Bushbaby growth hormone is much more similar to nonprimate growth hormones than to rhesus monkey and human growth hormones

Unlike other mammals, Old World primates have five growth hormone-like genes that are highly divergent at the amino acid level from the single growth hormone genes found in nonprimates. Additionally, there is a change in the interaction of growth hormone with its receptor in humans such that human growth hormone functions in nonprimates, whereas nonprimate growth hormone is ineffective in humans. A Southern blotting analysis of the genome of a prosimian, Galago senegalensis, revealed a single growth hormone locus. This single gene was PCR-amplified from genomic DNA and sequenced. It has a rate of nonsynonymous nucleotide substitution less than one fourth that of the human growth hormone gene, while the rates of synonymous substitution in the two species are less different. Human and rhesus monkey growth hormones exhibit variation at a number of amino acid residues that can affect receptor binding. The galago growth hormone is conservative at each of these sites, indicating that this growth hormone is functionally like nonprimate growth hormones. These observations indicate that the amplification and rapid divergence of primate growth hormones occurred after the separation of the higher primate lineage from the galago lineage.     

Episodic evolution of growth hormone in primates and emergence of the species specificity of human growth hormone receptor

Growth hormone (GH) evolution is very conservative among mammals, except for primates and ruminant artiodactyls. In fact, most known mammalian GH sequences differ from the inferred ancestral mammalian sequence by only a few amino acids. In contrast, the human GH sequence differs from the inferred ancestral sequence by 59 amino acids. However, it is not known when this rapid evolution of GH occurred during primate evolution or whether it was due to positive selection. Also, human growth hormone receptor (GHR) displays species specificity; i.e., it can interact only with human (or rhesus monkey) GH, not with nonprimate GHS: The species specificity of human GHR is largely due to the Leu-->Arg change at position 43, and it has been hypothesized that this change must have been preceded by the His-->Asp change at position 171 of GH. Is this hypothesis true? And when did these changes occur? To address the above issues, we sequenced GH and GHR genes in prosimians and simians. Our data supported the above hypothesis and revealed that the species specificity of human GHR actually emerged in the common ancestor of Old World primates, but the transitional phase still persists in New World monkeys. Our data showed that the rapid evolution of primate GH occurred during a relatively short period (in the common ancestor of higher primates) and that the rate of change was especially high at functionally important sites, suggesting positive selection. However, the nonsynonymous rate/synonymous rate ratio at these sites was <1, so relaxation of purifying selection might have played a role in the rapid evolution of the GH gene in simians, possibly as a result of multiple gene duplications. Similar to GH, GHR displayed an accelerated rate of evolution in primates. Our data revealed proportionally more amino acid replacements at the functionally important sites in both GH and GHR in simians but, surprisingly, showed few coincidental replacements of amino acids forming the same intermolecular contacts between the two proteins.     

Functional promiscuity of squirrel monkey growth hormone receptor toward both primate and nonprimate growth hormones

Primate growth hormone (GH) has evolved rapidly, having undergone approximately 30% amino acid substitutions from the inferred ancestral eutherian sequence. Nevertheless, human growth hormone (hGH) is physiologically effective when administered to nonprimate mammals. In contrast, its functional counterpart, the human growth hormone receptor (hGHR), has evolved species specificity so that it responds only to Old World primate GHs. It has been proposed that this species specificity of the hGHR is largely caused by the Leu --> Arg change at position 43 after a prior His --> Asp change at position 171 of the GH. Sequence analyses supported this hypothesis and revealed that the transitional phase in the GH:GHR coevolution still persists in New World monkeys. For example, although the GH of the squirrel monkey has the His --> Asp substitution at position 171, residue 43 of its GHR is a Leu, the nonprimate residue. If the squirrel monkey truly represents an intermediate stage of GH:GHR coevolution, its GHR should respond to both hGH and nonprimate GH. Also, if the emergence of species specificity was a result of the selection for a more efficient GH:GHR interaction, then changing residue 43 of the squirrel monkey growth hormone receptor (smGHR) to Arg should increase its binding affinity toward higher primate GH. To test these hypotheses, we performed protein-binding assays between the smGHR and both human and rat GHs, using the surface plasmon resonance methodology. Furthermore, the effects of reciprocal mutations at position 43 of human and squirrel monkey GHRs are measured for their binding affinities toward human and squirrel monkey GHs. The results from the binding kinetic assays clearly demonstrate that the smGHR is in the intermediate state of the evolution of species specificity. Interestingly, the altered residue Arg at position 43 of the smGHR does not lead to an increased binding affinity. The implications of these results on the evolution of the GH:GHR interaction and on functional evolution are discussed.     

Varying signals of the effects of natural selection during teleost growth hormone gene evolution.

The growth hormone (GH) gene of teleost fish exhibits a higher degree of variability compared with other vertebrate groups. However, the different selective constraints at the sequence level are not well understood. In this study, maximum-likelihood (ML) models of codon substitutions were used to investigate Darwinian adaptive evolution of the GH gene in teleost fishes. Complete GH gene sequences of 54 fish species were classified into 4 orders, and the variable nature of GH was examined by determining the dN and dS rate variation and the rates of molecular evolution for each teleost order. The results indicate that although the overall evolution rate for teleost GH is high ((1.15 +/- 0.01) x 10(-9) substitutions/(aa site x y)) compared with the "slow phases" in mammals ((0.21 to 0.28 +/- 0.05) x 10(-9)), the vital structure of this gene has been retained. While the majority of the amino acid changes appear to be due to relaxation of purifying selection, some positively selected sites were detected in regions with no specifically identified role in protein function. The positively selected regions observed in salmoniformes lineage suggests a possible role for positive selection driving functional divergence in paralogous forms of the GH gene after whole-genome duplication in this lineage.     

Influence of ovine growth hormone on water and NaCl absorption by the rat proximal jejunum and distal ileum

1. Ovine growth hormone appears to stimulate equally the absorption of water and NaCl in both the proximal jejunum and in the distal ileum. 2. Although the largest dose used of 0.8 mg/rat for 2 days produced greater increases in absorption rates than the lowest dose of 0.1 mg, a clear dose-related response was not obtained. 3. It is suggested that growth hormone may contribute significantly in the regulation of hydromineral metabolism in mammals at times other than during pregnancy and lactation.     

Molecular evolution of the avian growth hormone gene and comparison with its mammalian counterpart

The molecular evolution of all available avian growth hormone (GH) gene sequences was investigated using both maximum-likelihood and parsimony methods, and the patterns compared to those found in mammals. In contrast to the rapid bursts of evolution observed for mammalian GH, the evolutionary rate of the avian GH mature peptide appears to have been more constant. However several positively selected sites were identified at functionally important positions in the avian signal peptide by the site-specific likelihood method. This implies that sequence variation in the avian GH signal peptide may be adaptive, although more conservative parsimony methods failed to confirm this. Nevertheless, the differing patterns of avian and mammalian GH signal peptide molecular evolution are consistent with the apparently differing roles of GH in controlling growth in these taxonomic groups and support the hypothesis that signal peptide sequence variation may in fact be the basis for increased functional complexity.     

Study of goldfish (Carassius auratus) growth hormone structure-function relationship by domain swapping

Using goldfish as a model, the structure-function relationship of goldfish growth hormone was studied using the strategy of homologous domain swapping. Chimeric mutants were constructed by exchanging homologous regions between goldfish growth hormone (gfGH II) and goldfish prolactin (gfPRL) with their cloned complementary DNAs. Six mutants, with their domain-swapped, were generated to have different combinations of three target regions, including the helix a, helix d and the large section in between these helices (possess the helices b, c and other random coiled regions). After expression in E. coli and refolding, these mutants were characterized by using competitive receptor binding assay (RRA) and growth hormone responding promoter activation assay. The different activity profiles of mutants in Spi 2.1 gene promoter assays from that in RRA shows that, for gfGH, receptor binding dose not confer receptor signal activations. When either helices a or d of gfGH was maintained with other helices replaced by their gfPRL counterparts, both receptor binding and hence gene activation activities are reduced. In mutants with helices b and c in gfGH maintained, containing the gfGH middle section, and helices a and d swapped with gfPRL, the had reduced RRA activities but the promoter activation activities retained. In conclusion, as in the case of human GH, the gfGH molecule possesses two functional sites: one of them is composed of discontinuous epitopes located on the target regions of this study and is for receptor binding; another site is located on the middle section of the molecule that helices a and d are not involved, and it is for activation of GH receptor and intracellular signals.     

Functional heterodimerization of prolactin and growth hormone receptors by ovine placental lactogen

Although homo- or heterodimerization are common mechanisms for activation of cytokine receptors, cross-talk between two distinct receptors in this superfamily has been never shown. Here we show a physiologically relevant example indicating that such an interaction does occurs, thus raising the hypothesis that heterodimerization between distinct cytokine receptors may be a novel mechanism contributing to the diversity of cytokine signaling. These findings were documented using both surface plasmon resonance and gel filtration experiments and show that ovine placental lactogen (PL) heterodimerizes the extracellular domains (ECDs) of ruminant growth hormone receptor (GHR) and prolactin receptor (PRLR). We also show that PL or PL analogues that exhibit little or no activity in cells transfected with PRLRs and no activity in cells transfected with ovine GHRs exhibit largely enhanced activity in cells cotransfected with both PRLRs and GHRs. Furthermore, chimeric receptors consisting of cytosolic and transmembrane part of ovine GHR or ovine PRLR and ECDs of human granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) alpha or beta were constructed. Upon transfection into Chinese hamster ovary cells along with reporter luciferase gene and stimulation by GM-CSF, a significant increase in luciferase activity occurred when GM-CSFR-alpha-PRLR and GM-CSFR-beta-GHR or GM-CSFR-alpha-GHR and GM-CSRR-beta-PRLR were cotransfected. In conclusion, we show that ovine PL is capable of functional heterodimerization of GHR and PRLR and that when their cytosolic parts, coupled to the ECD of GM-CSF receptors, are heterodimerized by GM-CSF, they are capable of transducing biological signal.     

Prospects for a small molecule able to induce somatic growth through the growth hormone receptor

Summary This article reviews the prospects for a small-molecule agonist of the growth hormone receptor in the light of current successes in identifying small agonist molecules for other homomeric class 1 cytokine receptors. A variety of mutagenic analyses on both hormone and receptor, studies with monoclonal antibody agonists of the GH receptor, and the use of a constitutively dimerized GH receptor chimera which displays constitutive activity lead us to believe that such a development is possible. However, it is likely that a precise alignment of the lower cytokine receptor homology domains will be necessary in order to facilitate cross-activation of cytoplasmic Janus kinases bound to Box 1.     

Prospects for a small molecule able to induce somatic growth through the growth hormone receptor

This article reviews the prospects for a small-molecule agonist of the growth hormone receptor in the light of current successes in identifying small agonist molecules for other homomeric class 1 cytokine receptors. A variety of mutagenic analyses on both hormone and receptor, studies with monoclonal antibody agonists of the GH receptor, and the use of a constitutively dimerized GH receptor chimera which displays constitutive activity lead us to believe that such a development is possible. However, it is likely that a precise alignment of the lower cytokine receptor homology domains will be necessary in order to facilitate cross-activation of cytoplasmic Janus kinases bound to Box 1.     

Effects of growth hormone overexpression and growth hormone resistance on neuroendocrine and reproductive functions in transgenic and knock-out mice

Transgenic mice overexpressing growth hormone (GH) exhibit alterations in the function of the hypothalamic-pituitary-gonadal (HPG) axis and the H-P-adrenal axis. Alterations in the turnover of hypothalamic neurotransmitters, in plasma hormone levels, and in regulation of their release are associated with reproductive deficits, particularly in females. Results reported after publication of our minireview on this subject provided evidence that GH-transgenic mice have increased binding of GH to GH binding proteins in plasma, are hyperinsulinemic and insulin resistant, and have major alterations in energy budgets with increased allocation to growth. Reduced life span and fertility of these animals may be related to insufficient allocation of energy to reproduction and maintenance. Growth hormone resistance induced by transgenic expression of an antagonistic bGH analog or by targeted disruption (knock-out, KO) of the GH receptor (GH-R) gene leads to dramatic suppression of plasma levels of insulin-like growth factor-1 (IGF-1), and dwarf phenotype due to reduced growth and increased adiposity. In both models of GH resistance, there are marked reproductive deficits in females, decline of breeding performance of males, and alterations in the function of the HPG axis. In GH-R-KO females, puberty is delayed, and litter size is reduced. Fetal weights are reduced whereas placental weights are increased, and the weight of newborn pups is reduced despite an increase in the length of gestation. In GH-R-KO males, copulatory behavior and fertility are reduced, plasma PRL is elevated, and responses to luteinizing hormone releasing hormone (LHRH) in vivo and to LH in vitro are suppressed. However, reproductive deficits in GH-R-KO mice are very mild when compared to those described previously in IGF-KO animals. Apparently, the amounts of IGF-1 that may be produced locally in the absence of GH stimulation are sufficient for sexual maturation and fertility in both sexes, whereas quantitative deficits in reproductive function reflect absence of GH-dependent IGF-1 production and other consequences of eliminating GH signaling. The reproduction phenotype of the GH-R-KO mice is also mild when compared to dwarf mice that lack GH, prolactin (PRL), and thyroid stimulating hormone (TSH). This is presumably related to the presence of redundant mechanisms in the stimulatory control of the gonads by the pituitary and the ability of animals capable of producing PRL and TSH to compensate partially for the absence of GH signaling.     

Molecular evolution of growth hormone gene family in old world monkeys and hominoids

Growth hormone is a classic molecule in the study of the molecular clock hypothesis as it exhibits a relatively constant rate of evolution in most mammalian orders except primates and artiodactyls, where dramatically enhanced rate of evolution (25–50-fold) has been reported. The rapid evolution of primate growth hormone occurred after the divergence of tarsiers and simians, but before the separation of old world monkeys (OWM) from new world monkeys (NWM). Interestingly, this event of rapid sequence evolution coincided with multiple duplications of the growth hormone gene, suggesting gene duplication as a possible cause of the accelerated sequence evolution. Here we determined 21 different GH-like sequences from four species of OWM and hominoids. Combining with published sequences from OWM and hominoids, our analysis demonstrates that multiple gene duplications and several gene conversion events both occurred in the evolutionary history of this gene family in OWM/hominoids. The episode of recent duplications of CSH-like genes in gibbon is accompanied with rapid sequence evolution likely resulting from relaxation of purifying selection. GHN genes in both hominoids and OWM are under strong purifying selection. In contrast, CSH genes in both lineages are probably not. GHV genes in OWM and hominoids evolved at different evolutionary rates and underwent different selective constraints. Our results disclosed the complex history of the primate growth hormone gene family and raised intriguing questions on the consequences of these evolutionary events.     

Effect of growth hormone on fatty acid oxidation: growth hormone increases the activity of 2,4-dienoyl-CoA reductase in mitochondria

The effect of growth hormone on the beta-oxidation of saturated and unsaturated fatty acids was studied with mitochondria isolated from control rats, hypophysectomized rats, and hypophysectomized rats treated with growth hormone. Rates of respiration supported by polyunsaturated fatty acylcarnitines, in contrast to rates observed with palmitoylcarnitine or oleoylcarnitine, were slightly lower in hypophysectomized rats than in normal rats, but were higher in hypophysectomized rats treated with growth hormone. The effects were most pronounced with docosahexaenoylcarnitine, the substrate with the highest degree of unsaturation. Since uncoupling of mitochondria with 2,4-dinitrophenol resulted in lower rates of docosahexaenoylcarnitine-supported respiration, while substitution of ATP for ADP yielded higher rates, it appears that energy is required for the effective oxidation of polyunsaturated fatty acids. Growth hormone treatment of hypophysectomized rats caused a threefold increase in the activity of 2,4-dienoyl-CoA reductase or 4-enoyl-CoA reductase (EC 1.3.1.34) in mitochondria, but not in peroxisomes. The activities of other beta-oxidation enzymes remained virtually unchanged. Rates of acetoacetate formation from linolenoylcarnitine, but not from palmitoylcarnitine, were stimulated by glutamate in mitochondria from hypophysectomized rats and hypophysectomized rats treated with growth hormone. All data together lead to the conclusion that the mitochondrial oxidation of highly polyunsaturated fatty acids is limited by the availability of NADPH and the activity of 2,4-dienoyl-CoA reductase which is induced by growth hormone treatment.     

Bioactivity of growth hormone releasing hormone (1-29) analogues after SC injection in man

The bioactivity of growth hormone releasing hormone 1-29 [GHRH(1-29)NH2] has been compared with that of an agonist analogue [Ac-D-Tyr1,D-Ala2]-GHRH(1-29)NH2, in normal male volunteers. Using a submaximal dose of 3 micrograms/kg administered subcutaneously, peak growth hormone (GH) response and area under the GH curve were similar for the native and agonist analogue. In addition, no significant differences were found in peak GHRH(1-29) immunoreactivity, area under the GHRH(1-29) curves or plasma disappearance rates of the two peptides. The results suggest that, in keeping with the relative activities of other "superactive" analogues tested so far, the greatly enhanced activity of [Ac-D-Tyr1,D-Ala2]-GHRH(1-29)NH2 observed in the rat is not found in humans. It is possible that this species difference is due to differences in the interaction of GHRH peptides with the rat and the human somatotroph GHRH receptor.     

Effects of every-other-day feeding on prolactin regulatory mechanism in transgenic human growth hormone mice

Transgenic mice overexpressing human growth hormone (hGH) exhibit accelerated aging with functional hyperprolactinemia and greatly depressed endogenous prolactin. Calorie restriction (CR) is widely recognized as the most effective experimental intervention to delay aging. The aim of the present work was to analyze the effects of lifelong overexpression of hGH on prolactin-gene expression as well as the dopamine production at the pituitary level and discern whether this mechanism changes as a function of feeding patterns. Ten-month-old mice fed every other day (EOD) were killed after one day of fasting. The results confirmed typical phenotypic features of these transgenic mice: an increase in body weight, very high hGH plasma concentrations, and hyperinsulinemia. There was a marked inhibition of the expression of the prolactin gene, together with an increased tyrosine hydroxylase (TH) and the long isoform of dopamine receptor type 2 (D2LR) gene expression at the pituitary level. These parameters were not affected by the EOD feeding pattern. These data may suggest an autocrine or paracrine effect of dopamine at the hypophyseal level on prolactin secretion that is independent of the feeding pattern.     

Medaka receptors for somatolactin and growth hormone: phylogenetic paradox among fish growth hormone receptors

Somatolactin (SL) in fish belongs to the growth hormone/prolactin family. Its ortholog in tetrapods has not been identified and its function(s) remains largely unknown. The SL-deficient mutant of medaka (color interfere, ci) and an SL receptor (SLR) recently identified in salmon provide a fascinating field for investigating SL's function(s) in vivo. Here we isolated a medaka ortholog of the salmon SLR. The mRNA is transcribed in variable organs. Triglycerides and cholesterol contents in the ci are significantly higher than those in the wild type, providing the first evidence of SL's function in suppressing lipid accumulation to organs. Interestingly, phylogenetic comparisons between the medaka SLR and growth hormone receptor (GHR), which is also isolated in this study, in relation to GHRs of other fish, suggested that all GHRs reported from nonsalmonid species are, at least phylogenetically, SLRs. An extra intron inserted in medaka and pufferfish SLRs and flounder and sea bream GHRs also supports their orthologous relationship, but not with tetrapod GHRs. These results may indicate lineage-specific diversification of SLR and GHR functions among fish or just an inappropriate naming of these receptors. Further functional and comparative reassessments are necessary to address this question.     

Response of serine antiproteases to growth hormone therapy in growth hormone deficient children

Growth hormone regulates the hepatic mRNA levels of alpha 1-antitrypsin and two contrapsin-like mRNAs in the rat. To determine whether growth hormone regulates similar serine protease inhibitors in humans, we measured serum alpha 1-antitrypsin, alpha 1-antichymotrypsin, and antithrombin III by radioimmunodiffusion in 16 growth hormone deficient children before and after growth therapy. Of the 19 determinations made, 17/19 showed an increase in alpha 1-antitrypsin after administration of growth hormone, 198.6 +/- 39.1 mg/dl before growth hormone and 239.4 +/- 44 mg/dl after growth hormone (p = 0.005). Specificity of the response for alpha 1-antitrypsin was indicated by the fact that neither alpha 1-antichymotrypsin or antithrombin III values changed after growth hormone (p = 0.6 and 0.5, respectively). These data are compatible with the hypothesis that growth hormone regulates serine protease inhibitors in humans and suggests that investigation of other members of the serpin gene family might prove fruitful in defining additional growth hormone target genes.     

The molecular basis of growth hormone deficiency

A well-known law states that 'if a thing can go wrong it will go wrong'. This clearly applies to the hypothalamic-pituitary-somatic axis as to many other physiological and biochemical systems. Defects of this axis, giving rise to stunted growth, can occur at several different points, as has been discussed in detail in this review. Defects at the level of the brain can lead to inadequate production or secretion of the factors that control growth hormone secretion. Defects at the level of the pituitary can lead to failure to produce or secrete adequate quantities of growth hormone, or to production of inactive hormone. Defects at the level of target organs can lead to inability to respond to growth hormone or somatomedins. The axis involved in the production and effects of growth hormone is a complex one, and defects have been identified at most of the points that 'could go wrong', although in many cases the molecular details are far from fully understood. Increased understanding of the biochemistry and physiology of the hormonal control of growth, and of the impairments to which it is subject, should provide an improved basis for treatment of growth defects. Nevertheless, there remain many points at which our knowledge is very incomplete. The field is a rapidly moving one and further developments in both basic understanding and clinical treatment are to be expected during the next few years.     

Growth hormone receptors in lymphocytes of growth hormone-deficient children

Human growth hormone was injected intravenously into 18 growth hormone-deficient children and growth hormone binding sites in lymphocytes were investigated. Fresh circulating lymphocytes had a low initial value for the binding of growth hormone to solubilized receptors (3.45 +/- 1.46%) but after growth hormone injection, the binding rapidly increased to 14.8 +/- 4.2% at 2 1/2 h and 8.7 +/- 1.8% at 5 h. The sharp increase in binding is due to increase in the number of binding sites. Two control children who received chorionic gonadotropin had no change in lymphocyte growth hormone receptors. The methodological differences between the present study and previous attempts to identify human growth receptors in lymphocytes were (1) lymphocytes were separated and disrupted with Triton X-100 as quickly as possible (to avoid error from receptor leaking out of the cell) and (2) the receptors were assayed at 2 1/2 h after growth hormone administration (previous studies were 12-24 h later). One possible explanation for the data is that growth hormone receptor from liver is taken up by lymphocytes and rapidly released again, thus, contributing to the hormonal receptor economy in humans.