Abstract
The glucose-regulated protein 78 (GRP78) is a plasminogen (Pg) receptor on the cell surface. In this study, we demonstrate that GRP78 also binds the tissue-type plasminogen activator (t-PA), which results in a decrease in K(m) and an increase in the V(max) for both its amidolytic activity and activation of its substrate, Pg. This results in accelerated Pg activation when GRP78, t-PA, and Pg are bound together. The increase in t-PA activity is the result of a mechanism involving a t-PA lysine-dependent binding site in the GRP78 amino acid sequence (98)LIGRTWNDPSVQQDIKFL(115). We found that GRP78 is expressed on the surface of neuroblastoma SK-N-SH cells where it is co-localized with the voltage-dependent anion channel (VDAC), which is also a t-PA-binding protein in these cells. We demonstrate that both Pg and t-PA serve as a bridge between GRP78 and VDAC bringing them together to facilitate Pg activation. t-PA induces SK-N-SH cell proliferation via binding to GRP78 on the cell surface. Furthermore, Pg binding to the COOH-terminal region of GRP78 stimulates cell proliferation via its microplasminogen domain. This study confirms previous findings from our laboratory showing that GRP78 acts as a growth factor-like receptor and that its association with t-PA, Pg, and VDAC on the cell surface may be part of a system controlling cell growth.
Highlights
Glucose-regulated protein 78 (GRP78) is a plasminogen (Pg) and tissue-type plasminogen activator (t-PA) receptor on the cell surface
We demonstrate that glucose-regulated protein 78 (GRP78) binds the tissue-type plasminogen activator (t-PA), which results in a decrease in Km and an increase in the Vmax for both its amidolytic activity and activation of its substrate, Pg
We demonstrate that both Pg and t-PA serve as a bridge between GRP78 and voltage-dependent anion channel (VDAC) bringing them together to facilitate Pg activation. t-PA induces SK-N-SH cell proliferation via binding to GRP78 on the cell surface
Summary
Glucose-regulated protein 78 (GRP78) is a plasminogen (Pg) and tissue-type plasminogen activator (t-PA) receptor on the cell surface. We found that GRP78 is expressed on the surface of neuroblastoma SK-N-SH cells where it is co-localized with the voltage-dependent anion channel (VDAC), which is a t-PA-binding protein in these cells. We demonstrate that both Pg and t-PA serve as a bridge between GRP78 and VDAC bringing them together to facilitate Pg activation. Because t-PA activity is enhanced by VDAC [7] and because both t-PA and Pg activation are affected by the interaction of these ligands with either VDAC or GRP78, we hypothesized a functional cell surface interaction between these four components in the brain For these studies, we used the human neuroblastoma cell line SKN-SH as an in vitro model because these cells express VDAC on the cell surface [19]. Cross-linking studies show that both t-PA and Pg function as bridges between GRP78 and VDAC on the surface of SK-N-SH cells
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