Fluorescence lifetime imaging microscopy (FLIM) detects stimulus-dependent phosphorylation of the low density lipoprotein receptor-related protein (LRP) in primary neurons.
The low-density lipoprotein receptor-related protein (LRP) is a large, endocytic receptor involved in intracellular signalling. LRP acts as a co-receptor with the PDGF-receptor (PDGF-r) for platelet-derived growth factor (PDGF). PDGF-r and Src-kinases induce tyrosine-phosphorylation of LRP. We used fluorescence lifetime imaging microscopy (FLIM) to specifically detect LRP phosphorylation, measure its extent and localization in intact cells, and assess its effects upon LRP-APP interaction. Robust phosphorylation of LRP throughout the cell was observed after overexpression of Src-kinase. This depended on LRP's distal NPXY domain. By contrast, activation of the PDGF-r resulted in phosphorylation of the subpopulation of LRP at or near the cell surface. PDGF activation triggered phosphorylation of endogenous LRP in primary neurons. LRP is also a trafficking receptor for the Alzheimer-related molecule amyloid-precursor-protein (APP). PDGF stimulation did not affect LRP-APP interactions. This approach allows exquisite subcellular resolution of specific LRP post-translational changes and protein-protein interactions of endogenous proteins in intact cells.
Peltan
Ithan D
ID
Alzheimer's Disease Research Unit, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA.
Thomas
Anne V
AV
Mikhailenko
Irina
I
Strickland
Dudley K
DK
Hyman
Bradley T
BT
von Arnim
Christine A F
CA
D000682:Amyloid / Q000737:chemistry; D000818:Animals; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D026503:Low Density Lipoprotein Receptor-Related Protein-1 / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D009474:Neurons / Q000378:metabolism*; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D015398:Signal Transduction
D000682:Amyloid; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D010982:Platelet-Derived Growth Factor