Wen Y. Chen

A Growth Hormone (GH) Analog that Antagonizes the Lipolytic Effect but Retains Full Insulin-Like (Antilipolytic) Activity of GH

Abstract An analog of bovine growth hormone (bGH–M8: [Leu117, Arg119, Asp122]–bGH) with an idealized amphiphilic third αhelix has been proposed to be a functional antagonist of GH. In accordance with this proposition, bGH–M8 profoundly inhibited bGH–stimulated lipolysis by chicken adipose tissue <in vitro. bGH–M8 alone was a weak agonist in the lipolytic assay (1.9% the potency of bGH). The present evidence indicates that bGH–M8 is a competitive antagonist of the lipolytic action of GH based upon the following results: (i) increasing concentrations of bGH–M8 (antagonist) produce progressively greater inhibition of GH–stimulated lipolysis; (ii) increasing concentrations of bGH (agonist) are capable of overcoming this antagonism; and (iii) Schild plot analysis (slope = − 0.94) suggests a receptor antagonist with an equilibrium dissociation constant (K B) of 4.54 nM. In contrast to the antagonistic effects of bGH–M8 on bGH–stimulated lipolysis, bGH–M8 retained full insulin–like (“antilipolytic”) activity (i.e., inhibition of glucagon–induced lipolysis). bGH–M8 and bGH were similarly potent in eliciting antilipolytic effects < in vitro. Moreover, the antilipolytic effects of bGH–M8 and bGH were additive. Therefore, the third α–helix (particularly residues 117, 119, and 122) of bGH contains major structural determinants for the lipolytic effects of GH. The ability of bGH–M8 to act as an antagonist for at least one action of GH (lipolysis) while being a full agonist for another (antilipolysis) suggests that different domains of GH are responsible for its various biologic activities, possibly involving different binding sites and/or signal transduction mechanisms.

Expression of a functional porcine growth hormone receptor cDNA in mouse L cells

Porcine (p) growth hormone receptor (GHR) complementary DNA (cDNA) has been cloned and the primary amino acid structure was deduced from the nucleotide sequence. A comparison of pGHR to other GHRs revealed an approximately 70% similarity in amino acid sequence (Cioffi et al., 1990). Hybridization of this receptor cDNA to RNA samples isolated from various porcine tissues revealed a single RNA band of 4.2 kb. The full-length pGHR cDNA was subcloned into an eukaryotic expression vector, transcription of which was directed by the mouse metallothionein-I transcriptional regulatory sequence. Stable mouse L cell lines which express the pGHR cDNA were established. Approximately 80% of the cell lines were found to possess pGHR mRNA (approximately 2 kb) which corresponds to the length of the cloned pGHR cDNA. Binding studies showed that the stable cell lines were capable of specifically binding 125I-labeled pGH with a dissociation constant (Kd) of approximately 1.0 nM. The apparent molecular mass of the receptor, as determined by cross-linking studies, was found to be 118 kDa. Also, the receptor-ligand complex could be internalized. These results suggest that an active form of pGHR had been cloned and stably expressed in mouse L cells.

Human Growth Hormone Antagonist (G120R) Delivered by a Murine Yolk Sac Cell‐Derived Mini‐Organ Decreases the Growth Rate of Mice

Long‐term cultured murine embryonic yolk sac cells that are capable of forming capillary structures when cultured on base membrane proteins (Matrigel) were successfully transfected with a human growth hormone antagonist (G120R) gene. Cells that stably express relatively high levels of G120R were co‐implanted s.c. with Matrigel into BALB/c mice. G120R can be detected in the sera of those implanted mice for more than 14 days at levels from 4 ng/ml to 28 ng/ml. The insulin‐like growth factor‐1 levels in the sera of those implanted mice were significantly affected by the delivered G120R. One of the physiological effects of G120R delivered by this murine embryonic yolk sac cell‐derived mini‐organ system is to decrease the growth rate of the implanted mice. This gene delivery system can also be used as an alternative to transgenic animals to study protein function in vivo.