A cytoplasmic PPPSP motif determines megalin's phosphorylation and regulates receptor's recycling and surface expression.
Megalin is a large endocytic receptor expressed at the apical surface of several absorptive epithelia. It binds multiple ligands including apolipoproteins, vitamin and hormone carrier proteins and signaling molecules such as parathyroid hormone and the morphogen sonic hedgehog. An important characteristic of megalin is its high endocytic activity, which is mediated by tyrosine-based endocytic motifs within the receptor's cytoplasmic tail. This domain also harbors several putative consensus phosphorylation motifs for protein kinase (PK) C and casein kinase-II and one consensus motif for PKA and glycogen synthase kinase-3 (GSK3). Here we report that the cytoplasmic domain of megalin is constitutively phosphorylated depending on the integrity of a PPPSP motif, a putative GSK3 site, with a minor participation of the other phosphorylation motifs. Mutation of the serine residue within the PPPSP motif as well as blocking GSK3 activity, with two different inhibitors, significantly decreased the phosphorylation levels of the receptor. Both the megalin PPPAP mutant and the underphosphorylated wild-type receptor, by inhibition of GSK3 activity, were more expressed at the cell surface and more efficiently recycled, but they were not inhibited in their initial endocytosis rates. Altogether, these results show that the PPPSP motif and the GSK3 activity are critical to allow megalin phosphorylation and also negatively regulate the receptor's recycling.
Yuseff
María Isabel
MI
FONDAP Center for Cell Regulation and Pathology (CRCP), Departamento de Biología Celular y Molecular, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile and MIFAB, Santiago, Chile.
Farfan
Pamela
P
Bu
Guojun
G
Marzolo
María-Paz
MP
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D047390:Casein Kinase II / Q000037:antagonists & inhibitors / Q000378:metabolism; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004705:Endocytosis / Q000502:physiology*; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D018374:LLC-PK1 Cells; D018021:Lithium Chloride / Q000494:pharmacology; D026503:Low Density Lipoprotein Receptor-Related Protein-1 / Q000235:genetics / Q000378:metabolism; D026561:Low Density Lipoprotein Receptor-Related Protein-2 / Q000235:genetics / Q000378:metabolism*; D021962:Membrane Microdomains / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013552:Swine
D026503:Low Density Lipoprotein Receptor-Related Protein-1; D026561:Low Density Lipoprotein Receptor-Related Protein-2; D047428:Protein Kinase Inhibitors; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D047390:Casein Kinase II; D011493:Protein Kinase C; D038362:Glycogen Synthase Kinase 3; D018021:Lithium Chloride