Expression cloning screen for modifiers of amyloid precursor protein shedding.
Ectodomain shedding of the amyloid precursor protein (APP) is a key regulatory step in the generation of the amyloid beta peptide (Abeta), which is thought to provoke the pathogenesis of Alzheimer's disease. To better understand the cellular processes that regulate ectodomain shedding of APP we used human embryonic kidney 293 cells and applied a sib-selection expression cloning approach. In addition to a known activator of APP shedding -- protein kinase A -- the following cDNAs were identified: the endocytic proteins endophilin A1 and A3, the metabotropic glutamate receptor 3 (mGluR3), palmitoyl-protein thioesterase 1 (PPT1), Numb-like and the kinase MEKK2. Endophilins A1 and A3, as well as mGluR3 activated APP shedding relatively specifically. They had little or no effect on the shedding of the unrelated membrane proteins TNF receptor 2 and P-selectin glycoprotein ligand-1. In contrast, MEKK2 and PKA also increased shedding of TNF receptor 2, suggesting that these kinases contribute to a general program regulating ectodomain shedding. The strongest activator of APP shedding, endophilin A3, reduced the rate of APP endocytosis and specifically increased APP shedding by the protease alpha-secretase, as measured in an antibody uptake assay and by immunoblot analysis. This suggests that endophilin A3 is a novel modulator of APP trafficking affecting access of APP to alpha-secretase. In summary, this study shows that expression cloning is a suitable way to identify proteins controlling ectodomain shedding of membrane proteins.
Schöbel
Susanne
S
Adolf-Butenandt-Institut, Ludwig-Maximilians-University, Schillerstr. 44, 80336 Munich, Germany.
Neumann
Stephanie
S
Seed
Brian
B
Lichtenthaler
Stefan F
SF
D000217:Acyltransferases / Q000378:metabolism; D016564:Amyloid beta-Protein Precursor / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western / Q000379:methods; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology; D004789:Enzyme Activation / Q000502:physiology; D015870:Gene Expression / Q000502:physiology*; D015723:Gene Library; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021381:Protein Transport / Q000502:physiology; D047889:Receptors, Tumor Necrosis Factor, Type II / Q000378:metabolism; D013997:Time Factors; D014162:Transfection / Q000379:methods; D014168:Transferrin / Q000378:metabolism
D016564:Amyloid beta-Protein Precursor; D008562:Membrane Glycoproteins; C084882:P-selectin ligand protein; D047889:Receptors, Tumor Necrosis Factor, Type II; D014168:Transferrin; D049452:Green Fluorescent Proteins; D000217:Acyltransferases; C018764:2-acylglycerophosphate acyltransferase; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases