Characterization of vascular endothelial proteins associated with plasma cell membrane glycoprotein 1 and their influence on insulin receptor signaling

PC-1, a member of the multigene family of ecto-nucleotide pyrophosphatase/phosphodiesterases, is a disulfide bonded homodimeric 230-260 kDa class II transmembrane protein. The physiological function of PC-1 in most tissues is unknown but it plays an important role in the normal and pathological calcification of bone and cartilage by controlling levels of extracellular pyrophosphate which influences the formation of hydroxyapatite crystals. If and how PC-1 is involved in arterial calcification in atherosclerosis in not known yet. Interestingly, PC-1 content is increased in fibroblasts, muscle and adipose tissue from insulin-resistant subjects and its elevation correlates with in vivo insulin resistance even in the absence of either type 2 diabetes or obesity. PC-1 inhibits IR tyrosine kinase activity via direct interaction with a specific region in the insulin receptor alpha subunit preventing the insulin induced conformational change of the alpha subunit and the subsequent activation of the beta subunit tyrosine kinase. Thus, in PC-1 overexpressing cells IR tyrosine kinase activity and the phosphorylation of IRS-1, the major substrate for IR, are decreased. Since a polymorphism at position 173 (K173Q) of the PC-1 gene which is associated with insulin resistance lies in the somatomedin B like domain of PC-1 and differentially influences insulin receptor signaling, we speculate that the somatomedin B domain of PC-1 might be involved in the association of PC-1 with proteins which - in healthy cells - might compete with the insulin receptor for the binding to PC-1 or which modulate insulin receptor signaling. Factors that promote insulin resistance may, thus, interfere with these protein interactions and thus with PC-1 mediated regulation of insulin receptor signaling. The focus of the present study will be the isolation of PC-1 associated proteins and the identification as well as characterization of individual proteins of the complex by 2D gel electrophoresis followed by in-gel tryptic digest of the proteins and MALDI mass spectrometric analysis of the peptides. Additionally, we will quantitate their expression and determine whether these proteins dissociate from PC-1 or are modified following exposure of HUVEC to insulin. The precise knowledge of the proteins, the signaling machinery and the effector mechanisms participating in or preventing the effect of insulin on vascular endothelial cells is a prerequisite for defining means to interfere with insulin resistance.

PC-1, a member of the multigene family of ecto-nucleotide pyrophosphatase/ phosphodiesterases, is a disulfide bonded homodimeric 230-260 kDa class II transmembrane protein. The physiological function of PC-1 in most tissues is unknown but it plays an important role in the normal and pathological calcification of bone and cartilage by controlling levels of extracellular pyrophosphate which influences the formation of hydroxyapatite crystals. If and how PC-1 is involved in arterial calcification in atherosclerosis in not known yet. Interestingly, PC-1 content is increased in fibroblasts, muscle and adipose tissue from insulin-resistant subjects and its elevation correlates with in vivo insulin resistance even in the absence of either type 2 diabetes or obesity. PC-1 inhibits IR tyrosine kinase activity via direct interaction with a specific region in the insulin receptor alpha subunit preventing the insulin induced conformational change of the alpha subunit and the subsequent activation of the beta subunit tyrosine kinase. Thus, in PC-1 overexpressing cells IR tyrosine kinase activity and the phosphorylation of IRS-1, the major substrate for IR, are decreased. Since a polymorphism at position 173 (K173Q) of the PC-1 gene which is associated with insulin resistance lies in the somatomedin B like domain of PC-1 and differentially influences insulin receptor signaling, we speculate that the somatomedin B domain of PC-1 might be involved in the association of PC-1 with proteins which - in healthy cells - might compete with the insulin receptor for the binding to PC-1 or which modulate insulin receptor signaling. Factors that promote insulin resistance may, thus, interfere with these protein interactions and thus with PC-1 mediated regulation of insulin receptor signaling. The focus of the present study will be the isolation of PC-1 associated proteins and the identification as well as characterization of individual proteins of the complex by 2D gel electrophoresis followed by in-gel tryptic digest of the proteins and MALDI mass spectrometric analysis of the peptides. Additionally, we will quantitate their expression and determine whether these proteins dissociate from PC-1 or are modified following exposure of HUVEC to insulin. The precise knowledge of the proteins, the signaling machinery and the effector mechanisms participating in or preventing the effect of insulin on vascular endothelial cells is a prerequisite for defining means to interfere with insulin resistance.