Methotrexate-induced amplification of the bovine lutropin genes in Chinese hamster ovary cells. Relative concentration of the alpha and beta subunits determines the extent of heterodimer assembly.

Abstract

Methotrexate-induced gene amplification increased the expression of biologically active bovine luteinizing hormone (bLH) approximately 11-fold after stable transfection of a line of Chinese hamster ovary cells with genes encoding dihydrofolate reductase and the alpha and beta subunits of bLH. Subsequent analysis of the bovine genes revealed that while the alpha gene was amplified in response to methotrexate selection, the LH beta subunit gene remained unaffected. This effect was probably due to the linkage of the alpha subunit gene with the dihydrofolate reductase gene, the selectable and methotrexate-sensitive marker in the plasmid construct. Prior to methotrexate selection, the concentration of LH beta mRNA and the rate of LH beta synthesis exceeded that of alpha subunit mRNA and protein. Stepwise selection with methotrexate led to a progressive increase in the synthesis and secretion of biologically active bLH. Enhanced production of bLH correlated directly with similar increases in both the steady-state level of alpha subunit mRNA and the relative synthesis rate of alpha subunit protein. Despite progressive changes in alpha subunit concentration, formation of the alpha/beta heterodimer was always incomplete, even when the concentration of alpha subunit exceeded that of LH beta. Cumulatively, these results are consistent with a model in which the extent of steady-state combination of the subunits is determined by the mutual affinity and concentration of both subunits within the lumen of the secretory pathway. This stands in contrast to the long held view that the extent of glycoprotein hormone assembly is limited by the concentration of the beta subunit.