Proinsulin C-peptide : membrane interactions and intracellular signaling
Author: Zhong, Zhihui
Date: 2004-06-03
Location: Farmakologens föreläsningssal, Nanna Svartz väg 2, Karolinska Institutet
Time: 9.00
Department: Institutionen för kirurgisk vetenskap / 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
C-peptide 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.
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.
List of papers:
I. Zhong ZH, Pramanik A, Ekberg K, Jansson OT, Jornvall H, Wahren J, Rigler R (2001). "Insulin binding monitored by fluorescence correlation spectroscopy. " Diabetologia 44(9): 1184-8
Pubmed
II. Pramanik A, Ekberg K, Zhong Z, Shafqat J, Henriksson M, Jansson O, Tibell A, Tally M, Wahren J, Jornvall H, Rigler R, Johansson J (2001). "C-peptide binding to human cell membranes: importance of Glu27." Biochem Biophys Res Commun 284(1): 94-8
Pubmed
III. Shafqat J, Juntti-Berggren L, Zhong Z, Ekberg K, Kohler M, Berggren PO, Johansson J, Wahren J, Jornvall H (2002). "Proinsulin C-peptide and its analogues induce intracellular Ca2+ increases in human renal tubular cells. " Cell Mol Life Sci 59(7): 1185-9
Pubmed
IV. Zhong ZH, Davidescu A, Ehrén I, Ekberg K, Jornvall H, Wahren J, Chibalin A (2004). "C-peptide stimulates ERK1/2 and JNK MAP-kinases via activation of PKC in human renal tubular cells." (Submitted)
View record in Web of Science®
V. Zhong ZH, Kotova O, Davidescu A, Ehrén I Eber KJrnvall H, Wahren J, hibalin A (2004). "C-peptide stimulates Na+,K+-ATPase via activation of ERK1/2 MAP kinases in human renal tubular cells." (Submitted)
View record in Web of Science®
I. Zhong ZH, Pramanik A, Ekberg K, Jansson OT, Jornvall H, Wahren J, Rigler R (2001). "Insulin binding monitored by fluorescence correlation spectroscopy. " Diabetologia 44(9): 1184-8
Pubmed
II. Pramanik A, Ekberg K, Zhong Z, Shafqat J, Henriksson M, Jansson O, Tibell A, Tally M, Wahren J, Jornvall H, Rigler R, Johansson J (2001). "C-peptide binding to human cell membranes: importance of Glu27." Biochem Biophys Res Commun 284(1): 94-8
Pubmed
III. Shafqat J, Juntti-Berggren L, Zhong Z, Ekberg K, Kohler M, Berggren PO, Johansson J, Wahren J, Jornvall H (2002). "Proinsulin C-peptide and its analogues induce intracellular Ca2+ increases in human renal tubular cells. " Cell Mol Life Sci 59(7): 1185-9
Pubmed
IV. Zhong ZH, Davidescu A, Ehrén I, Ekberg K, Jornvall H, Wahren J, Chibalin A (2004). "C-peptide stimulates ERK1/2 and JNK MAP-kinases via activation of PKC in human renal tubular cells." (Submitted)
View record in Web of Science®
V. Zhong ZH, Kotova O, Davidescu A, Ehrén I Eber KJrnvall H, Wahren J, hibalin A (2004). "C-peptide stimulates Na+,K+-ATPase via activation of ERK1/2 MAP kinases in human renal tubular cells." (Submitted)
View record in Web of Science®
Issue date: 2004-05-13
Publication year: 2004
ISBN: 91-7349-886-6
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