Purification of the NF2 tumor suppressor protein from human erythrocytes.
BACKGROUND
Neurofibromatosis type 2 (NF2) is an autosomal dominant disease predisposing individuals to the risk of developing tumors of cranial and spinal nerves. The NF2 tumor suppressor protein, known as Merlin/Schwanomin, is a member of the protein 4.1 superfamily that function as links between the cytoskeleton and the plasma membrane.


METHODS
Upon selective extraction of membrane-associated proteins from erythrocyte plasma membrane (ghosts) using low ionic strength solution, the bulk of NF2 protein remains associated with the spectrin-actin depleted inside-out-vesicles. Western blot analysis showed a approximately 70 kDa polypeptide in the erythrocyte plasma membrane. Furthermore, quantitative removal of NF2 protein from the inside-out-vesicles was achieved using 1.0 M potassium iodide, a treatment known to remove tightly-bound peripheral membrane proteins.


RESULTS
These results suggest a novel mode of NF2 protein association with the erythrocyte membrane that is distinct from the known membrane interactions of protein 4.1. Based on these biochemical properties, several purification strategies were devised to isolate native NF2 protein from human erythrocyte ghosts. Using purified and recombinant NF2 protein as internal standards, we quantified approximately 41-65,000 molecules of NF2 protein per erythrocyte.


CONCLUSIONS
We provide evidence for the presence of NF2 protein in the human erythrocyte membrane. The identification of NF2 protein in the human erythrocyte membrane will make it feasible to discover novel interactions of NF2 protein utilizing powerful techniques of erythrocyte biochemistry and genetics in mammalian cells.
Jindal
Hitesh K
HK
Department of Medicine, St. Elizabeth's Medical Center, Tufts University School of Medicine, Boston, MA, USA.
Yoshinaga
Kazumi
K
Seo
Pil-Soo
PS
Lutchman
Mohini
M
Dion
Patrick A
PA
Rouleau
Guy A
GA
Hanada
Toshihiko
T
Chishti
Athar H
AH
D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000737:chemistry*; D004912:Erythrocytes / Q000737:chemistry; D006801:Humans; D007150:Immunohistochemistry; D025581:Neurofibromin 2 / Q000302:isolation & purification*
D025581:Neurofibromin 2