Specific binding of proinsulin C-peptide to human cell membranes.

Abstract

Recent reports have demonstrated beneficial effects of proinsulin C-peptide in the diabetic state, including improvements of kidney and nerve function. To examine the background to these effects, C-peptide binding to cell membranes has been studied by using fluorescence correlation spectroscopy. Measurements of ligand-membrane interactions at single-molecule detection sensitivity in 0.2-fl confocal volume elements show specific binding of fluorescently labeled C-peptide to several human cell types. Full saturation of the C-peptide binding to the cell surface is obtained at low nanomolar concentrations. Scatchard analysis of binding to renal tubular cells indicates the existence of a high-affinity binding process with K(ass) > 3.3 x 10(9) M(-1). Addition of excess unlabeled C-peptide is accompanied by competitive displacement, yielding a dissociation rate constant of 4.5 x 10(-4) s(-1). The C-terminal pentapeptide also displaces C-peptide bound to cell membranes, indicating that the binding occurs at this segment of the ligand. Nonnative D-C-peptide and a randomly scrambled C-peptide do not compete for binding with the labeled C-peptide, nor were crossreactions observed with insulin, insulin-like growth factor (IGF)-I, IGF-II, or proinsulin. Pretreatment of cells with pertussis toxin, known to modify receptor-coupled G proteins, abolishes the binding. It is concluded that C-peptide binds to specific G protein-coupled receptors on human cell membranes, thus providing a molecular basis for its biological effects.