Modderman P W, Beuling E A, Govers L A, Calafat J, Janssen H, Von dem Borne A E, Sonnenberg A
Department of Immunohaematology, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, University of Amsterdam, Academic Medical Center, The Netherlands.
Biochem J. 1998 Nov 15;336 ( Pt 1)(Pt 1):153-61. doi: 10.1042/bj3360153.
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.
P-选择素是血小板和内皮细胞的一种颗粒膜蛋白，在细胞激活后表达于质膜。为了确定其细胞质尾部的哪些残基在生物合成过程中对分选至储存颗粒很重要，我们在AtT-20细胞系中表达了P-选择素突变体，AtT-20是一种小鼠细胞系，其分泌颗粒中含有促肾上腺皮质激素（“ACTH”）。对通透细胞进行免疫荧光显微镜检查发现，野生型P-选择素以及在细胞质尾部14个不同位置进行丙氨酸替代的突变体集中在细胞突起的尖端，这些尖端含有大多数促肾上腺皮质激素颗粒。然而，对于Tyr777→Ala、Tyr777→Phe、Gly778→Ala、Phe780→Ala和Leu768/Asn769→Ala/Ala突变体，靶向细胞尖端的能力大大降低。免疫电子显微镜证实了这些突变体在促肾上腺皮质激素颗粒中的存在减少。用8-Br-cAMP刺激AtT-20转染细胞导致野生型P-选择素的膜表达显著增加，但五个突变体的表面表达仅略有增加。对完整细胞和通透细胞进行的抗体结合研究表明，这些突变体在细胞表面表达的P-选择素百分比明显高于野生型P-选择素（6%），Gly778和Phe780突变体约为20%，Leu768/Asn769突变体为63%。综上所述，这些结果表明Tyr777、Gly778和Phe780构成了一个非典型酪氨酸基序的一部分，该基序还需要Leu768和/或Asn769的存在来介导P-选择素分选至分泌颗粒。
