Genetic mapping of activity determinants within cellular prion proteins: N-terminal modules in PrPC offset pro-apoptotic activity of the Doppel helix B/B' region.
The PrP-like Doppel (Dpl) protein causes apoptotic death of cerebellar neurons in transgenic mice, a process prevented by expression of the wild type (wt) cellular prion protein, PrP(C). Internally deleted forms of PrP(C) resembling Dpl such as PrPDelta32-121 produce a similar PrP(C)-sensitive pro-apoptotic phenotype in transgenic mice. Here we demonstrate that these phenotypic attributes of wt Dpl, wt PrP(C), and PrPDelta132-121 can be accurately recapitulated by transfected mouse cerebellar granule cell cultures. This system was then explored by mutagenesis of the co-expressed prion proteins to reveal functional determinants. By this means, neuroprotective activity of wt PrP(C) was shown to be nullified by a deletion of the N-terminal charged region implicated in endocytosis and retrograde axonal transport (PrPDelta23-28), by deletion of all five octarepeats (PrPDelta51-90), or by glycine replacement of four octarepeat histidine residues required for selective binding of copper ions (Prnp"H/G"). In the case of Dpl, overlapping deletions defined a requirement for the gene interval encoding helices B and B' (DplDelta101-125). These data suggest contributions of copper binding and neuronal trafficking to wt PrP(C) function in vivo and place constraints upon current hypotheses to explain Dpl/PrP(C) antagonism by competitive ligand binding. Further implementation of this assay should provide a fuller understanding of the attributes and subcellular localizations required for activity of these enigmatic proteins.
Centre for Research in Neurodegenerative Diseases, Tanz Neuroscience Building, 6 Queen's Park Crescent West, University of Toronto, Toronto, Ontario M5S 3H2, Canada.
Bettina
B
Drisaldi
Janaky
J
Coomaraswamy
Peter
P
Mastrangelo
Bob
B
Strome
Jing
J
Yang
Joel C
JC
Watts
M Azhar
MA
Chishti
Melissa
M
Marvi
Otto
O
Windl
Rosemary
R
Ahrens
François
F
Major
Man-Sun
MS
Sy
Hans
H
Kretzschmar
Paul E
PE
Fraser
Howard T J
HT
Mount
David
D
Westaway
D000483:Alleles; D000818:Animals; D017209:Apoptosis; D016923:Cell Death; D045744:Cell Line, Tumor; D002531:Cerebellum / Q000378:metabolism; D002874:Chromosome Mapping; D003300:Copper; D004252:DNA Mutational Analysis; D004705:Endocytosis; D058851:GPI-Linked Proteins; D017353:Gene Deletion; D005998:Glycine / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007477:Ions; D051379:Mice; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D009154:Mutation; D009474:Neurons / Q000378:metabolism / Q000473:pathology; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D018621:PrPC Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D011328:Prions / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014162:Transfection; D019076:Transgenes
D058851:GPI-Linked Proteins; D007477:Ions; D018621:PrPC Proteins; D011328:Prions; C122036:Prnd protein, mouse; D049452:Green Fluorescent Proteins; D003300:Copper; D005998:Glycine