Expression of a mutated bovine growth hormone gene suppresses growth of transgenic mice.

Abstract

To determine the importance of the third a-helix in bovine growth hormone (bGH) relative to growthrelated biological activities, the following experimental approach was used: (i) mutagenesis ofhelix III of bGH to generate an idealized amphiphilic helix; (ii) in vitro expression analyses of the mutated bGH gene in cultured mouse L cells; (iii) mouse liver membrane binding studies ofwild-type and mutated bGH; and (iv) expression ofthe mutated gene in the transgenic mouse. An altered bGH gene (pBGHlOA6-M8) was generated that encodes the following changes: glutamate-117 to leucine, glycine-119 to arginine, and alanine-122 to aspartate. The plasmid pBGHlOA6-M8 was shown to be expressed in, and its protein product secreted by, mouse L cells. The altered hormone possessed the same binding affinity to mouse liver membrane preparations as wild-type bGH. Transgenic mice containing the mutated bGH gene, however, showed a significant growthsuppressed phenotype. The degree of suppression was directly related to serum levels of the altered bGH molecule. >90% amino acid sequence identity between bGH and pGH, it is likely that bGH has a similar three-dimensional structure. By aligning these a-helical structures into a two-dimensional Edmundson wheel projection (24), it is clear that an amphiphilic a-helical segment exists between amino acid residues 109 and 126 in the third a-helical region (ref. 25; Fig. 1). However, amino acids 117, 119, and 122 are positioned so that an idealized amphiphilic a-helix is not generated.In this study, we have used the following approach to determine whether this amphiphilic a-helix is important in the growth-related properties ofbGH: (i) mutagenesis ofthe third a-helix of bGH so as to generate an idealized amphiphilic a-helix (Fig. 1); (ii) expression analyses of the wild-type and mutated bGH genes in cultured mouse L cells; (iii) mouse liver membrane binding studies comparing wild-type and mutated bGH; and (iv) production of transgenic mice containing wild-type and mutated bGH genes.

Discovery and mechanism of action of pegvisomant

“…Data from in vitro GHR binding studies showed no significant difference between wild-type bGH and 3 D bGH in their GHR interactions, indicating that 3 D bGH had a binding affinity similar to wild-type GH. Surprisingly, however, when the gene encoding 3 D bGH analogue was expressed in transgenic mice, it resulted in growth retardation; the transgenic mice that expressed the mutated GH were significantly smaller compared with nontransgenic littermates (10). This result was contrary to the expectation that the 3 D bGH transgenic mice would be larger than normal.…”

“…(Reprinted, with permission, from (2).) activation of the receptor (10). Structural studies then demonstrated that dual binding sites on hGH to two GHRs are required for receptor dimerisation and activation, which results in growth-promoting activities (7,8).…”

“…A series of studies was thus undertaken to determine whether modification of these three residues in the GH molecule that generate a perfect amphipathic a-helix would alter the growth-related properties of bGH. A mutated bGH gene was engineered in which the codon for Glu-117 was replaced with Leu (E117L), Gly-119 was replaced with Arg (G119R), and Ala-122 was replaced with Asp (A122D) (3 D bGH) (10). The resulting molecule possessed a perfect amphipathic a-helix with all hydrophobic residues below and hydrophilic residues above the midline of the helix (Fig.…”

Putative pathophysiological role of growth factors and cytokines in experimental diabetic kidney disease

“…A series of highly specific antagonists of the GH action has recently been developed for the potential therapeutic use in various pathophysiological conditions, including diabetes. Initially it was shown that alteration of single amino acids in the third a-helix of bovine (b)GH (residues 109±126) results in a GH antagonist [68,69]. In vitro experiments showed that this new group of GH antagonists binds to the GH receptor with the same affinity as native GH but in vivo a phenotypic dwarf animal characterized by low circulating IGF-I concentrations and a proportional body composition develops when the GH antagonist is expressed in transgenic (TG) mice [68,69].…”

“…Initially it was shown that alteration of single amino acids in the third a-helix of bovine (b)GH (residues 109±126) results in a GH antagonist [68,69]. In vitro experiments showed that this new group of GH antagonists binds to the GH receptor with the same affinity as native GH but in vivo a phenotypic dwarf animal characterized by low circulating IGF-I concentrations and a proportional body composition develops when the GH antagonist is expressed in transgenic (TG) mice [68,69]. Studies in long-term diabetic GH antagonist TG mice, that express the GH antagonist (bGH-G119R or hGH-G120R), have shown that these animals are protected against development of diabetic renal changes [70,71].…”