Functional domains of human growth hormone necessary for the adipogenic activity of hGH/hPL chimeric molecules

Genetic analysis through construction of chimeric genes and their transfect ion in mammalian cells could provide a better understanding of biological f unctions of native or modified proteins, and would allow the design of new gene constructs encoding peptides that mimic or block ligand interaction wi th target tissues, To identify the hGH domains responsible for induction of adipose differentiation we constructed hGH/hPL chimeric molecules using ho mologous DNA mutagenesis, since hGH, but not human placental lactogen (hPL) , promotes adipose differentiation in mouse 3T3-F442A cells. We assayed the ir adipogenic activity in an autocrine/paracrine biological model consistin g of transiently transfected 3T3-F442A cells with the chimeric constructs. Plasmid DNAs carrying these constructs were transfected into growing 3T3-F4 42A cells, and cultures were further maintained for 7 days to differentiate into adipocytes. Secretion of transfected hGH/hPL chimeric proteins into t he medium was in the range of 5-25 ng/ml, Adipogenic activity was a propert y only of those chimeric proteins that contained hGH exon III together with either hGH exon II or hGH IV. Our results also suggest that hGH binding si te-2 is composed of two structural subdomains: subsite 2A encoded by exon I I of hGH and subsite-2B encoded by exon IV. We also suggest that full adipo genic activity requires the presence of binding site-1 and any of the subsi tes of binding site-2. This simple autocrine/paracrine biological model of gene transfection allows the analysis of specific biological activity of pr oducts encoded by modified genes.