Growth Hormone Analog Research Articles

Growth hormone (GH) induces tyrosine-phosphorylated proteins in mouse L cells that express recombinant GH receptors.

Porcine and bovine GH receptor (GHR) cDNAs were stably expressed in mouse L cells, which normally do not possess detectable levels of mouse GHR. Expression of the GHR cDNAs resulted in specific binding of 125I-labeled GH by these cell lines. To study GHR-related signaling events in these cells, protein tyrosine phosphorylation was examined. In GH-treated cells, a tyrosine-phosphorylated protein with a molecular mass of approximately 95 kDa (pp95) was increased dramatically (approximately 100-fold) relative to non-GH-treated cells. The amount of pp95 within the cells after GH treatment was positively correlated with the number of GHRs on the cells. Tyrosine phosphorylation of pp95 could not be induced by prolactin, insulin, insulin-like growth factor I, interleukin 2, epidermal growth factor, platelet-derived growth factor, or fibroblast growth factor. Phosphorylation of pp95 was found to be a rapid event that could be observed 60 sec after GH treatment. Also, pp95 appears to exist as a complex of two proteins, i.e., pp95 and pp96. The GH-induced response by these cells may be of use in screening GH analogs for biological activity.

Selective modification of human growth hormone by site-directed mutagenesis: implications for the diversity of biological actions.

Human growth hormone (GH) is an anabolic hormone required for normal growth. In addition, human GH affects the metabolism of proteins, carbohydrates and fats and possesses lactogenic effects. Although the GH receptor has recently been cloned, it is not clear whether the diverse biological activities of human GH are transduced through a single or through several types of related receptors. To address this question, 15 recombinant analogues of human GH were prepared and tested in bioassays in vitro and in vivo, in which the hormone action is mediated through lactogen or somatogen receptors. The results clearly suggest that recombinant analogues of human GH that recognize either somatogen or lactogen receptors, or both, but have selectively modified post-receptor effects, are helpful in elucidating the diverse biological activities of GH. These differences are most probably due to minor structural variability in GH and lactogen receptors in different organs and/or species. Genetic engineering of human GH may lead to production of modified analogues with changed and narrower specificities. One of the possible applications would be for a human GH analogue devoid of diabetogenic activity.

Implications of investigating the structure-function relationship of human growth hormone in clinical diagnosis and therapy.

Human growth hormone (GH) mediates longitudinal bone growth and also exerts a variety of other biological effects, e.g. lactogenic, insulin-like, diabetogenic, lipolytic, protein-anabolic and sodium/water-retaining effects. Investigation of the structure-function relationship of human GH has been attempted by epitope mapping using monoclonal antibodies and by systematic point mutations of the human GH molecule. The diagnosis of GH-related disorders is complicated by the fact that different commercial immunoassay kits give widely differing results. This phenomenon cannot be explained by lack of standardization, but has to be attributed to the epitope specificities of the antibodies employed in the assay techniques and the spectra of different molecular forms of human GH recognized by them. The information required by a clinician in ordering a GH determination is the bioactivity of all the human GH forms in the sample, rather than the immunoreactivity with a given set of antibodies. Therefore, the aim of future GH immunoassays must be the identification of antibodies that bind with high affinity only those forms of human GH that bind and activate the GH receptor. Epitope mapping by monoclonal antibodies appears to be the ideal approach to this goal. It has recently been shown that one molecule of human GH binds two molecules of GH receptor, and dimerization of the receptor by its ligand is a prerequisite for biological function. Improvements in understanding the structure-function relationship of the human GH molecule and of the interplay of the hormone with its receptor make it conceivable that recombinant analogues of human GH will be designed to inhibit the effects of GH excess in acromegaly.(ABSTRACT TRUNCATED AT 250 WORDS)

Site-directed mutagenesis of hGH at the 54–74 loop selectively modifies its lactogenic receptor-mediated biological activity

Four analogues of human growth hormone (hGH) mutated by site-directed mutagenesis at the 54–74 loop-MethGH(P59A), Met-hGH(P6lA), Met-hGH(P59A,P61A) and Met-hGH(Des 62–67) were analyzed for: (1) their biological activity mediated through lactogenic receptors using rat lymphoma Nb 2-llC cell proliferation and mouse mammary gland HC-11 cell β-casein synthesis bioassays and (2) their ability to interact with recombinant hGH binding protein (hGHBP). The analogues Met-hGH(P59A), Met-hGH(P61A) and Met-hGH(P59A,P61A) partially lost their activity relative to native hGH in the HC-11, but not in the Nb 2-llC cell bioassay. These analogues were nevertheless capable of forming a 1:2 complex with a recombinant hGH binding protein (hGHBP), despite the fact that the affinity of Met-hGH(P61A) and Met-hGH(P59A,P61A) analogues had decreased 8- and 14-fold, respectively. Met-hGH(Des 62–67) failed to form 1:1 or 1:2 complexes with hGHBP and did not compete with [ 125I]hGH for binding to hGHBP. It lost all biological activity in HC-11 cells, but retained 0.4% of its activity, in the Nb 2-11C cell proliferation bioassay. These results confirm the involvement of Pro-61 in the hGH binding and activity mediated through somatogenic receptors, while the activity mediated through two different types of lactogenic receptors was selectively modified. These findings emphasize the fact that lactogen receptors in different species or organs are not identical.

New Gaba-containing analogues of human growth hormone releasing hormone (1-30)-amide: II. Detailed in vivo biological examinations.

Analogues of human growth hormone-releasing hormone-(1-30)-amide [GH-RH(1-30)-amide] were tested for their ability to stimulate GH release in vivo by injecting the peptides intravenously (iv), subcutaneously (sc), and intramuscularly (im). The analogues involved derivatization with Nle27 and Gaba substituents at the C-terminus with or without D-amino acid(s) in the peptide chain. The potency of the analogues was compared to that of GH-RH(1-29)-amid testing their ability to release GH at 5, 15 and 30 min after the administration. In iv test the potency of the analogues was 1.2-2 times higher than that of the GH-RH(1-29)-amide, and no significant differences were detected between the potencies of the analogues with or without D-amino acid. In the sc test the analogue with D-Ala2, Nle27, and Gaba30 substitutions expressed 8.0-51.7 times higher potency than the GH-RH(1-29)-amide, however, the analogue with similar modifications but with L-Ala2 showed the same low potency (1.2-2.1) as in the iv test. Results from the im experiments were similar to those of SC test. The most potent analogues were those which had D-Ala2, Nle27, and Gaba30 substitutions with Gly15 or Leu15. Circular dichroism (CD) spectra of the analogues showed that Leu in position 15 increased the stability of the predominant alpha-helix conformation, which improved the absorption of the molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypoglycaemic activity of an analogue of human growth hormone [6-13] incorporating a d-ala-pro dipeptide unit

An analogue of human Growth Hormone [6–13] has been prepared incorporating a d-Ala 11-Pro 12 dipeptide unit to induce a type II′ β-turn conformation. Similar hypoglycaemic activity has been observed to that of the aspartimide-containing analogue Asu 11-hGH[6-13], indicating conservation of bioactive conformation properties.

Application of biotechnology and transgenic animals toward the study of growth hormone

Biotechnology has played a key role in our understanding of the molecular biology and physiology of growth hormone. The application of recombinant-DNA techniques and transgenic animals has resulted in the production of unlimited quantities of purified growth hormone and the development of growth hormone analogues. Ultimately, these analogues will be used to enhance growth and production characteristics in domestic livestock and treat certain growth and metabolic disorders in humans.

Hypoglycemic action of a novel constrained analog of human growth hormone-(6-13).

The amino-terminal region of human GH (hGH), in particular the amino acid sequence Leu-Ser-Arg-Leu-Phe-Asp-Asn-Ala[hGH-(6-13)], has been implicated as a functional region for the regulation of energy metabolism by exerting an insulin-potentiating action on insulin-sensitive tissues. Recent structural studies have revealed that the cyclization of the aspartate (Asp11) residue to form the alpha-aminosuccinimide (Asu11) ring is essential for the biological action of peptides related to this hGH fragment. The pharmacological application of these hGH-(6-13) peptides has been hindered by the vulnerability of the alpha-aminosuccinimide to hydrolytic modification leading to the loss of biological action. We have succeeded in stabilizing the structure of the Asu11-hGH-(6-13) peptide by replacing the alpha-aminosuccinimide ring with compatible and less rapidly metabolized gamma-lactam structures. In the present paper we report the bioactivity profile of an analog of hGH-(6-13) containing a gamma-lactam at residue position 11 that mimics the stereoelectronic and conformational characteristics of the alpha-aminosuccinimide ring. In vitro, the gamma-lactam11-hGH-(6-13) peptide analog increased [14C]glucose incorporation into glycogen in muscles and conversion to lipid in adipose tissues. In vivo, the gamma-lactam11-hGH-(6-13) peptide enhanced hypoglycemia during iv insulin tolerance tests. The results demonstrate that the gamma-lactam11-hGH-(6-13) peptide analog has similar biological properties to the Asu11-hGH-(6-13) peptide fragment, but with improved molecular stability and bioavailability.

Hypoglycemic action of a novel constrained analog of human growth hormone-(6-13)

Hypoglycemic action of a novel constrained analog of human growth hormone-(6-13)

A growth hormone (GH) analog can antagonize the ability of native GH to promote differentiation of 3T3-F442A preadipocytes and stimulate insulin-like and lipolytic activities in primary rat adipocytes.

The effect of amino acid substitutions introduced to the third alpha-helix in bovine GH (bGH) was investigated. A GH analog (bGH-M8), in which three amino acids were substituted to form an idealized amphiphilic alpha-helix, possessed the same specific binding affinity as wild-type bGH to cell membranes prepared from 3T3-F442A cells or rat adipocytes. However, bGH-M8 failed to stimulate preadipocyte differentiation, as measured by the level of glycerol-3-phosphate dehydrogenase activity. An equimolar concentration of bGH-M8 was inhibitory for this adipogenic effect caused by bGH at a concentration of 30 pM. bGH-M8 also failed to induce an insulin-like response and reduced lipolytic potency in rat primary adipocytes. A 10-fold excess of bGH-M8 abolished the effect of wild-type bGH in the insulin-like and lipolytic assays. Thus, bGH-M8 inhibited these actions of wild-type bGH and, therefore, appears to be a competitive antagonist. These results suggest that a major biologically active domain resides in the third alpha-helix of bGH, which is independent of amino acids important in the initial interaction of GH with its receptor.

A growth hormone (GH) analog can antagonize the ability of native GH to promote differentiation of 3T3-F442A preadipocytes and stimulate insulin-like and lipolytic activities in primary rat adipocytes

A growth hormone (GH) analog can antagonize the ability of native GH to promote differentiation of 3T3-F442A preadipocytes and stimulate insulin-like and lipolytic activities in primary rat adipocytes

Binding of Human Growth Hormone (GH)-Variant (Placental GH) to GH-Binding Proteins in Human Plasma*

Human GH-variant (hGH-V) is a natural GH analog arising from the hGH-V gene. It is expressed in the placenta and secreted into the maternal circulation during the second half of pregnancy. To gain information about its bioactivity in man, we examined the interaction of hGH-V with the high affinity GH-binding protein/receptor (GH-BP) in human plasma. hGH-V was equipotent with pituitary hGH (hGH-N) as a ligand for the GH-BP. hGH-N/hGH-V chimeric proteins, where the sequences encoded by exon 3 (amino acid residues 32-71, thought to be exposed on the molecule's surface and involved in receptor binding) were exchanged, also bound with similarly high affinities. A corresponding hGH-N/rat PRL chimeric protein had 25-fold reduced affinity for the GH-BP. We conclude that hGH-V is a potent somatogen in man, and that some of the manifestations of late pregnancy, such as increased insulin-like growth factor-I levels and coarsening of features, are probably related to the high circulating levels of hGH-V. GH-BP measurements in pregnancy must take into account BP saturation by endogenous hGH-V.

Functional Antagonism between Endogenous Mouse Growth Hormone (GH) and a GH Analog Results in Dwarf Transgenic Mice*

A dwarf transgenic mouse (DTM) line has been established in which mice express relatively high levels of a mutated bovine (b) GH gene. This bGH analog binds to mouse liver membrane preparations with an affinity similar to that of wild-type bGH. The mean growth ratio of these mice is approximately 0.7 relative to that of their nontransgenic littermates. Serum insulin-like growth factor-I (IGF-I) levels of DTM were found to be approximately half those in nontransgenic littermates. Liver GH receptor levels were up-regulated in DTM or wild-type bGH transgenic mice. Pituitary GH levels were negatively correlated with serum IGF-I concentrations. Wild-type bGH transgenic mice contain relatively high serum IGF-I and low pituitary GH levels, whereas DTM possess low serum IGF-I and high pituitary GH levels. The decrease in serum IGF-I resulting from the interaction between the bGH analog, the endogenous mouse GH, and GH receptor(s) apparently leads to a dwarf phenotype. These data suggest that this bGH analog has uncoupled GH ligand-receptor binding from IGF-I production and acts as a functional antagonist to the action of endogenous mGH.

4997916 Method for recovering a purified animal growth hormone or polypeptide analog thereof from a bacterial cell: Haim Aviv, Marian Gorecki, Avigdor Levanon, Amos Oppenheim, Tikva Vogel, Elisha Zeelon, Menachem Zeevi, Rehovot, Israel assigned to Bio-Technology General Corp

4997916 Method for recovering a purified animal growth hormone or polypeptide analog thereof from a bacterial cell: Haim Aviv, Marian Gorecki, Avigdor Levanon, Amos Oppenheim, Tikva Vogel, Elisha Zeelon, Menachem Zeevi, Rehovot, Israel assigned to Bio-Technology General Corp

Purification and properties of a recombinant DNA-derived ovine growth hormone analogue (oGH1) expressed in Escherichia coli

An Escherichia coli JM109 clone containing a plasmid, pOGHe101, based on pUC8 and the ovine GH (oGH) cDNA sequence, showed very high expression (up to 25% of total cell protein) of an oGH analogue (oGH1) after induction. oGH1 was found in the particulate fraction of induced bacteria, where electron-dense granules could be seen by electron microscopy. A simple method for the purification of oGH1 is described. The particulate fraction isolated from sonicated bacteria was dissolved in 6M guanidinium chloride containing dithiothreitol. After threefold dilution the proteins were reoxidized by gentle stirring overnight in air. Soluble renatured protein, recovered after dialysis, was further purified by ion-exchange and gel-filtration chromatography. Purified oGH1 had an Mr of 22,000, an isoelectric point of about 6.7 and an N-terminal sequence corresponding to that of oGH, with an extension of eight amino acids replacing the N-terminal alanine. oGH1 behaved similarly to authentic bovine GH in a radioimmunoassay, a radioreceptor assay and a weight-gain assay in hypophysectomized rats. Thus the renatured hormone appears to be correctly folded and the N-terminal extension has little or no effect on biological activity.

4831120 Method for recovering a purified animal growth hormone or polypeptide analog thereof from a bacterial cell

A method is provided for recovering a purified animal growth hormone or a polypeptide analog thereof having substantially the same amino acid sequence as, and the biological activity of, the corresponding naturally-occurring animal growth hormone from a bacterial cell in which the animal growth hormone or polypeptide analog has been produced by means of expression of a plasmid encoding the hormone or polypeptide analog which comprises: (a) disrupting the cell wall of the bacterial cell in a buffered neutral pH solution so as to produce a lysate containing precipitated hormone or polypeptide analog; (b) recovering the resulting precipitated hormone or polypeptide analog; (c) suspending the precipitated hormone or polypeptide analog so recovered in distilled water; (d) treating the resulting precipitate-containing suspension with a sodium hydroxide solution having an alkaline pH of about 11.8 so as to solubilize the precipitate and thus the hormone or polypeptide analog contained therein; (e) separating the solubilized hormone or polypeptide analog from other soluble components by gel filtration chromatography; and (f) subjecting the hormone or polypeptide analog thus separated to ion exchange chromatography to purify the hormone or analog and thereby recover purified hormone or polypeptide analog.

4831120 Method for recovering a purified animal growth hormone or polypeptide analog thereof from a bacterial cell: Haim Aviv, Marian Gorecki, Avigdor Levanon, Amos Oppenheim, Tikva Vogel, Elisha Zeelon, Menachem Zeevi, Rehovot, Israel assigned to Bio-Technology General Corp

4831120 Method for recovering a purified animal growth hormone or polypeptide analog thereof from a bacterial cell: Haim Aviv, Marian Gorecki, Avigdor Levanon, Amos Oppenheim, Tikva Vogel, Elisha Zeelon, Menachem Zeevi, Rehovot, Israel assigned to Bio-Technology General Corp

4831120 Method for recovering a purified animal growth hormone or polypeptide analog thereof from a bacterial cell: Haim Aviv, Marian Gorecki, Avigdor Levanon, Amos Oppenheim, Tikva Vogel, Elisha Zeelon, Menachem Zeevi, Rehovot, Israel assigned to Bio-Technology General Corp

4831120 Method for recovering a purified animal growth hormone or polypeptide analog thereof from a bacterial cell: Haim Aviv, Marian Gorecki, Avigdor Levanon, Amos Oppenheim, Tikva Vogel, Elisha Zeelon, Menachem Zeevi, Rehovot, Israel assigned to Bio-Technology General Corp

Intestinal adaptation in short-bowel syndrome.

After massive resection of the small intestine the remnant mucosa has an important capacity to enlarge the absorptive surface for the digestion, hydrolysis and absorption of nutrients. This intestinal adaptation is achieved by the interaction of various factors. Oral nutrients together with pancreatic biliary secretions stimulate the mucosa to become hyperplastic. Secondary to these luminal factors hormones play an important role in the adaptive process. Among the hormones, enteroglucagon is the most important growth promoting agent together with other growth factors such as epidermal growth factor, prostaglandin E2 and human growth hormone analogues, e.g. plerocercoid growth factor from the plerocercoid larvae of the tapeworm Spirometra mansonoides. The intestinal enterocyte is the target of these factors and within the cell the synthesis of polyamines, which are responsible for rapid growth, is the most essential step for the development of hyperplasia after resection. The rate limiting enzyme for polyamine synthesis ornithine decarboxylase (ODC) reacts to trophic stimuli with an increased activity. Thereafter rapid accumulation of tissue polyamines occurs. Blockade of ODC by specific inhibitors is accompanied by absence of intestinal hyperplasia after resection. Therefore it is concluded that ODC plays a key role in the intestinal adaptation of the remnant small bowel. To start and enhance intestinal hyperplasia after resection in patients with short bowel syndrome introduction of oral nutrition as soon as possible after operation is very important. On account of gastric acid hypersecretion the use of H2 receptor blocking agents is recommended. A decreased intestinal transit time is treated with loperamide.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of receptor affinity chromatography in purification of the growth hormone-like factor produced by plerocercoids of the tapeworm Spirometra mansonoides.

The plerocercoid stage of the tapeworm Spirometra mansonoides produces a functional analog of human growth hormone (hGH). Among the similarities between plerocercoid growth factor (PGF) and hGH is competition for the same receptors on rabbit liver membranes. To take advantage of this characteristic in a purification scheme for PGF, rabbit liver microsomes were solubilized in Triton X-100 and the hGH receptors were purified over an hGH affinity column. The purified receptors from six rabbit livers were coupled to Affi-Gel-10 to create a receptor affinity column which was used to purify PGF. Chromatography of crude PGF over the receptor column resulted in a 1044 fold increase in specific activity. SDS-PAGE in the presence of 2-mercaptoethanol showed that the affinity-purified PGF contained three protein bands with apparent Mrs of 27.5 K, 22 K, and 16.7 K. Injections of the partially-purified PGF into hypophysectomized rats produced a dose-dependent growth response and 400 ng eq of PGF each day for 10 days stimulated a growth response not significantly different from that produced by 250 micrograms of bovine GH each day. Receptor affinity chromatography was an effective method to purify small amounts of PGF in a single step with negligible loss of biological activity.