EP24.15 is associated with lipid rafts.
Metalloendopeptidase EC 3.4.24.15 (EP24.15, thimet oligopeptidase) is a neuropeptide-metabolizing peptidase expressed throughout the body, but primarily in the brain, gonads, and pituitary. For EP24.15 to have its greatest effect upon peptides in the periphery, it must be targeted and released into the extracellular space. Western blot analysis of fractions taken from discontinuous sucrose density gradients carried out on crude plasma membrane fractions from AtT-20 cells reveals colocalization of EP24.15 and flotillin-1, a known lipid raft marker. Further analysis revealed that an intracellular membrane marker and non-lipid raft, plasma membrane marker, failed to colocalize, supporting EP24.15/lipid raft association. Furthermore, EP24.15 immunoreactivity in lipid raft fractions generated from cells treated with methyl beta-cyclodextrin (MbetaCD) was greatly reduced. Finally, treatment with MbetaCD resulted in the accumulation of EP24.15 in the media of drug-treated cells over vehicle-treated cells, suggesting that a large percentage of EP24.15 associating with lipid rafts resides on the extracellular surface of the plasma membrane. With this exofacial localization, EP24.15 could have ample access to neuropeptides not only in the immediate microenvironment, but the ability to degrade or modify peptides bound for receptor interaction.
Jeske
Nathaniel A
NA
Department of Pharmacology, University of Texas Health Science Center, San Antonio, Texas 78229, USA.
Glucksman
Marc J
MJ
Roberts
James L
JL
D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism; D002458:Cell Fractionation; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D003505:Cyclodextrins / Q000494:pharmacology; D000091342:Endoplasmic Reticulum Chaperone BiP; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism; D006360:Heat-Shock Proteins; D021962:Membrane Microdomains / Q000378:metabolism*; D008565:Membrane Proteins; D008666:Metalloendopeptidases / Q000378:metabolism*; D051379:Mice; D018832:Molecular Chaperones / Q000378:metabolism; D009479:Neuropeptides; D010903:Pituitary Gland, Anterior / Q000187:drug effects; D018019:Receptors, Corticotropin-Releasing Hormone / Q000378:metabolism; D015398:Signal Transduction; D047392:beta-Cyclodextrins
D002352:Carrier Proteins; D003505:Cyclodextrins; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D018832:Molecular Chaperones; D009479:Neuropeptides; D018019:Receptors, Corticotropin-Releasing Hormone; D047392:beta-Cyclodextrins; C106354:flotillins; C108732:methyl-beta-cyclodextrin; D008666:Metalloendopeptidases; C022510:thimet oligopeptidase; D019205:GTP-Binding Protein alpha Subunits, Gs