Probing the structure of the LRP1 cytoplasmic domain

The low‐density lipoprotein receptor‐related protein (LRP1) has a small, 100 amino acid, cytoplasmic domain that contains two NPXY sequence motifs. To probe the accessibility of these sites to tyrosine phosphorylation, the GST‐fusion proteins were phosphorylated in vitro with v‐src kinase. Each NPXY site was mutated to NPXF and anti‐LRP1 and anti‐pTyr blots allowed identification of the phosphorylation sites. The site at Y4507 was phosphorylated in both denatured and native protein. The site at Y4473 was only phosphorylated when the protein was first denatured by boiling. This result suggested that the site at 4507 was always exposed, but the site at 4473 may be sequestered in a folded part of the protein. The cytoplasmic domain has no sequence similarity to any known protein, so its fold can not be predicted by homology. In order to probe which parts of the domain are folded, we expressed the protein as a GST‐fusion, and performed amide H/ 2 H exchange experiments with MALDI‐TOF mass spectrometry. We discovered that some regions of the protein do in fact show reduced amide exchange, including the first NPXY motif containing Y4473. Experiments on the phosphorylated form (pY4507) showed changes in amide exchange, such that the first NPXY motif became exposed. Overall, these results suggest that the first NPXY motif is not available as a potential binding or phosphorylation site until the downstream motif becomes phosphorylated. While the cytoplasmic domain of LRP1 does not appear to have any significant secondary structure, phosphorylation induced structural changes can be observed. NIH AG025343