Specific binding of proinsulin C-peptide to human cell membranes

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 mem branes has been studied by using fluorescence correlation spectroscopy. Mea surements of ligand-membrane interactions at single-molecule detection sens itivity in 0.2-fl confocal volume elements show specific binding of fluores cently labeled C-peptide to several human cell types. Full saturation of th e C-peptide binding to the cell surface is obtained at low nanomolar concen trations. Scatchard analysis of binding to renal tubular cells indicates th e 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 disp lacement, 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, i ndicating 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 w ith the labeled C-peptide, nor were cross-reactions 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, ab olishes the binding. It is concluded that C-peptide binds to specific G pro tein-coupled receptors on human cell membranes, thus providing a molecular basis for its biological effects.