The low-density lipoprotein receptor-related protein (LRP1) is a member of the low-density lipoprotein (LDL) receptor family. This family of receptors is classically viewed as endocytic receptors. New, emerging evidence though points to the fact that some of these receptors are functionally dynamic and can fulfill different roles within the cell.
One of these multipurpose LDL-R type receptors is LRP, which can function in both an endocytic role or in a signaling role. While the bulk of the receptor's endocytic machinery is located outside the cell, the signaling component of LRP is a small, 100 amino acid, cytoplasmic domain that contains two NPXY sequence motifs. NPXY motifs are potential sites of phosphorylation and are binding sites for PTB or SH2 domains. Much work to date has demonstrated an important role of the distal NPXY motif in LRP signaling, and it is the only site of known tyrosine phosphorylation in the receptor tail. The lack of observable phosphorylation to the proximal NPXY motif could be due to structural constraints.
To probe the accessibility of these sites to tyrosine phosphorylation, the GST-fusion proteins of wild-type and phosphorylated LRP cytoplasmic tail (LRP-CT) were studied by H/D exchange 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. 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.
In vitro kinase reactions using 32P-[ATP] and wild-type or mutant GST-LRP-CT, Tyr to Phe mutations at each NPXY motif (or Glu at distal Y4507) residue, and phosphopeptide mapping proved that both NPXY motifs can be phosphorylated. Further experiments showed that the Y4473 site can become phosphorylated in vivo. Overall, our data suggests a model in which the NPXY motifs of the LRP-CT are phosphorylated in a sequential manner. The distal motif is always accessible to potential kinases and is phosphorylated first (Y4507). This event effects distant residues within the protein causing the proximal NPXY motif to become more accessible. This allows phosphorylation to occur at Y4473.
This method of phosphorylation of the LRP-CT has implications for its interactions with other proteins. We studied the effect of LRP-CT tyrosine phosphorylation on its interactions with several proteins, one of which is a novel bindng partner that we identified using an affinity peptide search based on the LRP-CT NPXY4507 region and MS/MS. This protein, Shp2, a protein tyrosine phosphatase, requires LRP tyrosine phosphorylation to bind. Another known LRP-CT binding protein, Fe65, exhibited a slight preference for a phosphorylated binding site. However this preference may be due more to the "opening up" affect of phosphorylation, as distant residues from the NPXY motif appear to be important for binding. Finally, sorting nexin (Snx17) binding was regulated by LRP-CT phosphorylation. Interestingly, Snx17 binds to the proximal NPXY motif, but this binding is blocked by phosphorylation at Y4473. However, phosphorylation at Y4507 increases binding, presumably by making the Y4473 region more accessible.
