Expression of human growth hormone-releasing factor in transgenic mice results in increased somatic growth

The neurohumoral regulation of growth hormone secretion is mediated in part by two hypothalamic peptides that reach the anterior pituitary via the hypothalamo-hypophysial portal blood system. Somatostatin inhibits the release of growth hormone, whereas growth hormone-releasing factor (GRF) positively regulates both growth hormone synthesis and secretion. Two forms of human GRF, 40 and 44 amino acids long, have been characterized from extra-hypothalamic tumours as well as from the hypothalamus. Analysis of human GRF complementary DNA and genomic clones indicates that the GRF peptides are first synthesized as a 107- or 108-amino-acid precursor protein. To examine the physiological consequences of GRF expression, we have established strains of transgenic mice containing a fusion gene including the promoter/regulatory region of the mouse metallothionein-I (MT-I) gene and the coding region of the human GRF gene. We report that expression of the human GRF precursor protein in these animals results in measurable levels of human GRF and increased levels of mouse growth hormone in plasma and accelerated growth rates relative to control littermates. These results demonstrate a direct role for GRF in the positive regulation of somatic growth. Unexpectedly, female transgenic mice carrying the MT-GRF fusion gene are fertile, in contrast to female transgenic mice expressing human or rat growth hormone, which are generally infertile. These transgenic mouse strains should provide useful animal models for the study of several types of human growth disorders.     

Growth induced by pulsatile infusion of an amidated fragment of human growth hormone releasing factor in normal and GHRF-deficient rats

The discovery of human pancreatic growth hormone releasing factors (GHRFs) and subsequent characterization of human hypothalamic GHRF has led to studies on the role of these peptides in stimulating growth hormone (GH) release, and attempts to use GHRF peptides to increase growth rates in short children are already underway. However, there is no experimental evidence in animals that exogenous GHRF promotes growth in vivo. Although anaesthetized rats release GH reproducibly in response to GHRF injections, the responses in conscious male rats are much more variable, perhaps because of their highly episodic endogenous GH secretory pattern. In contrast, female rats secrete GH in a more continuous pattern and respond reproducibly to repeated injections of GHRF. We report here that it is possible to establish a 'male' type of GH secretory pattern in normal female rats by long-term pulsatile intravenous (i.v.) infusions of the active human GHRF fragment GHRF (1-29)NH2. We found that this treatment accelerates growth and increases pituitary GH content, whereas continuous infusions of this GHRF fragment at the same daily dose are ineffective. Pulsatile, but not continuous GHRF also stimulates growth in animals made GHRF-deficient by neonatal monosodium glutamate treatment. Thus exogenous GHRF will stimulate growth in both GHRF-deficient and normal animals provided it is administered in an appropriate pattern.     

cDNA sequence and chromosomal localization of human platelet-derived growth factor A-chain and its expression in tumour cell lines

The amino-acid sequence of the precursor of the human tumour cell line-derived platelet-derived growth factor (PDGF) A-chain has been deduced from complementary DNA clones and the gene localized to chromosome 7. The protein shows extensive homology to the PDGF B-chain precursor. Expression of the PDGF A-chain gene is independent of that of the PDGF B-chain in a number of human tumour cell lines, and secretion of a PDGF-like growth factor of relative molecular mass 31,000 correlates with expression of A- but not B-chain messenger RNA.     

Ghrelin induces adiposity in rodents

The discovery of the peptide hormone ghrelin, an endogenous ligand for the growth hormone secretagogue (GHS) receptor, yielded the surprising result that the principal site of ghrelin synthesis is the stomach and not the hypothalamus. Although ghrelin is likely to regulate pituitary growth hormone (GH) secretion along with GH-releasing hormone and somatostatin, GHS receptors have also been identified on hypothalamic neurons and in the brainstem. Apart from potential paracrine effects, ghrelin may thus offer an endocrine link between stomach, hypothalamus and pituitary, suggesting an involvement in regulation of energy balance. Here we show that peripheral daily administration of ghrelin caused weight gain by reducing fat utilization in mice and rats. Intracerebroventricular administration of ghrelin generated a dose-dependent increase in food intake and body weight. Rat serum ghrelin concentrations were increased by fasting and were reduced by re-feeding or oral glucose administration, but not by water ingestion. We propose that ghrelin, in addition to its role in regulating GH secretion, signals the hypothalamus when an increase in metabolic efficiency is necessary.     

Structure of a genomic clone encoding biologically active human relaxin

Relaxin is a peptide hormone synthesized in the corpora lutea of ovaries during pregnancy and is released into the blood stream prior to parturition. Its major biological effect is to remodel the mammalian reproductive tract to facilitate the birth process. Determination of the structure of human relaxin is thus a first step in opening up the possibility of clinical intervention in cases of difficult labour. However, the limited availability of human ovaries during pregnancy has prevented both direct amino acid sequence determination and isolation of cDNA clones obtained from relaxin producing tissue. Our approach has therefore been to screen directly for a human relaxin gene using an homologous porcine relaxin cDNA probe. We report here the successful identification of a genomic clone from which the structure of the entire coding region of a human preprorelaxin gene has been determined. Synthesis of biologically active relaxin has shown that the novel gene structure described herein codes for an authentic human relaxin. We believe this is the first successful synthesis of a biologically active hormone whose structure was predicted solely from the structure of a genomic clone.     

Growth hormone receptor and serum binding protein: purification, cloning and expression

A putative growth hormone receptor from rabbit liver and the growth hormone binding protein from rabbit serum have the same amino-terminal amino-acid sequence, indicating that the binding protein corresponds to the extracellular hormone-binding domain of the liver receptor. The complete amino-acid sequences derived from complementary DNA clones encoding the putative human and rabbit growth hormone receptors are not similar to other known proteins, demonstrating a new class of transmembrane receptors.     

Cloning and characterization of the cDNAs for human and rat corticotropin releasing factor-binding proteins.

Corticotropin-releasing factor (CRF), is a potent stimulator of synthesis and secretion of preopiomelanocortin-derived peptides. Although CRF concentrations in the human peripheral circulation are normally low, they increase throughout pregnancy and fall rapidly after parturition. Maternal plasma CRF probably originates from the placenta, which responds to the bioactive peptide and produces the peptide and its messenger RNA. Even though CRF concentrations in late gestational maternal plasma are similar to those in rat hypothalamic portal blood and to those that can stimulate release of adrenocorticotropic hormone (ACTH) in vitro, maternal plasma ACTH concentrations increase only slightly with advancing gestation and remain within the normal range. Several groups have now reported the existence of a CRF-binding protein in human plasma which inactivates CRF and which has been proposed to prevent inappropriate pituitary-adrenal stimulation in pregnancy. The binding protein was recently purified from human plasma. We have now isolated and partially sequenced the binding protein, allowing us to clone and characterize its complementary DNA from human liver and rat brain. Expression of the cDNAs for human and rat binding protein in COS7 cells showed that these proteins bind CRF with the same affinity as the native human protein. Both rat and human recombinant binding proteins inhibit CRF binding to a CRF antibody and inhibit CRF-induced ACTH release by pituitary cells in vitro.     

Molecular cloning of the receptor for human antidiuretic hormone.

Antidiuresis, the recovery of water from the lumen of the renal collecting tubule, is regulated by the hypothalamic release of antidiuretic hormone (ADH), which binds to specific receptors on renal collecting tubule cells, stimulates adenylyl cyclase and promotes the cyclic AMP-mediated incorporation of water pores into the luminal surface of these cells. We report here the isolation of the human ADH receptor gene using a genomic expression cloning approach. The gene was used to clone the complementary DNA from a human renal library. The deduced amino-acid sequence of the receptor yields a hydropathy profile characteristic of receptors with seven putative transmembrane regions. This and the comparison with other cloned receptors indicates that the ADH receptor is a member of the superfamily of G-protein-coupled receptors.     

Somatostatin selectively inhibits noradrenaline release from hypothalamic neurones

Somatostatin in a hypothalamic peptide hormone which inhibits growth hormone release from the anterior pituitary. However, biochemical and morphological investigations have revealed that somatostatin is located not only in the hypothalamus but also in other brain areas (for example the cerebral cortex) where it occurs and in nerve cell bodies and fibres from which it can be released in a Ca2+-dependent manner. It has therefore been suggested that the neuropeptide may have functions in the central nervous system other than its effect on growth hormone release; one possible action is that of a neuromodulator. Therefore, hypothalamic and cerebral cortical slices of the rat were used to examine whether somatostatin modifies the electrically or CaCl2-evoked release of tritiated monoamines from monoaminergic neurones. it is reported here that somatostatin inhibits 3H-noradrenaline release from the hypothalamus (but not from the cerebral cortex) but does not affect the release of 3H-dopamine and 3H-serotonin.     

Release of somatostatin-28(1-12) from rat hypothalamus in vitro

Following the discovery of the growth hormone release-inhibiting factor somatostatin from extracts of ovine hypothalamus, an N-terminally extended somatostatin of 28 amino acids has been identified in mammalian tissue. The original peptide, somatostatin-14 (SS14), corresponds to the C-terminus of somatostatin-28 (SS28). Both SS28 and SS14 have biological activity, occur in several rat brain regions, are present in cell bodies and nerve terminals and can be released in vitro upon depolarization in a calcium-dependent manner. Further, high-affinity binding sites were described for SS14, which also bind SS28 (refs 20-23). Recently, a dodecapeptide which corresponds to the N-terminus of somatostatin-28, somatostatin-28(1-12), has been characterized in rat hypothalamus. Radioimmunological and immunohistochemical studies have indicated the presence of SS28(1-12)-like immunoreactivity in several cortical and subcortical regions of the rat brain. This peptide was found to be unevenly distributed with the highest concentration in the hypothalamus, and preferentially localized to dendritic and axonal processes and terminals. These observations suggest that SS28(1-12) may be a neurotransmitter. In this study, we describe a calcium-dependent release of a SS28(1-12)-like peptide from hypothalamic slices in vitro. This finding supports a neurotransmitter function for this peptide.     

Primary structure of the precursor to the three major surface antigens of Plasmodium falciparum merozoites

Recently, a class of protein antigens of high relative molecular mass (Mt) which can induce protective immunity against blood-stage malaria has been identified. In Plasmodium falciparum the protein has a Mr of approximately 195,000 (P195). It is the precursor of three proteins of Mr 83,000 (83K), 42K and 19K which are the major surface antigens of merozoites; thus it may also be useful for immunization against P. falciparum. Three studies describing the isolation of single short complementary DNA clones for part of the P195 gene sequence have been reported. Here we describe the complete structure of the P195 gene determined from further DNA clones, its organization within genomic DNA and the location of the specific processing fragments within the primary amino-acid sequence.     

A prolactin-inhibiting factor within the precursor for human gonadotropin-releasing hormone

The cloned complementary DNA sequence encoding the human gonadotropin-releasing hormone (GnRH) precursor protein was used to construct an expression vector for the bacterial synthesis of the 56-amino acid GnRH-associated peptide (GAP). GAP was found to be a potent inhibitor of prolactin secretion and to stimulate the release of gonadotropins in rat pituitary cell cultures. Active immunization with peptides corresponding to GAP sequences led to greatly increased prolactin secretion in rabbits.     

Immunocytochemical localization in rat brain of a prolactin release-inhibiting sequence of gonadotropin-releasing hormone prohormone

The structure of a precursor protein for gonadotropin-releasing hormone (GnRH) of relative molecular mass 10,000 has recently been deduced from cloned complementary DNA sequences derived from human placental messenger RNA. The 56-amino-acid peptide representing residues 14-69 of this prohormone exhibits potent inhibition of prolactin secretion. To investigate whether the same prohormone is synthesized in mammalian brain and describe the anatomical distribution of the prolactin-inhibiting region of this molecule, we have generated antiserum to a synthetic peptide containing residues 40-53 of the human placental precursor. We report here that a substance recognized by this antibody is present in GnRH-containing neurones of the rat brain and appears to coexist with GnRH in secretory granules of nerve terminals in the median eminence. These results indicate homology between hypothalamic and placental prohormones for GnRH and are consistent with the suggestion elsewhere in this issue that a prolactin-inhibiting factor (PIF) is generated from this prohormone and cosecreted with GnRH by nerve terminals in the median eminence.     

Binding sites for growth hormone releasing factor on rat anterior pituitary cells

Growth hormone releasing factors (GRFs) have been isolated from human pancreatic tumours (hGRF) and rat hypothalamus (rhGRF). The response to GRF at the pituitary level can be modulated by other factors, including glucocorticoids, thyroid hormones, somatostatin and other neuropeptides and somatomedins. Glucocorticoids enhance GRF-induced growth hormone (GH) secretion in primary cultures of rat anterior pituitary cells, and the synthetic glucocorticoid dexamethasone has recently been shown to increase the amounts of GH released in freely moving rats in response to submaximal doses of intravenous GRF. To investigate whether somatotroph sensitivity to GRF is modulated at its receptor level, we have developed a radioreceptor assay using an iodinated analogue of hGRF as radioligand. We report here that the relative binding affinities of rGRF, hGRF and the two analogues are correlated with their in vitro biological potencies. Further, the number of GRF binding sites is drastically decreased in cells deprived of glucocorticoids either in vivo or in vitro.     

Molecular cloning and expression of cDNA for human granulocyte colony-stimulating factor

Granulocyte colony-stimulating factor (G-CSF) is a member of the CSF family of hormone-like glycoproteins that regulate haematopoietic cell proliferation and differentiation, and G-CSF almost exclusively stimulates the colony formation of granulocytes from committed precursor cells in semi-solid agar culture. Recently, Nomura et al. have established a human squamous carcinoma cell line (designated CHU-2) from a human oral cavity tumour which produces large quantities of CSF constitutively, and the CSF produced by CHU-2 cells has been purified to homogeneity from the conditioned medium. We have now determined the partial amino-acid sequence of the purified G-CSF protein, and by using oligonucleotides as probes, have isolated several clones containing G-CSF complementary DNA from the cDNA library prepared with messenger RNA from CHU-2 cells. The complete nucleotide sequences of two of these cDNAs were determined and the expression of the cDNA in monkey COS cells gave rise to a protein showing authentic G-CSF activity. Furthermore, Southern hybridization analysis of DNA from normal leukocytes and CHU-2 cells suggests that the human genome contains only one gene for G-CSF and that some rearrangement has occurred within one of the alleles of the G-CSF gene in CHU-2 cells.     

Identical V beta T-cell receptor genes used in alloreactive cytotoxic and antigen plus I-A specific helper T cells

T lymphocytes involved in the cellular immune response carry cell-surface receptors responsible for antigen and self recognition. This T-cell receptor molecule is a heterodimeric protein consisting of disulphide-linked alpha- and beta-chains with variable (V) and constant (C) regions. Several complementary DNA and genomic DNA clones have been isolated and characterized. These analyses showed that the genomic arrangement and rearrangement of T-cell receptor genes using VT, diversity (DT), joining (JT) and CT gene segments is very similar to the structure of the known immunoglobulin genes. We have isolated two cDNA clones from an allospecific cytotoxic T cell, one of which shows a productive V beta-J beta-C beta 1 rearrangement without an intervening D beta segment. This V beta gene segment is identical to the V beta gene expressed in a helper T-cell clone specific for chicken red blood cells and H-21. The other clone carries the C beta 2 gene of the T-cell receptor, but the C beta 2 sequence is preceded by a DNA sequence that does not show any similarity to V beta or J beta sequences.     

Ghrelin is a growth-hormone-releasing acylated peptide from stomach

Small synthetic molecules called growth-hormone secretagogues (GHSs) stimulate the release of growth hormone (GH) from the pituitary. They act through GHS-R, a G-protein-coupled receptor for which the ligand is unknown. Recent cloning of GHS-R strongly suggests that an endogenous ligand for the receptor does exist and that there is a mechanism for regulating GH release that is distinct from its regulation by hypothalamic growth-hormone-releasing hormone (GHRH). We now report the purification and identification in rat stomach of an endogenous ligand specific for GHS-R. The purified ligand is a peptide of 28 amino acids, in which the serine 3 residue is n-octanoylated. The acylated peptide specifically releases GH both in vivo and in vitro, and O-n-octanoylation at serine 3 is essential for the activity. We designate the GH-releasing peptide 'ghrelin' (ghre is the Proto-Indo-European root of the word 'grow'). Human ghrelin is homologous to rat ghrelin apart from two amino acids. The occurrence of ghrelin in both rat and human indicates that GH release from the pituitary may be regulated not only by hypothalamic GHRH, but also by ghrelin.