Abstract
Both the voltage-dependent anion channel and the glucose-regulated protein 78 have been identified as plasminogen kringle 5 receptors on endothelial cells. In this study, we demonstrate that kringle 5 binds to a region localized in the N-terminal domain of the glucose-regulated protein 78, whereas microplasminogen does so through the C-terminal domain of the glucose-regulated protein 78. Both plasminogen fragments induce Ca(2+) signaling cascades; however, kringle 5 acts through voltage-dependent anion channel and microplasminogen does so via the glucose-regulated protein 78. Because trafficking of voltage-dependent anion channel to the cell surface is associated with heat shock proteins, we investigated a possible association between voltage-dependent anion channel and glucose-regulated protein 78 on the surface of 1-LN human prostate tumor cells. We demonstrate that these proteins co-localize, and changes in the expression of the glucoseregulated protein 78 affect the expression of voltage-dependent anion channel. To differentiate the functions of these receptor proteins, either when acting singly or as a complex, we employed human hexokinase I as a specific ligand for voltage-dependent anion channel, in addition to kringle 5. We show that kringle 5 inhibits 1-LN cell proliferation and promotes caspase-7 activity by a mechanism that requires binding to cell surface voltage-dependent anion channel and is inhibited by human hexokinase I.
Highlights
Binding sites (LBS) responsible for Pg binding to extracellular matrix molecules [4] and cell receptors [5, 6]
We reported that K5 binding to the voltage-dependent anion channel (VDAC) on the surface of human umbilical vein endothelial cells (HUVEC) interfered with mechanisms controlling the regulation of intracellular Ca2ϩ producing a decrease in intracellular pH [11]
Because trafficking of VDAC to the cell membrane is associated with the heat shock proteins GRP75 and HSP70 [16, 17], we hypothesized a possible association between VDAC and glucose-regulated protein 78 (GRP78) in regulating the functions of VDAC on the cell surface
Summary
Excess serum was drained from the coverslips, and cells were incubated with the primary sheep anti-GRP78 or rabbit anti-VDAC1 IgGs in PBS containing 1% BSA, and 5 mM EDTA for 90 min at 4 °C. The cells were rinsed three times in PBS and incubated with a secondary antibody containing either a 1:400 dilution of an Alexa Fluor 488-conjugated donkey anti-sheep IgG, an Alexa Fluor 568-conjugated goat anti-rabbit IgG or a mixture of both for 90 min at 4 °C in the dark. Cells were washed three times with 400 l of PBS and incubated with a blocking solution containing 3% BSA and 5% nonimmune goat serum for 90 min at room temperature with gentle rocking. The day, wells were rinsed three times in PBS and incubated with 200 l of a solution containing a 1:800 dilution of an IRDye 800 DX-conjugated affinity-purified anti-sheep or anti-rabbit IgGs in blocking buffer.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
