Publications

The potential effects of insulin and insulin-like growth factor I (IGF-I) on mesangial cell (MC) metabolism and growth were examined. Radiolabeled insulin or IGF-I were incubated with cell membranes from rapidly proliferating (subconfluent) or nonproliferating (confluent) MC in the presence of increasing concentrations of unlabeled heterologous and homologous ligands (0-10(-6) M). Insulin binding to MC was specific and saturable, with Scatchard analysis of binding data showing the characteristic curvilinear plot. The predicted insulin binding maximum of 4.2 X 10(-12) M/100 micrograms protein for a theoretical high affinity site was consistent with a relatively low density of receptors, which were the same in proliferating and nonproliferating cell preparations. Specific binding of IGF-I to MC was also demonstrated. Binding data for membranes from proliferating cultures generated a linear Scatchard plot, which predicted a binding maximum of 3.5-9.7 X 10(-11) M/100 micrograms protein and a Kd of 2.0-3.2 X 10(-9) M. In contrast, membranes from nonproliferating cultures had no demonstrable specific binding of IGF-I. Covalent cross-linking of radiolabeled IGF-I to membranes from subconfluent cells demonstrated specific binding to a 145K membrane protein. A 95K membrane protein from a partially purified receptor preparation demonstrated autophosphorylation when incubated with 5 X 10(-9) M IGF-I. Incubation of MC with 10(-9) M IGF-I doubled cellular growth rates, an effect that could be duplicated only with high concentrations (10(-6) M) of insulin. These observations indicate that MC express predominantly receptors for IGF-I, and that growth stimulatory effects of physiological concentrations of IGF-I and pharmacological concentrations of insulin are probably mediated through the IGF-I receptor.

Acylation of cellular proteins with the fatty acids myristate or palmitate represents an important mechanism for the co- or posttranslational modification of proteins. Lipid A, the biologically active component of bacterial endotoxin, exerts a number of biochemical effects on responsive cell types. Evidence is presented that lipid A stimulates the synthesis and subsequent myristyl acylation of intracellular monocyte and glomerular mesangial cell proteins. Two of the myristylated monocyte proteins were identified by specific immunoprecipitation as the 33-kD IL 1 alpha and beta precursors; a similar myristylated protein was found in mesangial cells. The 17-kD secretory form of monocyte IL 1 beta did not contain covalently linked myristate. Myristyl acylation of the IL 1 precursor proteins may facilitate the processing or membrane localization of these proteins, which lack characteristic hydrophobic signal sequences. The acylated 33-kD IL 1 alpha may remain preferentially associated with the membrane in an active form, whereas limited proteolysis may convert the biologically inactive IL 1 beta precursor into the extracellular, nonacylated, active 17-kD protein.