Growth Hormone Analog Research Articles

Growth hormone treatment during suppression of early puberty in adopted girls

Girls adopted from developing countries often have early or precocious puberty, requiring treatment with gonadotropin‐releasing hormone (GnRH) analogues. During such treatment decreased growth velocity is frequent. The aim of this investigation was to study whether the addition of growth hormone (GH) to GnRH analogue treatment improves height velocity and final height in girls with early or precocious puberty. Forty‐six girls with early or precocious puberty adopted from developing countries were randomized for treatment with GnRH analogue or a combination of GH and GnRH analogue. After 2 y of treatment the mean growth in the GH/GnRH analogue group was significantly higher, 14.6 cm, compared to 10.9 cm in the control group. The increase in bone age did not differ, while the difference in predicted adult height increased by 2.7 cm in favour of the combination group. Although data on final height are not yet available, combined GH/GnRH analogue treatment for 2 y resulted in a higher growth velocity and predicted final height compared to GnRH analogue treatment alone. □Girls, gonadotropin‐releasing hormone, growth hormone, precocious, puberty

Vibrational spectroscopic studies of solid recombinant bovine growth hormone and related growth hormone analogs

Infrared and Raman spectra have been obtained for lyophilized recombinant bovine growth hormone (r-bGH), partially reduced, and completely reduced r-bGH, plus a tryptic digest fragment of r-bGH. Amide I and II data indicate r-bGH to have substantial helical character. Partially reduced r-bGH, in which the carboxyl terminal disulfide bridge (residues 181, 189) has been cleaved, has slightly less helical content than r-bGH. The spectral data indicate that breaking the carboxyl terminal cystine link produces only localized structural alterations. The additional cleavage of the second disulfide bridge (residues 53 164) leads to a further decrease in helix content, accompanied by increases in β-sheet and disordered structures. A tryptic digest r-bGH fragment (residues 96–133), which contains a small amount of biological activity (≈10%), has predominantly helical structure.

Production and characterisation of deletion mutants of ovine growth hormone.

A number of analogues of ovine growth hormone (GH), in which regions of the hormone had been deleted, were produced by site-directed mutagenesis, and characterised by radioimmunoassays and radioreceptor assays. These analogues were based on a previously described variant (oGH1) in which an 8-residue extension replaces the N-terminal alanine of pituitary-derived ovine GH. Three analogues with deletions near the N-terminus were studied, with shorter extensions of 7 or 1-2 residues (oGH14, oGH5) or with the N-terminal sequence Ala-Phe-Pro- of pituitary-derived ovine GH replaced by Thr-Met-Ile-Thr- (oGH11). These modifications had little effect on potency in radioimmunoassays based on a polyclonal antibody and five different monoclonal antibodies (MABs), or in a radioreceptor assay, indicating that the N-terminal sequence was not included in the epitope binding to any of the monoclonal antibodies, or a major epitope binding to the polyclonal antibody, or in receptor binding site 1. A variant in which residues 133-139 were deleted retained full binding to 4 of the 5 MABs, suggesting correct folding, but markedly reduced binding to MAB OA16, suggesting that the epitope for this MAB includes some or all of these residues. This variant also failed to displace about 35% of labelled hormone from the polyclonal antibody studied, suggesting that residues 133-139 may be involved in a major epitope for this antibody. This variant showed slightly lower receptor binding activity than ovine GH. Two other deletion variants - oGH1Delta33-46 (equivalent to the naturally occurring 20K variant of human GH) and oGH1Delta180-191 (lacking the C-terminal 12 residues) showed poor folding efficiency and solubility, and low binding to all MABs except OA15, which has a linear epitope. The results suggest that these variants were incorrectly folded, but interestingly they did retain some activity in the receptor-binding assay (respectively about 5% and 0.5% of the activity of ovine GH itself).

Immune function in transgenic mice overexpressing growth hormone (GH) releasing hormone, GH or GH antagonist.

Effects of life-long exposure to high levels of homologous or heterologous growth hormone (GH) and effects of GH resistance on selected parameters of immune function were studied in adult male transgenic mice overexpressing GH releasing hormone (GHRH), bovine (b) GH or an antagonistic bGH analog. In metallothionein I (MT)-bGH transgenic mice with high peripheral levels of bovine GH, there were significant increases in the absolute weight of the thymus and the spleen and in the mitogenic responses of splenocytes to concanavalin A (ConA), lipopolysaccharide (LPS) and phytohemagglutinin (PHA), as compared to age-matched normal animals. There were no significant differences between MT-bGH transgenic and normal mice in splenocyte viability or in delayed-type hypersensitivity measured by the allergic contact dermatitis response to oxazolone. Similar results, including significant stimulation of splenocyte responses to ConA, LPS, and PHA, were obtained in MT-hGHRH transgenic mice in which overexpression of GHRH leads to striking pituitary enlargement and massive elevation of peripheral levels of homologous (mouse) GH. In MT-bGH-antagonist transgenic mice in which overexpression of an antagonistic bGH analog interferes with the actions of endogenous GH, spleen weight was reduced but proliferative responses of splenocytes to ConA, LPS, and PHA were not affected. It is concluded that overexpression of heterologous or homologous GH in transgenic mice can lead to significant stimulation of some parameters of immune function, whereas antagonism of GH action by expression of an antagonistic GH analog does not affect splenocyte responses to mitogens.

A vibrational spectroscopic assignment of the disulfide bridges in recombinant bovine growth hormone and growth hormone analogs

Disulfide stretching vibrations for bovine growth hormone (bGH) occur in a vibrational envelope centered at 540 cm −1 which spans 480–580 cm −1. A multitude of vibrational bands present in this envelope, that are not related to disulfide stretching, emphasize the need for model compounds when assigning S–S stretching modes. Raman spectroscopic data for bGH analogs, in which one or both of the two disulfide bridges have been selectively cleaved, have been used to characterize the S–S stretching envelope for the two cystine links in bGH. The Raman data for the r-bGH analogs indicate that the number of disulfide bonds present in r-bGH is determined, not by the observance of the presence or absence of a single spectral peak, but by the relative intensity of vibrational envelope from 520–560 cm −1. Cleavage of disulfide bridges in bGH results in a general decrease in vibrational spectral intensity in the 520–560 cm −1 range. This decrease in intensity is proportional to the number of cystine links severed.

ALTERATION OF THE INSULIN-LIKE GROWTH FACTOR SYSTEM OF MITOGENS IN HYPERPLASTIC BLADDERS OF PARAPLEGIC RATS

Smooth muscle cells of the bladder retain the ability to proliferate in response to injury or mechanical stimulation. Insulin-like growth factor (IGF) I is a mitogenic and hypertrophic agent for smooth muscle cells. The purpose of this study is to examine if spinal cord injury could lead to bladder hypertrophy via the IGF system. The study involved spinal cord transection of female Sprague Dawley rats (approximately 250 to 300 gm.). Six weeks following surgery the urinary bladder was collected. Northern and Western blotting, and IGF-I receptor (IGF-IR) affinity labeling were used to determine the expression of the IGF system, IGFBP levels, and IGF-IR levels respectively. Chronic spinal cord injury leads to an increase in the wet weight of the bladder and in the level of proliferation cell nuclear antigen expression. IGF-I mRNA levels increase, while IGFBP 3 and 5 mRNA and protein levels dramatically decrease. The gene expression of IGFBP 2 and 4 varies from rat to rat, and IGF-IR expression slightly increases. Our results suggest that following spinal cord injury, overexpression of IGF-I and underexpression of IGFBP 3 and 5 may lead to hyperplasia of the smooth muscle layer of the bladder.

Molecular and biochemical aspects of chitin synthesis inhibition.

Chitin, is a beta-1,4-linked aminopolysacharide homopolymer of GlcNAc that occurs as a glycoprotein in the exoskeleton of arthropods, the cell wall of fungi and in various components of diverse invertebrates. It is synthesized in two different ways: in fungi the chitin synthase enzyme occurs as an inactive zymogen in vesicles called chitosomes and requires proteolytic activation; in arthropods this enzyme is membrane-bound and catalyzes the addition of GlcNAc units to a dolichol carrier. Chitin is degraded by three different chitinases, the endochitinase that degrades chitin into oligosaccharides of differing chain lengths, the exochitinase that degrades oligosaccharides into diacetylchitobiose and chitobiase, which degrades diacetylchitobiose into GlcNAc monomers. Inhibition of chitin synthesis as well as degradation can both result in deleterious effects that are often similar. Chitin synthesis can be blocked during the various steps by a variety of antibiotics, metabolic inhibitors, insect growth regulators, alkaloids and hormone analogs. During the molting process in arthropods, genes are sequentially expressed and repressed by developmental hormones. When these hormones or their analogs are administered temporally out of sequence, it can result in the blocking of cuticle formation, including chitin synthesis. With the advent of biotechnology and the availability of both complementary DNA and antibody probes, it is possible to develop high throughput assays for discovering new chemicals that can block chitin formation. Chitin synthesis inhibitors as well as inhibitors of chitin degradation that produce similar effects are promising agents for controlling insect pests, fungal pathogens and helminthic parasites.

A study of the scale-up of reversed-phase liquid chromatography

Abstract A general rate model for liquid chromatography which considers nonlinear isotherms and various mass transfer effects, including axial dispersion, interfacial film mass transfer and intraparticle diffusion is applied to the modeling and scale-up of reversed-phase liquid chromatography. The model is solved with a fortran program which is run on a personal computer. With a few simple experiments using a small analytical column, the binding characteristics and the porosity of the packing particles are determined. Mass transfer parameters are evaluated using existing correlations in the literature without any experimentation. Human growth hormone and a recombinant human growth hormone analog (hGHG120R) were used as protein samples in experiments. Gradient elution profiles of a preparative column are predicted without a posteriori data.

Final height after combined growth hormone and gonadotropin-releasing hormone analog therapy in short healthy children entering into normally time puberty

Final height after combined growth hormone and gonadotropin-releasing hormone analog therapy in short healthy children entering into normally time puberty

Selective modification at the N-terminal region of human growth hormone that shows antagonistic activity

A new analogue of recombinant human growth hormone (hGH), hGH des(1–6,14) was expressed in Escherichia coli, refolded and purified to homogeneity. The mutation decreased the hormone's ability to bind lactogenic and somatogenic receptors through its site 1, and almost completely abolished its ability to bind these receptors through site 2, as evidenced by both binding and gel-filtration experiments. More specifically, the binding to prolactin receptors (PRLRs) from various species or their soluble recombinant extracellular domains (ECDs) was decreased 1.5–4-fold, whereas the binding to hGH receptor (hGHR) was decreased 10–85-fold. These changes caused an almost total loss of hormone agonistic activity in several in vitro bioassays and subsequently, the hGH des(1–6,14) analogue acquired antagonistic properties. This antagonistic activity was dependent upon modification of site 1. In those cases in which the binding was reduced only slightly, e.g. binding to rabbit PRLRs, hGH des(1–6,14) acted as a strong antagonist, whereas in others in which the binding of site 1 was reduced to a higher degree, such as other PRLRs and, in particular, hGHR, the antagonistic activity was correspondingly weaker. Circular dichroism spectra of the analogue suggested that these changes do not result from a decrease in overall α-helix content, but rather from minor local structural modifications at the N-terminus.

Recombinant analogues of prolactin, growth hormone, and placental lactogen: correlations between physical structure, binding characteristics, and activity.

The availability of recombinant growth hormones, prolactins, placental lactogens and a few soluble extracellular domains of their receptors have extended our ability to study the interaction of somatogenic and lactogenic hormones with their receptors. Modifications of their respective cDNAs have enabled the preparation of sufficient amounts of the corresponding proteins. The present review summarizes two aspects of these interactions: (a) the relationship between binding, the apparent ability to dimerize the receptors and biological activities in vitro and in vivo; and (b) the effect of mutations on selective changes in the ability of human growth hormone and bovine placental lactogen to interact with somatogenic and lactogenic receptors. In view of this summary, strategies for preparing a second generation of biologically relevant recombinant hormones are discussed.

Differential in vivo activities of bovine growth hormone analogues.

In rodents, bovine (b) growth hormone (GH) binds only to GH receptors, while human (h) GH binds to both GH and PRL receptors. The phenotypic consequences of expression of bGH and hGH in transgenic mice are different and, in some cases, opposite. In the present study, site-directed in vitro mutagenesis of the bGH gene was used systematically to eliminate its differences from hGH at one, two, three or four suspected of conferring lactogenic activity: D11, H18, S57 and T60, respectively (corresponding to sites 12, 19, 57 and 60 of the bGH molecule). The resulting bGH analogues were expressed in cell lines and in transgenic mice. All of the seven bGH analogues produced retained their ability to bind to GH receptors and exhibited somatogenic activity in vitro and in vivo. However, none of them were able to bind to PRL receptors or to elicit detectable lactogenic response in vitro. Transgenic animals expressing any of the generated analogues were characterized by gigantism and splanchnomegaly. The effects of expression of each of the double, triple or quadruple mutants on the seminal vesicle weight resembled the effects of wild-type hGH and differed from the effects of expression of wild-type bGH. There were differences between the effects of the expression of different bGH analogues on plasma PRL levels and on the PRL response to pharmacological blockade of catecholamine synthesis. Plasma LH levels in ovariectomized females were suppressed by several of the analogues tested, an effect not seen in animals expressing wild-type bGH or hGH. Dopamine turnover in the median eminence of male mice was also altered in animals expressing different bGH analogues but not in those expressing wild-type bGH or hGH. In ovariectomized females, the effects of different bGH analogs on the turnover of dopamine and norepinephrine in the median eminence included changes resembling those detected in animals expressing hGH, as well as alterations differing from the effects of both bGH and hGH. The results indicate that biological actions of these bGH analogues cannot be characterized simply in terms of enhanced or reduced somatogenic or lactogenic activity and raise a possibility that different sites, domains or features of tri-dimensional structure of GH are involved in its actions on different cellular targets.

Growth Hormone (GH) and a GH Antagonist Promote GH Receptor Dimerization and Internalization

It has previously been shown that a human growth hormone (hGH) analog, hGH-G120R, acts as a GH antagonist (Chen, W. Y., Wight, D. C. , Wagner, T. E., and Kopchick, J. J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 5061-5065; Chen, W. Y., White, M. E., Wagner, T. E., and Kopchick, J. J. (1991) Endocrinology 129, 1402-1408; Chen, W. Y., Chen, N-Y., Yun, J., Wang, X. Z., Wagner, T. E., and Kopchick, J. J. (1994) J. Biol. Chem. 269, 15892-15897). In this study, we report the ability of hGH and hGH-G120R to be internalized by GH receptor expressing cells. Additionally, results of chemical cross-linking experiments revealed that both native hGH and hGH-G120R form complexes similar in size to that expected for hGH when bound to recombinant hGH-binding protein (bp). The molecular mass of the complex was determined to be approximately 280 kDa which is consistent with multiple receptors interacting with the ligand. The predominant radiolabeled band detected was a complex of approximately 140 kDa which probably represents one GH molecule bound to one GH receptor. The cross-linked complexes were not detected in the presence of excess unlabeled hGH or hGH-G120R and were not observed in cells which do not express detectable levels of GH receptors. Also, GH induced tyrosine phosphorylation of a complex of proteins of approximately 95 kDa in these cells whereas hGH-G120R did not. Thus, we have separated the hGH or hGH-G120R/GHR binding and internalization capabilities from the ability to stimulate tyrosine phosphorylation of intracellular proteins.

Diabetic nephropathy: molecular analysis of extracellular matrix and clinical studies update.

Progressive glomerulosclerosis associated with decreasing kidney function, resulting in end-stage renal failure, is the major finding in diabetic nephropathy. There are multiple components of the extracellular matrix, and neither the exact composition nor the factors responsible for its increase in diabetic nephropathy is known. We examined matrix synthesis and degradation at the level of gene expression and ECM composition, in the glomeruli of diabetic mice transgenic for a mutated bGH and their littermate controls, and in renal biopsies of patients with diabetic nephropathy. We found that streptozotocin-treated C57BL/SJL mice developed histological lesions closely resembling human diabetic glomerulosclerosis, whereas there were no glomerular lesions in diabetic mice transgenic for a growth hormone analogue which competes with native GH and results in dwarfism. We found that diabetic glomerulosclerosis in mice was associated with an increase in type IV collagen and laminin synthesis, and that these changes are selectively inhibited in dwarf mice transgenic for the mutant GH analogue. Preliminary examination of glomeruli isolated from renal biopsies revealed a homogeneous elevation in the alpha 2/alpha 3IV ratio among diabetics. These data suggested that diabetic nephropathy is characterized by modifications in ECM gene regulation in both mouse models and in diabetics, and that GH may promote the susceptibility to this complication. Finally, the data from the Diabetes Control and Complications Trial is updated, emphasizing the importance of close glucose control in the amelioration of an elevated albumin excretion rate as a continuous function of the level of control.

Effect of Salts on the Structure of a Potent Analog of Growth Hormone Releasing Hormone As Determined by Optical Spectroscopy

Optical spectroscopic methods (circular dichroism, analytical ultracentrifugation, and static light scattering) were employed to study the solution behavior of an N‐terminal‐acylated 76‐residue analog of growth hormone releasing hormone (GHRH). The GHRH analog had a 30% helical configuration in aqueous acidic solution, unlike other GHRH analogs that had a random coil configuration in aqueous solutions, and self‐associated. High concentrations (7 M) of urea were required to obtain monomeric peptide, but such urea concentrations unfolded the peptide. Therefore, the folding and self‐association were related events. The self‐association and helix content were increased by the addition of various cations (e.g., Na+, Ca2+). In 7M urea, when these cations were added, the peptide started to refold toward its aqueous conformation in a pH dependent manner; at low pH (2.5–3.5) the peptide folded to approximately 50% of the native configuration. At neutral pH (>6) only small changes were seen when salts were added. When CaCl2was added to a solution containing 7M urea at pH 2.5, the self‐association of the peptide increased with the concentration of CaCl2. Therefore, the salt dependent self‐association observed in aqueous solvents was present in 7M urea. Residues 45–76 in this GHRH analog, which are not present outside the hypothalamus, are probably needed to interact with a folding chaperonein vivo and provide stability for successful membrane transport and maintenance of biological activity.

Combination treatment with growth hormone and gonadotropin-releasing hormone analogs in short normal girls

To improve final adult height, we treated with growth hormone (0.65 ± 0.07 [mean ± SD] IU · kg - 1 · wk -1) and gonadotropin-releasing hormone analogs (66 ± 9 μg · kg - 1 every 28 days) a group of seven short normal girls in early puberty with a chronologic age (CA) of 11.50 ± 0.95 years, predicted adult height (PAH) lower (0.003 < p < 0.001) than mean target height, and without any endocrine abnormalities. The results were compared with those obtained in a similar group of seven untreated girls considered as control subjects. The mean period of combined therapy was 2.01 ± 0.52 years; in two subjects treatment is still in progress. The value of height standard deviation score for bone age (BA) improved from −1.69 ± 0.47 to −1.04 ± 0.56 ( p = 0.001); height age (HA)/BA ratio also increased from 0.83 ± 0.05 to 0.90 ± 0.04 ( p <0.01), as did PAH (from 146.8 ± 4.4 to 152.9 ± 3.6 cm; p <0.002). The ratio of gain in HA to gain in BA was 2.08 ± 0.78. Pubertal stages showed an arrest in five cases and a regression in the other two girls. After administration of gonadotropin-releasing hormone analogs was interrupted, in four of five girls growth hormone was administered alone for a further period of 6 to 18 months to improve their physiologic growth spurt. The present height in five girls is higher than PAH before therapy. In the treated girls the height values for BA, for BA/CA and HA/BA ratios, and for PAH were higher (0.002 < p <0.04) than those in control subjects. This preliminary study demonstrates that combination therapy with growth hormone and gonadotropin-releasing hormone analogs in short, endocrinologically normal girls may be useful in improving both height prognosis and predicted adult height. Further studies are necessary to reach definitive conclusions regarding the efficacy of this kind of therapy. (J P EDIATR 1995;126:468-73)

Effect of newly developed analogue of growth hormone releasing peptide [D-Ala-D-beta Nal-Ala-Trp-D-Phe-Lys-NH2 (KP-102)] on growth hormone secretion in adult male rats

The effect of a newly developed hexapeptide; D-Ala-D-beta Nal-Ala-Trp-D-Phe-Lys-NH2 (KP-102) on GH secretion was studied in adult male rats. KP-102 induced a large plasma GH response at a dose of 0.5 micrograms/kg BW in conscious rats, although GH-releasing abilities of KP-102 varied markedly depending on the phase of GH secretion. Among urethan-anesthetized rats, KP-102 alone exerted a small influence on GH secretion, but it produced a large plasma GH response in the presence of exogenous GH-releasing factor (GRF). The rest of studies were performed on urethan anesthetized rats. During intermittent administration of GRF, the somatotropes became refractory to a large bolus dose of GRF, but KP-102 induced a marked increase of plasma GH. The GH response to KP-102 alone or KP-102 with GRF was significantly augmented when cysteamine HCl was previously administered. Although KP-102 and GRF acted synergistically on GH secretion in control animals, they acted additively in cysteamine-administered rats. Taken together, these findings suggest that the KP-102-induced GH secretion largely depends on GRF and the secretagogue potentiates the GRF effect by antagonizing the SS action at the level of somatotropes. It is concluded that KP-102 alone or in combination with GRF provides a means of stimulating GH secretion in the face of elevated inhibitory tone mediated by SS.

In vitro and in vivo studies of the antagonistic effects of human growth hormone analogs.

A bovine growth hormone (bGH) analog, bGH-G119R, has been shown to act as a functional antagonist of GH activity both in vitro and in vivo. In the present study, human (h) GH analogs with alterations in the third alpha-helix (G120A, G120R) and N terminus (I4A) were generated. These two regions have been reported to form binding site 2 in hGH, which is involved in in vitro dimerization of the GH receptor (GHR). The biological activities of these hGH analogs were tested in vitro by a radioreceptor assay and an assay to test the ability of the molecules to induce tyrosine phosphorylation of a 93-kDa protein in a human lymphocyte line, IM-9. The growth rate of transgenic mice that express different hGH analogs was used as an in vivo test of the activity of the molecules. The results demonstrated that G120R is a potent hGH antagonist both in vitro and in vivo. Transgenic mice expressing G120R demonstrated a growth-suppressed phenotype. However, I4A, which has been demonstrated to be a potent inhibitor of in vitro GHR dimerization, exhibited full growth promoting activity in transgenic mice. Thus, the ability of hGH analogs to induce GHR dimerization in vitro and the ability to promote growth in vivo are not directly correlated.

Growth hormone (GH) binding and effects of GH analogs in transgenic mice.

Overexpression of human (h) or bovine (b) growth hormone (GH) in transgenic mice is associated with marked (2- to 12-fold) and significant increase in hepatic binding of GH and prolactin (PRL). This is due to an increase in the number of GH and PRL receptors (GHR, PRLR) per mg of microsomal protein without changes in binding affinity. Comparison of results obtained in transgenic animals expressing bGH with a mouse metallothionein (MT) or a rat phosphoenolpyruvate carboxykinase (PEPCK) promoter suggests that effects of bGH on hepatic GHR and PRLR do not require GH overexpression during fetal life and, within the dose range tested, the effects on PRLR are not dose dependent. The increase in hepatic GHR was accompanied by significant increases in plasma GH-binding protein (GHBP) and in mean residence time of injected GH. Thus, life-long elevation of peripheral GH levels alters the availability of both free GH and GHR. Site-directed in vitro mutagenesis was used to produce hGH and bGH analogs mutated within one of the sites involved in binding to GHR and PRLR. Mutating hGH to produce amino acid identity with bGH at Position 11, 18 (within Helix 1), 57, or 60 (within the loop between Helix 1 and 2) did not affect binding to GHR in vitro, or somatotropic activity in transgenic mice in vivo but reduced lactogenic activity in Nb2 cells by 22%-45%. Mutations of bGH designed to produce amino acid identity with hGH at one to four of the corresponding positions in the bGH molecule did not interfere with binding to GHR or somatotropic activity in vivo, and failed to produce significant binding to PRLR but resulted in alterations in the effects on the hypothalamic and anterior pituitary function in transgenic mice. Apparently region(s) outside the domains examined are essential for lactogenic activity of hGH, and different portions of the GH molecule are responsible for its diverse actions in vivo.

In vitro and in vivo studies of antagonistic effects of human growth hormone analogs.

A bovine growth hormone (bGH) analog, bGH-G119R, has been shown to act as a functional antagonist of GH activity both in vitro and in vivo. In the present study, human (h) GH analogs with alterations in the third alpha-helix (G120A, G120R) and N terminus (I4A) were generated. These two regions have been reported to form binding site 2 in hGH, which is involved in in vitro dimerization of the GH receptor (GHR). The biological activities of these hGH analogs were tested in vitro by a radioreceptor assay and an assay to test the ability of the molecules to induce tyrosine phosphorylation of a 93-kDa protein in a human lymphocyte line, IM-9. The growth rate of transgenic mice that express different hGH analogs was used as an in vivo test of the activity of the molecules. The results demonstrated that G120R is a potent hGH antagonist both in vitro and in vivo. Transgenic mice expressing G120R demonstrated a growth-suppressed phenotype. However, I4A, which has been demonstrated to be a potent inhibitor of in vitro GHR dimerization, exhibited full growth promoting activity in transgenic mice. Thus, the ability of hGH analogs to induce GHR dimerization in vitro and the ability to promote growth in vivo are not directly correlated.