The cytoplasmic domains of many membrane proteins have short sequences, usually including a tyrosine or a di-leucine, that function as sorting signals. P-selectin is an adhesion receptor for leukocytes that is expressed on activated platelets and endothelial cells. Its 35-residue cytoplasmic domain contains signals for sorting into regulated secretory granules, for endocytosis, and for movement from endosomes to lysosomes. The domain has a membrane-distal sequence, YGVFTNAAF, that resembles some tyrosine-based signals. We studied the effects of deletions and mutations in the cytoplasmic tail of human P-selectin on its internalization in clathrincoated pits of transfected Chinese hamster ovary cells. Mutations and deletions in the putative tyrosine-based motif did not clearly implicate these residues as critical components of a short internalization signal. Indeed, a construct containing a truncated 18-residue cytoplasmic domain with a single substitution (K761A/H773Stop) was internalized nearly three times as fast as wild-type P-selectin; this construct contained no di-leucine, tyrosine, or other known sorting motif. Substitution of residues throughout the cytoplasmic domain affected the internalization rate of P-selectin. Furthermore, the cytoplasmic domain of P-selectin mediated faster internalization when attached to the extracellular and transmembrane domains of the low density lipoprotein receptor than when attached to the corresponding domains of P-selectin. Thus, we were unable to identify a short internalization signal in the cytoplasmic tail of P-selectin. Residues throughout the cytoplasmic domain, and perhaps the transmembrane sequence to which the domain is attached, affect the efficiency of internalization.

P-selectin (CD62), formerly called GMP-140 or PADGEM, is a membrane protein located in secretory storage granules of platelets and endothelial cells. To study the mechanisms responsible for the targeting of P-selectin to storage granules, we transfected its cDNA into COS-7 and CHO-Ki cells, which lack a regulated exocytic pathway, or into AtT20 cells, which are capable of regulated secretion. P-selectin was expressed on the plasma membrane of COS-7 and CHO-Kl cells but was concentrated in storage granules of AtT20 cells. Immunogold electron microscopy indicated that the electron-dense granules containing P-selectin in AtT20 cells also stored the endogenous soluble hormone ACTH. Activation of AtT20 cells with 8-Br-cAMP increased the surface expression of P-selectin, consistent with agonist-induced fusion of granule membranes with the plasma membrane. Deletion of the last 23 amino acids of the 35-residue cytoplasmic domain resulted in delivery of Pselectin to the plasma membrane of AtT20 cells. Replacement of the cytoplasmic tail of tissue factor, a plasma membrane protein, with the cytoplasmic domain of P-selectin redirected the chimeric molecule to granules. We conclude that the cytoplasmic domain of P-selectin is both necessary and sufficient for sorting of membrane proteins into the regulated pathway of secretion.

P-selectin, a cell adhesion protein participating in the early stages of inflammation, contains multiple sorting signals that regulate its cell surface expression. Targeting to secretory granules regulates delivery of P-selectin to the cell surface. Internalization followed by sorting from early to late endosomes mediates rapid removal of P-selectin from the surface. We show here that the P-selectin cytoplasmic domain bound AP-2 and AP-3 adaptor complexes in vitro. The amino acid substitution L768A, which abolishes endosomal sorting and impairs granule targeting of P-selectin, reduced binding of AP-3 adaptors but not AP-2 adaptors. Turnover of P-selectin was 2.4-fold faster than turnover of transferrin receptor in AP-3-deficient mocha fibroblasts, similar to turnover of these two proteins in AP-3-competent cells, demonstrating that AP-3 function is not required for endosomal sorting. However, sorting P-selectin to secretory granules was defective in endothelial cells from AP-3-deficient pearl mice, demonstrating a role for AP-3 adaptors in granule assembly in endothelial cells. P-selectin sorting to platelet a-granules was normal in pearl mice, consistent with earlier evidence that granule targeting of P-selectin is mechanistically distinct in endothelial cells and platelets. These observations establish that AP-3 adaptor functions in assembly of conventional secretory granules, in addition to lysosomes and the 'lysosome-like' secretory granules of platelets and melanocytes. P-selectin, a cell adhesion protein that functions in leukocyte recruitment early in the inflammatory response, is targeted to secretory granules in platelets and endothelial cells, and delivered to the cell surface upon regulated granule exocytosis in response to inflammatory stimuli (1). Cell surface Pselectin is rapidly internalized in endothelial cells and transfected cell lines (2,3), then undergoes selective sorting from early endosomes to late endosomes, resulting in rapid recycling through late endosomes and the trans-Golgi network (TGN) (4). Rapid internalization followed by endosomal sorting provides temporal regulation for cell surface expression of P-selectin following granule exocytosis, and also results in rapid delivery of P-selectin to lysosomes when it is not packaged in secretory granules (5-7). These three sorting activities are critical to regulating the adhesive function of this protein.The 35-amino acid cytoplasmic domain of P-selectin ( Figure 1) mediates each of the three sorting activities. Rapid internalization of P-selectin occurs in clathrin-coated pits (3). Although a conventional internalization signal was not identifiable by alanine-scanning mutagenesis, the cytoplasmic domain of P-selectin has recently been shown to associate with the medium (m) subunit of the AP-2 adaptor through an extended tyrosine-containing motif (8). Endosomal sorting of P-selectin is mediated by a sorting determinant that functions independently of the rapid internalization activity, and can be abolished by the single amino acid substi...

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