PRL and GH are hormones with a wide spectrum of actions. Specific receptors are widely distributed in a number of classical target organs, but other tissues that are not known targets also contain measurable binding sites or receptor mRNA. The most likely explanation is that PRL and GH cause effects that have not yet been characterized in certain tissues. Cloning of the cDNAs encoding PRL and GH receptors has led to the discovery that the receptors, like the hormones themselves, form a gene family. Multiple receptor forms have been identified, including a short form, which for PRL is a membrane-bound receptor or for GH is a soluble BP, and a long form, which for both PRL and GH is a membrane-bound receptor. PRL and GH receptors, and the mRNAs encoding them, can be up- and down-regulated. GH induces an up-regulation of both GH and PRL receptors, whereas PRL stimulates an increase of only its own receptor. High concentrations of either hormone induce a homologous down-regulation of receptor expression. An assay has been developed to measure the functional activity of different forms of PRL receptor by cotransfecting a milk protein fusion gene specific to PRL coupled to a reporter-gene along with the cDNA of the PRL receptor. Although the short form represents the major form present in rat mammary gland, only the long form of receptor is able to stimulate milk protein gene transcription. For GH, increased expression of the receptor in some target cells is accompanied by a modest enhancement of the response to GH. No single second messenger mediating the action of either PRL or GH has been identified. Several potential components of the signal transduction pathways have been identified, but as yet none has clearly been shown to be able to mimic the effect of PRL or GH. Because of the wide range of biological actions associated with PRL, and the existence of various forms of PRL receptors, it is doubtful that one unifying mechanism of action will be found for this hormone. No human or animal model of a genetic defect of the PRL receptor has thus far been published. Mutations in the GH receptor gene have been demonstrated in Laron type dwarfism. Different exon deletions or point or nonsense mutations resulting in modifications in the extracellular, GH binding region of the GH receptor have been reported.(ABSTRACT TRUNCATED AT 400 WORDS)

Laron dwarfism is associated with resistance to growth hormone (GH). To investigate its genetic basis, we used genetic linkage to test whether the disorder results from a defect in the gene for the human GH receptor. Denaturing gradient gel electrophoresis and sequencing of specific GH-receptor-gene fragments allowed us to characterize specific intragenic DNA markers in 35 control subjects of Mediterranean descent, for use in linkage studies. In two Mediterranean families in which the parents were consanguineous and some of the children had Laron dwarfism, the disease trait and DNA polymorphisms were inherited together. Moreover, an analysis of the GH-receptor-gene RNA transcripts in lymphocytes from one of these families allowed us to identify a thymidine-to-cytosine substitution that generated a serine in place of a phenylalanine at position 96 in the extracellular coding domain of the mature protein. This defect probably affects the receptor adversely and is probably responsible for the lack of plasma GH-binding activity in the patients. This mutation was not found in the GH-receptor genomic sequences of seven unrelated subjects with Laron dwarfism who belonged to different population groups. An analysis of the GH-receptor markers in these patients indicated that different gene frameworks (polymorphic sites within the single gene) were associated with the mutant alleles. We conclude that Laron dwarfism is due to abnormalities in the gene for GH receptor, which may differ from family to family.

The GH receptor (GHR) is a member of the cytokine receptor family. Short isoforms resulting from alternative splicing have been reported for a number of proteins in this family. RT-PCR experiments, in human liver and cultured IM-9 cells, using primers in exon 7 and 10 of the GHR, revealed three bands reflecting alternative splicing of GHR mRNA: the predicted product at 453 bp and two other products at 427 and 383 bp. The 427-bp product (GHR1-279) utilized an alternative 3'-acceptor splice site 26 bp downstream in exon 9; the predicted C-terminal residues are six frameshifted exon 9 codons ending in an inframe stop codon. The 383-bp product (GHR1-277) resulted from skipping of exon 9; the predicted C-terminal residues are three frame-shifted exon 10 codons ending in an in-frame stop codon. RNase protection experiments confirmed the presence of the GHR1-279 variant in IM-9 cells and human liver. The proportion of alternative splice to full length was 1-10% for GHR1-279 and less than 1% for GHR1-277. The function of GHR1-279 was examined after subcloning in an expression vector and transient transfection in 293 cells. Scatchard analysis of competition curves for [125l]-hGH bound to cells transfected either with GHR full length (GHRfl) or GHR1-279 revealed a 2-fold reduced affinity and 6-fold increased number of binding sites for GHR1-279. The increased expression of GHR1-279 was confirmed by cross-linking studies. The media of cells transfected with GHR1-279 contained 20-fold more GH-binding protein (GHBP) than that found in the media of cells transfected with the full-length receptor. Immunoprecipitation and Western blotting experiments, using a combination of antibodies directed against extracellular and intracellular GHR epitopes, demonstrated that GHRfl and GHR1-279 can form heterodimers and that the two forms also generate a 60-kDa GHBP similar in size to the GHBP in human serum. Functional tests using a reporter gene, containing Stat5-binding elements, confirmed that while the variant form was inactive by itself, it could inhibit the function of the full-length receptor. We have demonstrated the presence of a splice variant of the GHR in human liver encoding a short form of the receptor similar in size to a protein previously identified in human liver and choroid plexus. Expression studies in 293 cells support the hypothesis that while the expression of the splice variant accounts for only a small proportion of the total GHR transcript, it produces a short isoform that modulates the function of the full-length receptor, inhibits signaling, and generates large amounts of GHBP. The differential expression of GHR receptor short forms may regulate the production of GHBP, and truncated receptors may act as transport proteins or negative regulators of GHR signaling.

Previous evidence indicates that GH modulates thymic cell migration. In this study we approached this issue in vivo, studying thymocyte migration in GH transgenic animals and in normal mice treated intrathymically with GH. Extracellular matrix and chemokines are involved in thymocyte migration. In this respect, thymocyte adhesion to laminin was higher in GH-treated animals than controls, and the numbers of migrating cells in laminin-coated Transwells was higher in GH-transgenic and GH-injected mice. Additionally, CXC chemokine ligand 12 (CXCL12)-driven migration was higher in GH-Tg and GH-treated animals compared with controls. Interestingly, although CXCR4 expression on thymocytes did not change in GH-Tg mice, the CXCL12 intrathymic contents were higher. We found that CXCL12, in conjunction with laminin, would additionally enhance the migration of thymocytes previously exposed to high concentrations of GH in vivo. Lastly, there was an augmentation of recent thymic emigrants in lymph nodes from GH-Tg and GH-injected animals. In conclusion, enhanced thymocyte migration in GH transgenic mice as well as GH-injected mice results at least partially from a combined action of laminin and CXCL12. Considering that GH is presently being used as an adjuvant therapeutic agent in immunodeficiencies, including AIDS, the concepts defined herein provide important background knowledge for future GH-based immune interventions.

A technique using high pressure liquid chromatography gel filtration was used to evaluate GH-binding proteins (BP) in human plasma; eluate was monitored for radioactivity in a gamma-detection system connected to a computer. Plasma (200 microL) was incubated with [125I]human (h) GH (200,000 cpm) at 4 C for 20 h. The main GH-BP (peak II) was well separated from free [125I]hGH (peak III) and from a higher mol wt complex (peak I), which was minor. In our control plasma, the specific binding of [125I]hGH to peak II BP (II-BP) was 32.2 +/- 0.6% of the radioactivity. Scatchard analyses indicate an association constant of 3.6-7.4 X 10(8) M-1 and a binding capacity ranging from 24-86 ng/mL for peak II-BP in five normal adult plasma samples. Peak I material, separated from plasma of boys with pubertal delay, bound hGH with a low affinity (3 x 10(6) M-1) and a very high capacity (2 micrograms/mL). In cross-linking experiments, peak I appeared as two proteins of 165 and 174 kD; these mol wt were much higher than that of peak II-BP, previously estimated at 53,000. hGH complexed to peak II-BP remained fully immunoreactive with use of the anti-hGH antibodies of our assay. In plasma containing 10-20 micrograms/L hGH, the proportion of bound hormone (peak II) was 44.5 +/- 2.3%, whereas the amount of hGH in peak I was very low or undetectable. Specific binding of hGH to II-BP was lowest during the first year of life and highest in adulthood. No sex difference was found. I-BP is differentially regulated, since its binding activity was significantly lower in adults than in prepubertal children. Normal values for age should be taken into account to interpret GH-binding activity, particularly in children 2 yr of age or younger. Our GH binding assay offers important gains in terms of rapidity and resolution; it has permitted a clear separation and characterization of the two GH-binding components present in human plasma.

The GH receptor (GHR) is a member of the cytokine/hematopoietic growth factor family, and protein tyrosine phosphorylation has been implicated in the signaling cascade of these receptors. It was recently shown that the tyrosine kinase JAK2 is associated with the GHR. GH induces the activation of JAK2, which phosphorylates itself and the receptor. Mitogen-activated protein (MAP) kinase activation and transcriptional stimulation of specific genes, such as Spi 2.1, have also been reported to be induced by GH. To identify functionally important regions in the cytoplasmic domain of the GHR, we compared the actions of the wild-type receptor, two truncated mutants, and one internal deletion mutant (similar to the intermediate Nb2 form of the PRL receptor) in transfectants of the Chinese hamster ovary cell line. A region of 46 amino acids adjacent to the membrane was found to be sufficient for activation of both JAK2 and MAP kinases. This region contains a proline-rich sequence (box 1) conserved in the cytokine receptor family that is important for signal transduction. For transcriptional activity, the C-terminal region of the GHR is required, and we found that the last 80 terminal residues contain sequences allowing activation of the Spi 2.1 promoter. Tyrosine phosphorylation of the receptor also requires the C-terminal portion of the GHR cytoplasmic domain, and we found that GHR tyrosine phosphorylation appears to be linked to activation of the Spi 2.1 transcription pathway. Thus, the GHR could be composed of at least 2 functional regions: the 46 proximal amino acids required for activation of JAK2 and sufficient to stimulate the MAP kinase pathway, and an additional carboxy-terminal region necessary for transcriptional activation.

Increasing evidence has placed hormones and neuropeptides among potent immunomodulators, in both health and disease. Herein, we focus on the effects of growth hormone (GH) upon the thymus. Exogenous GH enhances thymic microenvironmental cell-derived secretory products such as cytokines and thymic hormones. Moreover, GH increases thymic epithelial cell (TEC) proliferation in vitro, and exhibits a synergistic effect with anti-CD3 in stimulating thymocyte proliferation, which is in keeping with the data showing that transgenic mice overexpressing GH or GH-releasing hormone exhibit overgrowth of the thymus. GH also influences thymocyte traffic: it increases human T-cell progenitor engraftment into the thymus; augments TEC/thymocyte adhesion and the traffic of thymocytes in the lymphoepithelial complexes, the thymic nurse cells; modulates in vivo the homing of recent thymic emigrants, enhancing the numbers of fluroscein isothiocyanate (FITC) cells in the lymph nodes and diminishing them in the spleen. In keeping with the effects of GH upon thymic cells is the detection of GH receptors in both TEC and thymocytes. Additionally, data indicate that insulin-like growth factor (IGF)-1 is involved in several effects of GH in the thymus, including the modulation of thymulin secretion, TEC proliferation as well as thymocyte/TEC adhesion. This is in keeping with the demonstration of IGF-1 production and expression of IGF-1 by TEC and thymocytes. Also, it should be envisioned as an intrathymic circuitry, involving not only IGF-1, but also GH itself, as intrathymic GH expression is seen both in TEC and in thymocytes, and that thymocyte-derived GH could enhance thymocyte proliferation. Finally, the possibility that GH improve thymic functions, including thymocyte proliferation and migration, places this molecule as a potential therapeutic adjuvant in immunodeficiency conditions associated with thymocyte decrease and loss of peripheral T cells. The crosstalk between the neuroendocrine and the immune systems is now well-demonstrated. Similar ligands and receptors used by these systems allow the existence of a physiological intra-and intersystem communication circuitry, which seems to play a relevant role in homeostasis. Increasing evidence has placed hormones and neuropeptides among potent immunomodulators, in both health and disease (reviewed in [1±4]). More particularly, these molecules modulate the physiology of the thymus [5]. Herein, we focus on the effects of growth hormone (GH) upon the microenvironmental and the lymphoid compartments of the thymus. Nevertheless, before discussing such effects, it seems worthwhile to provide background data concerning the immunomodulatory function of GH, as well as the general features regarding the thymic microenvironment and its role upon intrathymic T-cell differentiation.

A survey to identify children and adolescents with primary growth hormone insensitivity syndrome (GHIS) yielded 38 patients who were positively identified using a scoring system that included five criteria: height, basal growth hormone (GH), GH binding protein, basal insulin‐like growth factor 1 (1GF‐I) and the increase of IGF‐I after 4 days of GH administration (IGF generation test). Because of an overlap of the accepted and excluded groups with respect to points scored, an attempt was made to improve the scoring system. The new criteria were: height below –3 SDS, basal GH 4 mU/I or above, GH binding below 10%, basal IGF‐I and basal IGF binding protein‐3 (IGFBP‐3) below the 0.1 centile for age, an increase of IGF‐I in the IGF generation test less than 15 μg/1, and the increase of IGFBP‐3 less than 0.4 mg/1. With this scoring system, a clear separation between the accepted and the excluded groups was obtained. IGFBP‐3 was included to give the GH‐dependent parameters of the IGF system more weight and because the accuracy of IGFBP‐3 in the IGF generation tests was greater than the accuracy of IGF‐I, when the group of patients with GHIS was compared with a group of patients with GH deficiency. Unexpectedly, the IGF generation test was unable to segregate both cohorts completely. In the GHIS‐positive group, a significant correlation was found between basal IGF‐I or IGFBP‐3 levels corrected for age (SDS) and height SDS (r= 0.49, p < 0.002 and r= 0.61, p < 0.0001, respectively). There was also a significant correlation between the changes of IGF‐I or IGFBP‐3 in the IGF generation test and height SDS. That is, the patients with a slight response to GH were those with the least growth retardation, suggesting the existence of partial GH insensitivity.