Determinants in the cytoplasmic domain of P-selectin required for sorting to secretory granules

P-selectin is a granule membrane protein of platelets and endothelial cells that is expressed at the plasma membrane after cell activation. To determi ne which residues in its cytoplasmic tail are important for sorting to stor age granules during biosynthesis, we expressed P-selectin mutants in AtT-20 , a murine cell line with secretory granules that contain the hormone corti cotropin ('ACTH'). Immunofluorescence microscopy of permeabilized cells rev ealed that wild-type P-selectin and mutants with alanine substitutions at 1 4 different positions in the cytoplasmic tail were concentrated in the tips of the cellular processes, which contain the majority of corticotropin gra nules. However, targeting to the cell tips was greatly decreased for Tyr(77 7) --> Ala, Tyr(777) --> Phe, Gly(778) --> Ala, Phe(780) --> Ala and Leu(76 8)/Asn(769) --> Ala/Ala mutants. The reduced presence of these mutants in c orticotropin granules was confirmed by immunoelectron microscopy. Stimulati on of AtT-20 transfectants with 8-Br-cAMP resulted in a significant increas e in membrane expression of wild-type P-selectin, but in only a marginal in crease in the surface expression of the five mutants. Antibody binding stud ies with intact and permeabilized cells demonstrated that the percentage of P-selectin that is expressed on the surface of the cells was considerably higher for these mutants than for wild-type P-selectin (6%), ranging from s imilar to 20 % for the Gly(778) and Phe(780) mutants to 63 % for the Leu(76 8)/Asn(769) mutant. Taken together, these results indicate that Tyr(777), G ly(778) and Phe(780) form part of an atypical tyrosine-based motif, which a lso requires the presence Leu(768) and/or Asn(769) to mediate sorting of P- selectin to secretory granules.