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The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein binds and internalizes
M Z Kounnas1, R E Morris, M R Thompson
1Biochemistry Laboratory, American Red Cross, Rockville, Maryland 20855.
Abstract:
The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2 MR/LRP) is a large cell-surface glycoprotein consisting of a 515-kDa and an 85-kDa polypeptide; this receptor is thought to be responsible for the binding and endocytosis of activated alpha 2-macroglobulin and apoE-enriched beta-very low density lipoprotein. A similar high molecular weight glycoprotein has been identified as a potential receptor for Pseudomonas exotoxin A (PE). We demonstrate that the alpha 2 MR/LRP and the PE-binding glycoprotein have a similar mobility upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis and are immunologically indistinguishable. Furthermore, affinity-purified alpha 2 MR/LRP binds specifically to PE but not to a mutant toxin defective in its ability to bind cells. The 39-kDa receptor-associated protein, which blocks binding of ligands to alpha 2 MR/LRP, also prevents binding and subsequent toxicity of PE for mouse fibroblasts. The concentration of receptor-associated protein that was required to reduce binding and toxicity to 50% was approximately 14 nM, a value virtually identical to the KD measured for the interaction of receptor-associated protein with the purified receptor. Overall, the studies strongly suggest that the alpha 2 MR/LRP is responsible for internalizing PE.
Insights
The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2 MR/LRP) acts as the cell surface receptor for Pseudomonas exotoxin A (PE). This receptor mediates the internalization of PE, leading to cellular toxicity.
Area of Science:
- Cell biology
- Molecular biology
- Toxicology
Background:
- The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2 MR/LRP) is a cell-surface glycoprotein involved in endocytosis.
- A distinct glycoprotein was identified as a potential receptor for Pseudomonas exotoxin A (PE).
Purpose of the Study:
- To determine if alpha 2 MR/LRP functions as the receptor for Pseudomonas exotoxin A (PE).
- To investigate the role of alpha 2 MR/LRP in PE binding, internalization, and toxicity.
Main Methods:
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to compare protein mobility.
- Immunological assays to assess protein identity.
- Ligand binding studies using purified alpha 2 MR/LRP and PE.
- Toxicity assays in mouse fibroblasts using PE and a receptor-associated protein inhibitor.
Main Results:
- The PE-binding glycoprotein exhibited similar SDS-PAGE mobility and was immunologically indistinguishable from alpha 2 MR/LRP.
- Purified alpha 2 MR/LRP specifically bound to PE.
- A receptor-associated protein inhibited both PE binding to alpha 2 MR/LRP and PE-induced toxicity in fibroblasts.
Conclusions:
- The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2 MR/LRP) is the primary cell-surface receptor responsible for Pseudomonas exotoxin A (PE) internalization.
- Alpha 2 MR/LRP mediates PE binding and subsequent cellular toxicity.