The Membrane-Associated Guanylate Kinase Protein MAGI-1 Binds Megalin and Is Present in Glomerular Podocytes

Abstract. The transmembrane endocytic receptor glycoprotein 330/megalin (hereafter referred to as megalin) is localized to the apical membrane domain of epithelial cells, where it is involved in the uptake of proteins from extracellular sources. The cytoplasmic domain of megalin contains amino acid motifs that have the potential to bind to other proteins, which may influence its localization or function. The yeast two-hybrid system was used to search for proteins that bind to the cytoplasmic tail of megalin, and a protein fragment from a mouse embryonic cDNA library that contained a single PDZ domain was identified. This protein, which was named g lycoprotein 330- as sociated p rotein (GASP), appears to be a truncated mouse counterpart of the human and rat proteins atrophin-1-interacting protein-1 and synaptic scaffolding molecule, respectively. The interaction of GASP with megalin is mediated by the PDZ domain of GASP binding to the DSDV motif found at the carboxyl-terminus of megalin. A mutant version of megalin that lacks the terminal valine is unable to bind to GASP, illustrating the PDZ domain-dependent interaction between these two proteins. A close homolog of GASP, i.e. , membrane-associated guanylate kinase with inverted orientation-1 (MAGI-1), is more ubiquitous in its tissue distribution (including kidney) and is also able to specifically bind to megalin via its fifth PDZ domain. Immunofluorescence studies of adult kidney revealed that MAGI-1 is expressed in the glomerulus of the kidney, in a manner that parallels the expression of the podocyte-specific protein glomerular epithelial protein 1. Western analysis of endogenous MAGI-1 from glomerular preparations suggests that it is associated with the cytoskeleton and seems to be expressed in a different form, compared with cell line-derived endogenous MAGI-1. The association of megalin with MAGI-1 may allow the assembly of a multiprotein complex, in which megalin may serve a nonendocytic function in glomerular podocytes.