We have cloned a member of the steroid hormone receptor superfamily of ligand-activated transcription factors. The receptor homologue is activated by a diverse class of rodent hepatocarcinogens that causes proliferation of peroxisomes. Identification of a peroxisome proliferator-activated receptor should help elucidate the mechanism of the hypolipidaemic effect of these hepatocarcinogens and aid evaluation of their potential carcinogenic risk to man.

Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators

I. Introduction II. Molecular Aspects A. PPAR isotypes: identity, genomic organization and chromosomal localization B. DNA binding properties C. PPAR ligand-binding properties D. Alternative pathways for PPAR activation E. PPAR-mediated transactivation properties III. Physiological Aspects A. Differential expression of PPAR mRNAs B. PPAR target genes and functions in fatty acid metabolism C. PPARs and control of inflammatory responses D. PPARs and atherosclerosis E. PPARs and the development of the fetal epidermal permeability barrier F. PPARs, carcinogenesis, and control of the cell cycle IV. Conclusions

Peroxisome proliferator-activated receptors: nuclear control of metabolism.

A serine/threonine kinase, named protein kinase B (PKB) for its sequence homology to both protein kinase A and C, has previously been isolated. PKB, which is identical to the kinase Rac, was later found to be the cellular homologue of the transforming v-Akt. Here we show that PKB is activated by stimuli such as insulin, platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Activation of PKB was inhibited by the phosphatidylinositol-3-OH kinase (PI(3)K) inhibitor wortmannin and by coexpression of a dominant-negative mutant of PI(3)K. PDGF receptor mutants that lack detectable associated PI(3)K activity also fail to induce PKB activation, PKB kinase activity is correlated with phosphorylation of PKB on serine. Finally, we show that a constructed Gag-PKB fusion protein, homologous to the v-akt oncogene, displays significantly increased ligand-independent kinase activity. Furthermore, this activity is sufficient to activate the p70 S6-kinase (p70S6K). These results suggest a role for PKB in PI(3)K-mediated signal transduction.

Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction

Small synthetic molecules termed growth hormone secretagogues (GHSs) act on the pituitary gland and the hypothalamus to stimulate and amplify pulsatile growth hormone (GH) release. A heterotrimeric GTP-binding protein (G protein)-coupled receptor (GPC-R) of the pituitary and arcuate ventro-medial and infundibular hypothalamus of swine and humans was cloned and was shown to be the target of the GHSs. On the basis of its pharmacological and molecular characterization, this GPC-R defines a neuroendocrine pathway for the control of pulsatile GH release and supports the notion that the GHSs mimic an undiscovered hormone.

https://science.sciencemag.org/content/sci/273/5277/974.full.pdf

A Receptor in Pituitary and Hypothalamus That Functions in Growth Hormone Release

The PPARs: from orphan receptors to drug discovery.

The ability to engineer specific modifications within the coding region of a structural gene is a powerful tool with which to study the relationship between protein structure and function. The process of site-directed mutagenesis involves a) mutation of the target gene, b) verification of the mutation by sequencing and c) expression of the mutated gene. We describe here a vector, pSG5 (Fig.lA), in which each of these steps may be performed. pSG5 was constructed essentially by combining the eukaryotic expression vector, pKCR2 (1), and the high copy plasmid vector Bluescribe Ml3+ (Stratagene). The principle features of pSG5 are a) unique EcoRI and BamHI restriction enzyme sites for insertion of cDNAs, b) production of single stranded DNA containing the antisense strand of the structural gene for mutagenesis and sequencing, c) high yields of double stranded DNA, d) in vivo expression from the SV40 early promoter and e) in vitro expression from the T7 promoter situated just upstream of the cDNA insertion site. Expression from pSG5 was tested after insertion of a cDNA encoding the human oestrogen receptor (2) (ER) into the EcoRI site to produce pSG5-ER. Fig.IB shows an SDS polyacrylamide gel of the 66Kd ER protein synthesised in a rabbit reticulocyte cell-free translation cocktail programmed with mRNA transcribed from the T7 promoter of either Bluescribe-ER (BSM-ER (3)) or pSG5-ER. The ER when expressed in HeLa cells stimulates transcription from oestrogen-responsive 'reporter' genes (4) (vitellogenin-TK-globin (VTG) in Fig. 1C). Using quantitative SI nuclease analysis with an internal reference gene (RXF in Fig.lC), the ER expressed from either pSG5-ER or pKCR2-ER stimulates gene transcription equally well indicating that equivalent levels of ER are expressed in vivo using either of these two vectors.

Isabelle Issemann

Papers

A versatile in vivo and in vitro eukaryotic expression vector for protein engineering

A role for fatty acids and liver fatty acid binding protein in peroxisome proliferation

The molecular mechanism of peroxisome proliferator action: a model for species differences and mechanistic risk assessment