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The growth hormone-releasing hormone receptor (GHRHR) is a member of the family of G protein-coupled receptors that is expressed on pituitary somatotrope cells and mediates the actions of GHRH in stimulating growth hormone (GH) synthesis and secretion. We report that the Ghrhr gene is located in the middle of mouse chromosome 6 in the same region as the little mutation. Mice homozygous for this mutation have reduced GH secretion and a dwarf phenotype. A missense mutation was identified in the extracellular domain of the little GHRHR that disrupts receptor function, suggesting that the growth deficit in these mice results from a defect in the GHRHR. Similar alterations in GHRHR might explain some isolated GH deficiencies in humans.

The pituitary gonadotropins FSH and LH are key hormones for regulating gametogenesis and steroidogenesis in the ovary and testis. The cell surface receptors that mediate the biological activities of these hormones are thought to be expressed in a cell-specific fashion in the ovary and are regulated as animals progress through the reproductive cycle. Using cloned receptor cDNAs, we have examined the expression and hormonal regulation of the ovarian FSH and LH receptor mRNAs in the rat. A quantitative reverse transcription-polymerase chain reaction amplification scheme was used to measure relative levels of the FSH and LH receptor mRNAs, while in situ hybridization was used to localize FSH and LH receptor transcripts. In immature animals, low levels of FSH receptor mRNA are observed in the granulosa cells of small follicles, while low levels of LH receptor mRNA are found in the thecal cells of these same follicles. After stimulation with PMSG, levels of both mRNAs increase, and the LH receptor mRNA is localized in both the granulosa and thecal cells of large follicles. Further treatment of PMSG-primed animals with hCG results in down-regulation, particularly of the LH receptor mRNA in granulosa cells. In adult animals, LH receptor mRNA levels change dramatically during the estrous cycle, particularly after the preovulatory LH surge. FSH receptor mRNA levels show a similar pattern of change, but the FSH receptor mRNA is of lower abundance and is not as highly regulated as the LH receptor mRNA. FSH receptor mRNA is confined to the granulosa cells of healthy developing follicles, whereas LH receptor mRNA is localized predominantly to thecal cells of small follicles on estrous morning, then appears in the granulosa cells of growing follicles by diestrous morning. LH receptor mRNA is also found in interstitial tissues and corpora lutea throughout much of the estrous cycle. Our results indicate that the gonadotropin receptor genes are regulated in a complex fashion during the recruitment, maturation, and ovulation of the ovarian follicle.

Cortisol treatment for 6 or 12 days had no effect on serum FSH in intact males and animals castrated for 1 or 7 days, but pituitary FSH was increased by the steroid in both intact and castrate groups. In contrast, cortisol inhibited serum LH in both intact and castrated animals while only increasing pituitary levels of LH in 7 day castrates. Cortisol also increased the mRNA for FSH beta without affecting alpha or LH beta mRNAs. These data suggest that the selective increase in pituitary content of FSH may be due to a selective increase in FSH beta mRNA following exposure to cortisol.

The molecular characterization of GHRH and the GHRH receptor provides a framework for understanding the hypothalamic regulation of pituitary somatotroph function. The signaling events discerned from our investigation of GHRH receptor structure and function form the basis of a model for GHRH action, which is shown in Fig. 20. GHRH interaction with its seven transmembrane domain Gs-coupled receptor on the somatotroph (step 1) leads to the release of growth hormone from secretory granules (step 2), which is likely to involve a G protein-mediated interaction with ion channels, and to a stimulation of intracellular cAMP accumulation (step 3) (Mayo, 1992; Lin et al., 1992; Gaylinn et al., 1993). In several cell types tested, elevated cAMP leads to the phosphorylation and activation of the transcription factor CREB by protein kinase A (Gonzalez and Montminy, 1989; Sheng et al., 1991), and one target gene for CREB action is the pituitary-specific transcription factor Pit-1 or GHF-1 (step 4) (Bodner et al., 1988; Ingraham et al., 1988; McCormick et al., 1990). Pit-1 is a prototypic POU domain protein that is required for the appropriate regulation of the growth hormone gene in somatotroph cells, thus providing a pathway by which a GHRH signal can lead to increased growth hormone synthesis in the pituitary (step 5). In addition, Pit-1 is likely to directly regulate the synthesis of the GHRH receptor (step 6), in that the receptor is not expressed in the pituitary of dw/dw mice that lack functional Pit-1 (Lin et al., 1992), and a cotransfected Pit-1 expression construct can activate the GHRH receptor promoter in transiently transfected CV1 cells (Lin et al., 1993). It remains to be determined whether additional direct regulation of the GHRH receptor gene in response to the cAMP signaling pathway occurs (step 7). The inhibitory peptide somatostatin presumably interacts with this same signaling pathway through G protein-mediated suppression of the cAMP pathway (Tallent and Reisine, 1992; Bell and Reisine, 1993). In agreement with the importance of this signaling system for normal growth, a transgene encoding a nonphosphorylatable mutant CREB protein, which blocks the function of the endogenous CREB protein, is able to cause somatotroph hypoplasia and dwarfism in mice when its expression is targeted to pituitary somatotrophs (Struthers et al., 1991). Several steps in the signaling pathway leading to growth hormone secretion are subject to disruption, resulting in growth hormone deficiency.(ABSTRACT TRUNCATED AT 400 WORDS)

The gonadotropins, luteinizing hormone (LH) and follicle stimulating hormone (FSH), play major roles in regulating the mammalian reproductive cycle by influencing the gametogenic and steroidogenic functions of the gonads (1, 2). The simultaneous release of LH and FSH from anterior pituitary gonadotrophs is known to be positively regulated by the hypothalamic decapeptide GnRH (3,4). More recently, several gonadal peptides that specifically modulate FSH synthesis and secretion have been isolated and characterized. These include the inhibins, activins, and follistatin (5-12).

To pursue questions concerning the regulation of somatic growth in a species amenable to both genetics and germ-line manipulation, we have isolated and characterized a full-length cDNA clone encoding mouse GH-releasing hormone (mGHRH). A GHRH cDNA clone isolated from a mouse placental library contains an open-reading frame of 309 basepairs that predicts a 103 amino acid mouse GHRH precursor protein. The mature mouse GHRH is predicted to be 42 amino acids with a free carboxyl-terminus. Although the mGHRH precursor sequence is clearly related to those determined for rat and human, the mature mGHRH peptide differs at seven of its 42 positions from all previously characterized GHRH peptides. RNA blot analysis of mouse tissues indicates that the mature 750 nucleotide mGHRH mRNA is found in hypothalamus and placenta, while testis contains a larger GHRH-related transcript. In situ hybridization analysis of GHRH gene expression in the mouse brain indicates that GHRH mRNA is localized predominantly to the arcuate nucleus of the hypothalamus. In the placenta, GHRH mRNA levels are developmentally regulated and peak on days 16-17 of gestation. GHRH mRNA is localized predominantly to trophoblast giant cells and to cytotrophoblasts of the placental labyrinth.