Role of the membrane-proximal O-glycosylation site in sorting of the human receptor for neurotrophins to the apical membrane of MDCK cells.
We have analyzed the respective roles of the stalk and/or the O-glycosylation sites in apical sorting by producing partially deleted mutants in this region of the human receptor for neurotrophins (P75(NTR)). The mere presence of O-glycosylations was not sufficient for efficient delivery to the apical surface since changing the stalk domain of P75(NTR) for the heavily O-glycosylated stalk from human decay-accelerating factor led to random distribution of the chimera. The presence of O-glycosylations, however, was a prerequisite for exit from the ER and protection from intracellular cleavage since a P75(NTR) containing the non O-glycosylated stalk of the human placental alkaline phosphatase was not transported to the cell surface but was cleaved and secreted from the basolateral side. Deletion of the membrane-proximal part of the stalk showed a more dramatic reversal of polarity of P75(NTR) than the deletion of the distal part. Furthermore, moving the first putative O-glycosylation site (T216) two amino acids away from the membrane resulted in a loss of apical polarity of P75(NTR), suggesting that an important clue for apical sorting resides in this part of the stalk. This loss of apical polarity paralleled a loss of association of P75(NTR) mutants with Lubrol rafts. These data indicate that the position of O-glycans in the proximal part of the stalk domain of P75(NTR) is crucial for apical sorting and may regulate association with apical rafts.
Breuza
Lionel
L
Laboratoire de Génétique et Physiologie du Développement, UMR 6545, IBDM, Faculté des Sciences de Luminy, Université de la Méditerranée, Marseille, France.
Garcia
Martine
M
Delgrossi
Marie-Hélène
MH
Le Bivic
André
A
D000818:Animals; D001665:Binding Sites; D018938:Caco-2 Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D011134:Polysaccharides / Q000378:metabolism*; D020800:Receptor, Nerve Growth Factor; D017475:Receptors, Nerve Growth Factor / Q000378:metabolism*
D011134:Polysaccharides; D020800:Receptor, Nerve Growth Factor; D017475:Receptors, Nerve Growth Factor