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B Enberg, A Hulthén, C Möller, G Norstedt, and S M Francis


The mechanism by which GH transmits a signal to the nucleus via its membrane-bound receptor is unknown. To study this process, Buffalo rat liver (BRL), rat hepatoma (FAO), human hepatoma (HepG2) and Chinese hamster ovary (CHO) cell lines were transfected with GH receptor cDNA, and stable clones expressing GH receptor mRNA and protein were selected. From previous in vivo studies it is known that GH regulates the expression of the rat hepatic serine protease inhibitor (SPI) 2.1 gene at the transcriptional level. However, in all the cell lines tested, SPI gene expression was less than 0·2% of that measured in rat liver, and GH did not affect the expression of the endogenous SPI gene in GH receptor-expressing cells.

A 45 bp GH-responsive element (GHRE) has previously been defined in the SPI 2.1 gene. A construct containing six repeats of this GHRE was assembled with the thymidine kinase promoter and a chloramphenicol acetyl transferase (CAT) reporter gene. Transient transfection of this reporter gene resulted in GH stimulation of CAT activity in all GH receptor-transfected cell lines. A 33-fold induction was measured in the GH receptor-expressing BRL cells. Induction of CAT activity was observed after 8 h of GH treatment in the BRL-GHR638 cell line. Stable BRL cell lines expressing GH receptors with carboxy-terminal truncations (GHR380 and GHR454) did not show increased CAT activity on GH stimulation. This suggests that more than half of the intracellular domain of the GH receptor is required to activate transcription of the SPI 2.1 gene.

It is concluded that the use of GH receptor-expressing cell lines in combination with the GH-regulated reporter system described here provides a good model for studying intracellular signalling after GH stimulation.

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G. L. Francis, M. Ross, F. J. Ballard, S. J. Milner, C. Senn, K. A. McNeil, J. C. Wallace, R. King, and J. R. E. Wells


An efficient expression system in Escherichia coli for several biologically active insulin-like growth factor-I (IGF-I) fusion peptide analogues is described. These novel IGF-I fusion protein analogues have properties that make them very useful reagents in the investigation of IGF-I action. The analogues comprise an IGF-I sequence and the first 11 amino acids of methionyl porcine growth hormone (pGH) and include [Met1]-pGH(1–11)-Val-Asn-IGF-I, which contains the authentic IGF-I sequence, and two analogues, [Met1]-pGH(1–11)-Val-Asn-[Gly3]-IGF-I and [Met1]-pGH(1–11)-Val-Asn-[Arg3]-IGF-I, where Glu3 in the human IGF-I sequence has been replaced by Gly or Arg respectively. The three peptides are referred to as Long IGF-I, Long [Gly3]-IGF-I or Long [Arg3]-IGF-I depending on the IGF-I sequence present. Production of the purified fusion peptides was aided by folding the reduced and denatured fusion peptide sequence under conditions that gave very high yields of biologically active product. Introduction of a hydrophobic N-terminal extension peptide appears to facilitate the correct folding of the IGF-I analogues compared with that obtained previously when folding normal-length IGFs. The biological activities of the IGF-I fusion peptides were compared with authentic IGF-I and the truncated analogue, des(1–3)IGF-I. In L6 rat myoblasts, all the analogues were more potent than authentic IGF-I in their abilities to stimulate protein and DNA synthesis and inhibit protein breakdown. In H35 hepatoma cells, where the IGFs act through the insulin receptor, the Long IGF-I analogues maintained a similar potency relative to IGF-I as was observed in the L6 myoblasts. The order of biological potency in cell lines secreting IGF-binding proteins (IGFBPs) into the medium was Long [Arg3]IGF-I-des(1–3)IGF-I>Long [Gly3]-IGF-I>Long IGF-I>IGF-I. In chicken embryo fibroblasts, a cell line that does not secrete detectable IGFBPs into the medium, Long [Arg3]-IGF-I, was less potent than IGF-I. Investigation of receptor and IGFBP association by these analogues reinforced our previous findings that N-terminal analogues of IGF-I show increased biological potency due to changes in the degree of their IGFBP interactions.