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 determine which residues in its cytoplasmic tail are important for sorting to storage granules during biosynthesis, we expressed P-selectin mutants in AtT-20, a murine cell line with secretory granules that contain the hormone corticotropin ('ACTH'). Immunofluorescence microscopy of permeabilized cells revealed that wild-type P-selectin and mutants with alanine substitutions at 14 different positions in the cytoplasmic tail were concentrated in the tips of the cellular processes, which contain the majority of corticotropin granules. However, targeting to the cell tips was greatly decreased for Tyr777-->Ala, Tyr777-->Phe, Gly778-->Ala, Phe780-->Ala and Leu768/Asn769-->Ala/Ala mutants. The reduced presence of these mutants in corticotropin granules was confirmed by immunoelectron microscopy. Stimulation of AtT-20 transfectants with 8-Br-cAMP resulted in a significant increase in membrane expression of wild-type P-selectin, but in only a marginal increase in the surface expression of the five mutants. Antibody binding studies 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 approximately 20% for the Gly778 and Phe780 mutants to 63% for the Leu768/Asn769 mutant. Taken together, these results indicate that Tyr777, Gly778 and Phe780 form part of an atypical tyrosine-based motif, which also requires the presence Leu768 and/or Asn769 to mediate sorting of P-selectin to secretory granules.
Modderman
P W
PW
Department of Immunohaematology, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, University of Amsterdam, Academic Medical Center, The Netherlands. p.modderman@clb.nl
Beuling
E A
EA
Govers
L A
LA
Calafat
J
J
Janssen
H
H
Von dem Borne
A E
AE
Sonnenberg
A
A
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D003593:Cytoplasm / Q000378:metabolism* / Q000648:ultrastructure; D003594:Cytoplasmic Granules / Q000378:metabolism*; D017931:DNA Primers; D051379:Mice; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D019007:P-Selectin / Q000737:chemistry / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000737:chemistry
D017931:DNA Primers; D019007:P-Selectin; D014443:Tyrosine