequence for Gn-RH and the subsequent use of this DNA in the isolation of cloned human placental cDNA encoding a 92 amino acid precursor protein for Gn-RH (7). The placental protein, whose structure was solely deduced from nucleotide sequencing, contains an enzymatic processing site that separates the Gn-RH structure from a 56 amino acid sequence initially termed GAP for "Gn-RHassociated peptide." Immunocytochemical studies revealed antigenic determinants of this peptide in rat brain. In fact, such antigenic determinants were found by us in Gn-RH containing neurons only and coexisted with Gn-RH in secretory granules of nerve terminals in the median eminence (8).The 56 amino acid peptide synthesized by genetically programed Escherichia coli effected gonadotropin release similar to Gn-RH and possessed potent prolactin secretion inhibitory action when tested in vitro by using primary rat anterior pituitary cell cultures (9). Active immunization with synthetic peptides derived from the 56 amino acid sequence led to greatly increased serum levels of prolactin in rabbits (9). These findings identified the 56 amino acid sequence as a dominant prolactin secretion-inhibitory factor (PIF) and demonstrated that the hypothalamic control of gonadotropin and prolactin secretion is coupled through the synthesis of a common precursor for Gn-RH and PIF by neurosecretory cells (9). This concept assumes that the same precursor protein is biosynthesized in placenta and hypo...

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