Proinsulin C-peptide : Membrane interactions and intracellular signaling

University dissertation from Stockholm : Karolinska Institutet, Department of Surgical Science

Abstract: In the past decade, new evidence has emerged of a physiological role for C-peptide. C-peptide replacement improves renal and nerve function and augments regional blood flow in type I diabetic patients. Data demonstrate that C-peptide initiates its effects by binding to a G-protein coupled receptor, subsequently activating Ca 2' and MAPK-dependent intracellular signaling pathways and eventually stimulates Na+,K+-ATPase and eNOS. However, the precise molecular mechanisms of Cpeptide action are not fully understood. The aim of this thesis is to study the interactions of Cpeptide with cell membranes and the intracellular signaling pathways that C-peptide activates. Fluorescence correlation spectroscopy (FCS) has been evaluated for the study of ligand-receptor interactions by examining the binding characteristics of insulin. The findings indicate thatFCS is a highly sensitive technique, suitable for the study of hormone-membrane binding. FCS was subsequently employed to investigate the structural requirements of the C-terminal pentapeptide for C-peptide binding. The capacity of stepwise Ala-substituted pentapeptide analogues to displace bound rhodamine-labeled C-peptide from human renal tubular cells (HRTC) was determined. The results showed that Glu27 is essential for displacement, while Gly28 had little effect, and the residues Ser29, Leu30 and Gln31 had intermediate effects. The effects of C-peptide and related peptides on intracellular Ca2+ concentrations, [Ca 2+]i, in human renal tubular cells were studied using the indicator fura-2/AM. The results showed that human Cpeptide and its C-terminal pentapeptide, but not the des(27-3 1)C-peptide or scrambled C-peptide, elicit a marked increase in [Ca 21 +] i . This effect was abolished by pretreatment of the cells withpertussis toxin (PTX), indicating the involvement of a G-protein in the signal transduction. Using specific inhibitors and phospho-specific antibodies, we examined the intracellular signaling pathways activated by C-peptide using Western blot analysis. The results show that human C-peptide increases phosphorylation of ERK1/2, JNK and PKB/Akt, but not p38 MAPK in HRTC. MEK ½ inhibitor PD98059 blocks C-peptide's effect on ERK1/2 phosphorylation. Furthermore, C-peptide causes translocation of PKC delta and epsilon from the cytosol to the cell membrane of the HRTC. All stimulatory effects of C-peptide were abolished by pertussis toxin. C-peptide stimulation also causes translocation of the small GTPase RhoA from the cytosol to the cell membrane. A phospholipase C inhibitor abolished C-peptide's effect on phosphorylation of ERK 1/2, JNK and PKC delta. The effect of C-peptide on activation of Na+,K+-ATPase was evaluated using ouabain-sensitive 86 Rb+ uptake in HRTC. C-peptide caused 86 Rb+ uptake to increase by 40%, while the C-terminal pentapeptide gave a less marked but significant effect. The effect was inhibited by PD98059, GF109203X and PTX. Using 32p labeling, bioinformatic analysis and subcellular fractionation, we found evidence that C-peptide leads to phosphorylation of the Na+,K+-ATPase alpha-subunits on threonine residues, and causes translocation of alpha1, and beta1-subunits of Na+,K+-ATPase from an endosomal fraction to the basolateral membranes. These effects were inhibited by PD98059. In conclusion, the C-terminal pentapeptide is involved in membrane binding of C-peptide and Glu27 is of importance in this process. C-peptide, in physiological concentration, triggers intracellular signaling by binding to a G-protein coupled receptor, which leads to the stimulation of phospholipase C and subsequent activation of PKC delta and epsilon, followed by the activation of small GTPase RhoA and, eventually, MAPK ERK1/2 and JNK. ERK1/2 stimulates Na+,K+-ATPase, which undergoes phosphorylation of the alpha subunit at its threonine residues and translocation from an endosomal fraction to the basolateral membrane. PKB/Akt activation and Ca 2+ influx are also involved in the Cpeptide signaling transduction system. The present results support the hypothesis that replacement of C-peptide together with insulin may retard or prevent the development of long-term complications of type I diabetes.

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