Megalin Cytoplasmic Tail Phosphorylation and Function in Kidney Proximal Tubular Cells

A hallmark of progressive renal disease in man is the presence of abnormal urinary constituents termed proteinuria. These substances include proteins, of which albumin predominates and are derived from the serum but are present in the urine because of abnormal glomerular permeability. Such macromolecules are bioactive and interact with proximal tubular cells (PTC) to activate intracellular signalling cascades. The result is a PTC phenotype that stimulates localised renal inflammation and fibrosis whicht are characteristic pathological changes of proteinuric nephropathy. A PTC albumin receptor is a complex that includes megalin, a member of the low-density lipoprotein (LDL-R) family. The paradigm regarding LDL-R family members is that they are cargo receptors which transport ligand from one epithelial surface to another. The cytoplasmic domains of some members of the LDL-R mediate important signalling functions. Therefore, it is postulated that the cytoplasmic tail of megalin (MegCT) might link PTC albumin exposure with signalling effects within the cell. As signalling cascades and protein-protein interactions are regulated by protein phosphorylation studies in this thesis were designed to address whether MegCT is phosphorylated. Potential agents of MegCT phosphorylation were used to stimulate PTC in culture and stimulated cell lysate tested for its ability to phosphorylate a MegCTGST fusion protein. Human serum albumin (HSA), epidermal growth factor (EGF) and activators of Protein kinase C (PKC) were all identified to increase the substantial basal phosphorylation of MegCT. Using specific kinase inhibitors phosphoinositide 3-kinase (PI 3-kinase), epidermal growth factor receptor (EGF-R) and PKC were all identified as important mediators of MegCT phosphorylation. MegCT immunoprecipitated from intact cells demonstrated an identical pattern of phosphorylation. To overexpress MegCT a MegCT-CD8 chimaeric protein was developed and demonstrated to associate with the cell membrane and phosphorylate identically to MegCT-GST. Using mass spectrometry of phosphorylated peptides derived from MegCT-GST six sites of MegCT phosphorylation were identified under basal and stimulated conditions. By measuring FITC-albumin uptake agents stimulating MegCT phosphorylation were identified to be functionally associated with attenuated albumin endocytosis. In summary this is the first description of the regulated phosphorylation of MegCT in PTC and indicates a number of sites of potential pharmacological inhibition to abrogate the progression of proteinuric nephropathy.