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.     

Mutations that modulate receptor–hormone congruency as a cause of the primate GH receptor species specificity

Peptide hormones depend on reliable recognition by their receptors. Any mutation that compromises recognition of hormone and receptor molecules is dangerous, the carrier animal would not procreate and the mutation would be lost. Although, most of the hormones from one mammalian species are active when injected into another, the incompatibility of human GH receptor toward nonprimate GHs is a notable exception. It is reported that the coevolution of GH and GHR in primates includes two crucial steps (Mol. Biol. Evol. 18 (2001) 945). The first was mutation of GH His→Asp at position 171 that happened before the split of Old world and New world monkeys. The second event was Leu→Arg change at position 43 in the GH receptor molecule that happened in the ancestor of Old world monkeys. The proposed model is based on the possibility that certain mutations can modify the surface of one of interacting molecules to form a confined empty space, a niche in the otherwise congruent hormone/receptor interface. Although affinity between molecules is probably slightly reduced, recognition and function are not compromised in this special case. Further mutations of hormone and receptor molecules are allowed under the condition that they remain confined to the niche space. Mutations that do not compromise hormone function can be passed to offsprings. If the consequent mutation of one molecule change its shape to fill the niche space, further mutations without function loss will become less probable. Without the niche space, the phase of fast evolution is closed and both genes become conserved. In this setting, accumulated mutations before the niche closing mutation are the cause of species specificity. To become a dominant variety, carrier animals must possess survival advantage in comparison to the carriers of other less advantageous mutations.     

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.     

Mutations that modulate receptor-hormone congruency as a cause of the primate GH receptor species specificity

Peptide hormones depend on reliable recognition by their receptors. Any mutation that compromises recognition of hormone and receptor molecules is dangerous, the carrier animal would not procreate and the mutation would be lost. Although, most of the hormones from one mammalian species are active when injected into another, the incompatibility of human GH receptor toward nonprimate GHs is a notable exception. It is reported that the coevolution of GH and GHR in primates includes two crucial steps (Mol. Biol. Evol. 18 (2001) 945). The first was mutation of GH His→Asp at position 171 that happened before the split of Old world and New world monkeys. The second event was Leu→Arg change at position 43 in the GH receptor molecule that happened in the ancestor of Old world monkeys. The proposed model is based on the possibility that certain mutations can modify the surface of one of interacting molecules to form a confined empty space, a niche in the otherwise congruent hormone/receptor interface. Although affinity between molecules is probably slightly reduced, recognition and function are not compromised in this special case. Further mutations of hormone and receptor molecules are allowed under the condition that they remain confined to the niche space. Mutations that do not compromise hormone function can be passed to offsprings. If the consequent mutation of one molecule change its shape to fill the niche space, further mutations without function loss will become less probable. Without the niche space, the phase of fast evolution is closed and both genes become conserved. In this setting, accumulated mutations before the niche closing mutation are the cause of species specificity. To become a dominant variety, carrier animals must possess survival advantage in comparison to the carriers of other less advantageous mutations.     

Identification of ligand binding determinants of the prolactin receptor.

The prolactin (PRL) receptor, a lactogen- and primate somatogen-binding protein, is a member of an expanding superfamily (cytokine/growth hormone (GH)/PRL) of single membrane-spanning receptors. Two features commonly shared among this group of proteins are the presence of two pairs of cysteines, generally found in the N-terminal region of the extracellular domain, and a WSxWS (WS) motif, frequently located proximal to the transmembrane domain. We have recently shown the 4 cysteines to be critical to the maintenance of the structural and functional integrity of the PRL-receptor. In the present study, we prepared a set of eight chimeric rat PRL/human GH receptors and several alanine mutants, to assess the importance of the Cys-rich domain (residues 12-68) in confering specificity to PRL binding. The role of the WS motif in high affinity binding was also investigated. Binding of 125I-labeled ovine PRL or human GH to membrane preparations from COS-7 cells transiently expressing the mutant receptors have defined a region within the first disulfide loop (residues Arg13, Asp16, Glu18) and the set of lactogen-specific sequences between the two pairs of cysteines as key determinants of PRL-binding specificity, which converge to form a patch on a two-dimensional model of the PRL receptor. We also demonstrate that, although PRL- and GH-specific determinants overlap in certain areas, they are not identical. Finally, substitution of the WS motif with alanine residues precludes high affinity binding to ovine PRL and human GH and suggests that this structural element may provide a target site for the interaction of an accessory protein necessary for the formation of a high-affinity receptor complex.     

Site-directed mutations of human growth hormone that selectively modify its lactogenic activity and binding properties

Two novel analogs of human (h) GH, 1) Des-7-hGH (Arg8Met, Asp11Ala) in which the Arg8 was substituted by Met and Asp11 by Ala, and 2) bovine (b) GH/hGH hybrid II (MetAla 1-13/14-191, Ala11Asp) composed of 13 N-terminal amino acid of bGH and elongated by two amino acids (Met-Ala-1-13) and 14-191 amino acids of hGH, were constructed and expressed in Escherichia coli. CD spectra indicated that the alpha-helix content of the purified proteins was similar to that of the native hormone. Both analogs retained their full ability to stimulate the proliferation of Nb2 lymphoma cells, and their binding to the lactogen receptors in homogenate of Nb2 cells and in microsomal fraction from bovine lactating mammary gland was only slightly reduced. However, their ability to bind to the somatogen receptors in intact IM-9 lymphocytes and bovine liver was reduced by 7- to 11-fold (bGH/hGH hybrid II) and 20- to 30-fold (Des-7-hGH). Both analogs were able to down-regulate the respective lactogen and somatogen receptors in intact Nb2 and IM-9 cells. The galactopoietic activity of both analogs in the lactating bovine mammary explants bioassay was almost completely abolished, and the bGH/hGH hybrid II exhibited a remarkable antagonistic activity. These results further indicate that the lactogen receptors in different species or organs are not identical. We have shown that the new recombinant analogs of hGH that recognize both somatogen and lactogen receptors but have modified postreceptor effects are helpful in elucidating these differences.     

Identification of novel sites in the ovine growth hormone receptor involved in binding hormone and conferring species specificity.

Using site-directed mutagenesis we mutated the extracellular domain of the ovine growth hormone receptor (oGHR) to the corresponding amino acids in the rat GHR at two different sites: site A is between Thr28 and Leu34 and represents a major immunogenic epitope, while site B is between Ser121 and Asp124 and is involved in the interaction of the human GHR with growth hormone (GH). Native and mutant receptors were bacterially expressed and refolded, and then RIA and GH-binding assays were carried out on the purified recombinant proteins. Mutations at the N-terminal site A of oGHR led to greatly reduced binding to bovine GH and, in addition, to significant loss of recognition by a polyclonal antiserum to bovine GHR which recognizes site A as a major epitope. The crystal structure of human GH bound to human GHR did not resolve this extreme N-terminal region of the receptor but our data indicate that the N-terminal loop undertakes a 180 degrees turn bringing it into close proximity to the hormone-binding domain in a fashion analogous to the prolactin receptor. A fourfold decrease in affinity for binding bovine GH was also observed after mutation of site B. However, this change from the ovine sequence to the equivalent sequence in the rat GHR at site B caused a 2.4-fold increase in the affinity of binding to rat GH. Taken together, the changes in binding affinity of the site-B mutant for rat and bovine GH demonstrate that this site is involved in conferring species specificity for binding GH.     

Three adjacent serines in the extracellular domains of the CaR are required for L-amino acid-mediated potentiation of receptor function

The extracellular calcium (Ca(2+)(o))-sensing receptor (CaR) is a key player in Ca(2+)(o) homeostasis. The activity of CaR can be potentiated by various l-amino acids. In this study, we examined whether conserved amino acid residues involved in the binding of glutamate to metabotropic glutamate receptors (mGluRs) also participate in the potentiation of the activity of CaR by l-phenylalanine. Ser-170 corresponding to Thr-188 in rat mGluR1a appears to be important for the modulating actions of phenylalanine. In the presence of phenylalanine, a mutant CaR with a single mutation S170A showed no significant decrease in its EC(50) for stimulation by Ca(2+)(o) and a modest increase in its maximal activity. In addition, mutating Ser-169 and Ser-171 together with Ser-170 yielded a more complete block of the phenylalanine modulation than did the single mutation. The presence of the triple mutation, S169A/S170A/S171A, also eliminated phenylalanine potentiation of the activities of heterodimeric receptors in which one of the monomeric receptors had intact triple serines (A877Stop). The putative amino acid binding site of the CaR is probably close to or structurally dependent on the Ca(2+)(o) binding sites of the receptor, because mutant CaRs with mutations in the putative amino acid binding site exhibited severely reduced responses to Ca(2+)(o).     

Determination of a region of the HisJ binding protein involved in the recognition of the membrane complex of the histidine transport system of Salmonella typhimurium

Site-directed mutagenesis has been utilized to examine the nature of the interaction of the histidine-binding protein (HisJ) with the membrane-bound components of the histidine transport system. In order to examine a region of the HisJ protein involved in the interaction with the membrane components, a number of charged amino acids in the vicinity of the genetically isolated interaction mutant hisJ5625 (R176C) were mutated. It was found that residues Asp171, Arg176, and Asp178 could be independently altered without affecting the histidine-binding affinity of the HisJ protein. However, the alteration of residues Asp171 and Arg176 greatly reduced the interaction of the HisJ protein with the membrane protein complex, whereas altering residue Asp178 had no effect on this interaction. Simultaneously, altering residues Asp183 and Glu184 resulted in a completely defective protein. The ability of a his-J5625 suppressor HisP protein (HisP(T205A)) to suppress the newly created site-directed mutants was also examined. This suppressor demonstrated specificity toward the amino acid present at position 176 and was also able the suppress the mutation created at position 171.     

Similarities and Differences Among Prolactins and Growth Hormones and Their Receptors

SYNOPSIS. Data from the literature and from our own studieson the receptors for prolactin (PRL) and growth hormone (GH)are reviewed and analyzed. Receptors for PRL have been studiedin a wider range of species and in a greater diversity of targetorgans than have the binding sites for GH. Although GHs arestructurally more highly conserved among the vertebrates thanare PRLs, the available data indicate that there is greaterdiversity among GH receptors than there is among PRL receptors.In general, GH receptors show greater species specificity butless hormone specificity than do PRL receptors. The reason forthe greater diversity among GH receptors as compared to PRLreceptors is unknown; it bears no relationship to phylogeny. Data on the binding of purified preparations of mammalian PRL,GH and placental lactogen (PL) to renal and hepatic receptorsfor PRL and GH, respectively, of several vertebrate speciesare reviewed. The species and hormone specificity of the bindingof the hormones to the two typesof receptors showed no consistentpattern. To explain this disarray, we propose that the receptorbinding domains of PRL and GH were present in their common ancestralgene and that they havebeen retained to variable degrees byall of the descendant members of the PRL-GH family. We furtherpropose that hormone and species specificity of binding is determinedby hindering features on the hormones and on the receptors,rather than by merely the presence or absence of the appropriatebinding determinants.     

The specificity of binding of growth hormone and prolactin to purified plasma membranes from pregnant-rabbit liver.

The binding of 125I-labelled human growth hormone (hGH) to a purified plasma membrane preparation from the liver of pregnant rabbit, and to receptors solubilized from this fraction with Triton X-100, was dependent on time, temperature, the cations used and the receptor concentration. Solubilization did not affect the binding properties of the receptors at low concentrations of Triton X-100. Some somatogenic hormones, such as bovine GH, and some lactogenic hormones, such as ovine prolactin, displaced 125I-labelled hGH from purified plasma membranes and solubilized receptor preparations, but GHs and prolactins from various other species were rather ineffective. The results indicate that although there are binding sites for hGH in these pregnant rabbit liver membranes, few of these are specifically somatogenic or lactogenic. The binding properties of the purified plasma membranes are similar to those of a microsomal preparation studied previously, suggesting that the complex nature of the binding of hGH is not due to the heterogeneity of cellular membranes used to study binding, but is a property of the receptors associated with plasma membranes.     

Conserved and unique amino acid residues in the domains of the growth hormones. Flounder growth hormone deduced from the cDNA sequence has the minimal size in the growth hormone prolactin gene family

Growth hormone (GH), prolactin (PRL), and placental lactogen (PL) constitute a protein family whose genes are considered to have evolved from a common ancestral gene. GHs isolated from various vertebrate species are known to possess highly conserved structural and functional features. In the present study we have cloned and sequenced flounder growth hormone (fGH) cDNA to predict the primary structure of the hormone. The preprotein of fGH is composed of 190 amino acids, and mature fGH is found to be extraordinarily small, having 171 or 173 amino acid residues. The estimated molecular masses of mature fGH are 19.4 to 19.7 kDa. This minimal size of fGH enabled an extended analysis of the essential domains and of amino acid residues required in hormone-specific activities. fGH conserves and shares 37 residues with 20 other vertebrate GHs. These common residues are seen to cluster in five distinct domains (GD1 to GD5). In human PL (hPL), which has low growth-promoting activity, 35 of these 37 residues are conserved, while the other 2 residues in the GD1 domain (Arg-16 and Leu-20) are replaced by Gln and Ala, respectively. In a less active variant of human GH, hGH-V, only 1 residue (His-21) of the 37 residues is replaced by Tyr. Besides these 3 residues, 6 other residues unique to the GHs and some PLs, that is, Ala-24 (GD1), Ser-54 (GD2), Ser-78 (GD3), Leu-106, Leu-116, and Asp-122 (GD4), appear to be important for specific binding of the GHs. The GD5 domain, at the carboxyl-terminal ends of the GHs is considered to be involved mainly in the formation and stabilization of GH molecules.     

Natural variation at position 45 in the D1 domain of lineage III killer cell immunoglobulin-like receptors (KIR) has major effects on the avidity and specificity for MHC class I

Alternative lysine and methionine residues at position 44 in the D1 domain determine the specificities of human lineage III killer cell immunoglobulin-like receptors (KIR) for the C1 and C2 epitopes of HLA-C. KIR having glutamate 44 are also present in orangutans (Popy2DLB) and chimpanzees (Pt-2DL9) but notably absent from humans. Popy2DLB exhibits broad specificity for both the C1 and C2 epitopes, whereas Pt-2DL9 has narrow specificity for C2. Mutation of phenylalanine 45 in Popy2DLB to the cysteine residue present in Pt-2DL9 was sufficient to narrow the Popy2DLB specificity to be like that of Pt-2DL9. In contrast, replacement of cysteine 45 in Pt-2DL9 by phenylalanine had no effect on its C2 specificity, but reduced the avidity. In a similar manner, replacement of phenylalanine 45 with cysteine in Popy2DLA, which has lysine 44 and recognizes C1, maintained this specificity while reducing avidity. Position 45 is exceptionally variable, exhibiting twelve residues that distinguish KIR of different lineages and species. Our study demonstrates the potential for variation at position 45 to modulate KIR avidity and specificity for HLA-C. The various effects of position 45 mutation are consistent with a model in which a Popy2DLB-like receptor, having glutamate 44 and broad specificity for C1 and C2, facilitated the evolution of the C2 epitope from the C1 epitope and C2-specific KIR from C1-specific KIR. With the acquisition of C2 and C2-specific receptors, the selection against this broadly specific receptor led to its loss from the human line and narrowing of its specificity on the chimpanzee line.     

Growth hormone-binding proteins in high-speed cytosols of multiple tissues of the rabbit

Soluble, specific binding protein(s) for growth hormone (GH) have been identified and partially characterized in high-speed cytosolic preparations from a number of rabbit tissues. The binding of 125I-labelled human GH to proteins in liver, heart, adipose tissue, skeletal muscle and kidney cytosols was dependent on time and cytosolic protein concentration. By Scatchard analysis, the binding affinities (KA: (2-7) X 10(9) M-1) were somewhat higher than those generally reported for membrane GH receptors. The binding proteins had a greater specificity for somatotrophic hormones than lactogenic hormones, although the kidney appeared to have, in addition, a lactogen-binding protein. By gel filtration, the Mr of the cytosolic GH-binding protein was approximately 100 000 in all tissues. None of the binding proteins was detectable by the poly(ethylene glycol) precipitation method used widely for soluble hormone receptors. The cytosolic GH-binding proteins also cross-reacted with a monoclonal antibody to the rabbit liver membrane GH receptor. These results indicate the ubiquitous presence of apparently naturally soluble GH-binding proteins in the cytosolic fractions of several tissues in the rabbit. Of great interest is their presence in muscle, where GH receptors or binding proteins have not previously been detected, despite muscle being recognized as a classical GH target tissue.     

Mutational analysis of human DNase I at the DNA binding interface: implications for DNA recognition,catalysis, and metal ion dependence.

Human deoxyribonuclease I (DNase I), an enzyme used to treat cystic fibrosis patients, has been systematically analyzed by site-directed mutagenesis of residues at the DNA binding interface. Crystal structures of bovine DNase I complexed with two different oligonucleotides have implicated the participation of over 20 amino acids in catalysis or DNA recognition. These residues have been classified into four groups based on the characterization of over 80 human DNase I variants. Mutations at any of the four catalytic amino acids His 134, His 252, Glu 78, and Asp 212 drastically reduced the hydrolytic activity of DNase I. Replacing the three putative divalent metal ion-coordinating residues Glu 39, Asp 168, or Asp 251 led to inactive variants. Amino acids Gln 9, Arg 41, Tyr 76, Arg 111, Asn 170, Tyr 175, and Tyr 211 were also critical for activity, presumably because of their close proximity to the active site, while more peripheral DNA interactions stemming from 13 other positions were of minimal significance. The relative importance of these 27 positions is consistent with evolutionary relationships among DNase I across different species, DNase I-like proteins, and bacterial sphingomyelinases, suggesting a fingerprint for a family of DNase I-like proteins. Furthermore, we found no evidence for a second active site that had been previously implicated in Mn2+-dependent DNA degradation. Finally, we correlated our mutational analysis of human DNase I to that of bovine DNase I with respect to their specific activity and dependence on divalent metal ions.     

Correlative studies between the presence of thyrotropin-releasing hormone (TRH) receptors and the in vitro stimulation of growth-hormone (GH) secretion in human GH-secreting adenomas

Specific receptors for TRH were characterized on cellular membranes of 6 out of 13 somatotrophic adenomas obtained from acromegalic patients. These receptors had the same dissociation constant (Kd: 62 +/- 10 nM) as those found in human PRL-secreting adenomas, but their maximal number of binding sites (Bmax: 76 +/- 24 fmol/mg of protein) was six fold smaller. A good correlation was found between the presence of TRH receptors and the in vitro TRH-induced stimulation of GH secretion. The increase in GH release varied from 25 to 200%. It was thus concluded that these receptors are functional. However, why only some of the human somatotrophic adenomas possess TRH receptors and respond to TRH in vitro needs further investigations.     

Site-directed mutagenesis of human pancreatic secretory trypsin inhibitor

Arg-42 or Lys-43 or Arg-44 of human pancreatic secretory trypsin inhibitor (PSTI) was replaced by Thr or Ser by site-directed mutagenesis, and the inactivation rates of the mutants after mixing with human trypsin were compared with that of the natural form. The inactivation rate decreased for one mutant (Arg-44----Ser), whereas no change was observed for another (Arg-42----Thr) and an increase was observed for a third (Lys-43----Thr). Kinetic studies on the interactions between human trypsin and synthetic peptides, comprising the regions of Phe39-Ser47 of the respective PSTI species, showed that human trypsin cleaved the Arg42-Lys43 bond preferentially to the Arg44-Gln45 bond. However, it is cleavage of the latter bond that is thought to cause inactivation of human PSTI. These results suggest that the Arg44-Gln45 bond of human PSTI is responsible for its inhibitory activity, and inactivation of human PSTI is probably caused by deletion of the dipeptide Lys43-Arg44.     

Computational studies of pandemic 1918 and 2009 H1N1 hemagglutinins bound to avian and human receptor analogs

The purpose of this work was to study the binding properties of two pandemic influenza A virus 1918 H1N1 (SC1918) and 2009 H1N1 (CA09) hemagglutinin (HA) with avian and human receptors. The quantum chemical calculations have been performed to analyze the interactions of 130 loop, 190 helix, 220 loop region, and conserved residues 95,145,153–155, of pandemic viruses’ HA with sialo-trisaccharide receptor of avian and human using density functional theory. The HA’s residues Tyr 95, Ala 138, Gln 191, Arg 220, and Asp 225 from the above regions have stronger interaction with avian receptor. The residues Thr 136, Trp 153, His 183, and Asp 190 of HA are important and play a significant role to bind with human receptor. The residues Tyr 95, Ala 138, Lys 145, Trp 153, Gln 192, and Gln 226 of HA of CA09 virus have found more interaction energies with human than avian receptors. Due to mutations in the active residues of HA of CA09 virus comparing with SC1918, the binding capabilities of HA with human have been increased. The molecular dynamics simulation was made to understand the different dynamical properties of HA and molecular interactions between HA of these two viruses with sialo-trisaccharide receptors of avian and human receptors. The interaction energy of HA of CA09 virus with human receptor decreases due to the human receptor far away from conserved residue region of HA protein. This reveals that the conserved residues particularly Lys 145 play major contribution to interaction with human receptor in HA of CA09 virus.     

The role of insulin-like growth factor-I in neuroendocrine function and the consequent effects on sexual maturation: inferences from animal models

It is known that growth hormone (GH) plays an important role in growth and development.Additionally, emerging evidence suggest that it also influences hypothalamic-pituitary-gonadal function. We have found that GH from different species has different effects in mice. In rodents, human GH (hGH) binds to both GH and prolactin (PRL) receptors; it has both somatotrophic and lactotrophic effects. Since PRL has a profound effect on neuroendocrine function, the results obtained from hGH treatment or from transgenic animals expressing the hGH gene reflect PRL-like effects of this hormone. However, bovine GH (bGH) is purely somatogenic and therefore the effects of bGH represent the function of the natural GH produced in rodents. Furthermore, our studies in mice and rats have shown that not all effects of GH are stimulatory and the duration of exposure of the hypothalamo-hypophyseal-gonadal system to GH might influence the secretions of gonadotropins and gonadal steroids. In humans, excess productions of GH in acromegaly and GH resistance in Laron syndrome adversely affect reproduction. Similarly, it has been demonstrated that in transgenic mice expressing various GH genes, in insulin-like growth factor-I (IGF-I) gene-knockout mice, in GH receptor gene-disrupted (GHR-KO) mice, and in Ames dwarf mice the onset of puberty and/or fertility is altered. Therefore, excess or subnormal secretion of GH can affect reproduction. We have shown that the hypothalamic-pituitary functions are affected in transgenic mice expressing the GH genes, Ames dwarf mice and in GH receptor gene knockout mice. The majority of the GH effects are mediated via IGF-I and the aforementioned effects may be due to the GH-induced IGF-I secretion or due to the absence of this peptide production. It is important to realize that the syntheses and actions of IGF binding proteins are controlled by IGF-I. Furthermore, some IGF binding proteins can inhibit IGF-I action. Therefore, the concentrations of IGF binding proteins and the ratio of these binding proteins and IGF-I within the body might play a pivotal role in modulating IGF-I effects on the neuroendocrine-gonadal system.     

Isolation and characterization of growth hormone from a marine fish, bonito (Katsuwonus pelamis)

Growth hormone (GH) was extracted under alkaline conditions (pH 10) from pituitary glands (6.3 g) of bonito (Katsuwonus pelamis), and subsequently purified by gel filtration, ion exchange chromatography, and reversed-phase HPLC. The GH was monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and by immunoblotting with yellowtail GH antiserum at each step of purification. GH activity was determined by an in vivo bioassay. The yield of this hormone was 4.8 mg/g wet tissue. Intraperitoneal injection of bonito GH at doses of 0.1 and 1 micrograms/g body wt at 7-day intervals resulted in a significant increase in body weight and length of juvenile rainbow trout. Bonito GH antiserum exhibited both species and hormone specificity in radioimmunoassay. However, the bonito GH antiserum as well as yellowtail GH antiserum exhibited hormone specificity but not species specificity in immunoblotting. A molecular weight of 21,000 and an isoelectric point of 7.0 for bonito GH were estimated by SDS-PAGE and gel electrofocusing, respectively. The complete amino acid sequence of 185 residues was determined by sequencing fragment peptides prepared by chemical and enzymatic cleavages. Sequence comparison of bonito GH with other GHs revealed that there is a significant deletion in the middle of the molecule.