Overexpressed SNX17 and endogenous P-selectin colocalize in endosomes of HUVECs in secretagogue stimulated cells only. (A) P-selectin and SNX17-GFP do not colocalize in resting HUVECs. HUVECs were nucleofected with SNX17-GFP (5 μg). One day later, cells were stained with anti-P-selectin antibody (AK6). (B) Endocytosed anti-P-selectin antibody colocalizes with SNX17. One day postnucleofection with SNX17-GFP (5 μg) HUVECs were stimulated with histamine (10^-4 M) for 15 min in the presence of anti-P-selectin antibody AK6 (10 μg/ml). After a 30-min incubation in RM (MATERIALS AND METHODS) cells were fixed and processed for microscopy. (C-F) Localization of SNX17 overexpressed in HUVECs. HUVECs were nucleofected with SNX17-GFP (5 μg) and 1 day postnucleofection cells were processed for microscopy and stained with the indicated marker: (C) TfnR, (D) M6PR, (E) CD63, and (F) LAMP1. For all images primary antibodies were visualized with relevant donkey secondary antibodies conjugated with Texas Red as described in MATERIALS AND METHODS. Bars, 10 μm.

Overexpressed SNX17 is localized to early endosomes in HEK-293 cells. One day postnucleofection with SNX17-GFP (5 μg) HEK-293 cells were fixed, processed for microscopy and stained with the indicated markers: (A) TfnR, (B) LBPA, (C) EEA1, and (D) TGN46. Primary antibodies were visualized with relevant donkey secondary antibodies conjugated with Texas Red as described in MATERIALS AND METHODS. Bars, 10 μm.

SNX17 overexpression reduces the clipping of HRP-P-selectin. (A) HEK-293 cells nucleofected with either ssHRP^P-selectin () or SNX17-GFP and ssHRP^P-selectin (□) were seeded onto 60-mm plates. At 1 to 3 days after nucleofection cells were lysed and Triton X-114 partitioned, and HRP activity in aqueous and detergent phases was determined as described in MATERIALS AND METHODS. The data are presented as percentage of total activity present in the aqueous phase (i.e., clipped HRP). (B) HEK-293 cells were nucleofected with either ssHRP^P-selectin or SNX17-GFP and ssHRP^P-selectin at a 2:1 (); 5:1 (□) and 10:1 (▪) ratio of DNA. Cells were seeded onto 60-mm dishes and lysed and Triton X-114 partitioned, and HRP activity in aqueous and detergent phases was determined at 1 and 2 days postnucleofection. Data are shown as difference between the percentage of HRP activity in aqueous phase for cells nucleofected with ssHRP^P-selectin and that for cells nucleofected with SNX17-GFP and ssHRP^P-selectin. (C) HUVEC were nucleofected with either ssHRP^P-selectin () or SNX17-GFP and ssHRP^P-selectin (□) and seeded onto 100-mm plates. Cells were then treated as in A. Each bar represents the mean ± SD of six independent determinations.

SNX17 and SNX1 both bind to P-selectin but only SNX17 has a major effect on HRP clipping. (A) HEK-293 cells were transfected with ssHRP^P-selectin or with ssHRP^{P-selectin763} either in combination with or separately with pDHH-SNX1 or pSNX17-HH, which encode the respective His-tagged SNX's. Binding was then determined in lysates from cells coexpressing the two potential partners (prelysis) or in extracts from cells expressing the two components separately that were mixed after lysis (postlysis). His-tagged complexes were isolated using Probond, eluted from the beads, and the HRP activity were determined as in MATERIALS AND METHODS. Data are presented as a percentage of the binding of SNX17 and to ssHRP^P-selectin for either method, (±SE) and is representative of two independent experiments analyzed in triplicate. (B) Elutants from A were subjected to SDS-PAGE followed by Western blotting with an anti-His antibody. Bands were visualized using an HRP-conjugated anti-mouse secondary followed by ECL detection. (C) HEK-293 cells were nucleofected with ssHRP^P-selectin or ssHRP^{P-selectin763} in combination with a control plasmid, pSNX17-GFP, or pDHH-SNX1 and were subjected to TX114 partitioning. The HRP activity in the aqueous and detergent phases was determined as in MATERIALS AND METHODS. The data from six independent determinations are expressed as a percentage of the HRP-clipping activity of SNX17.

Both PI3 kinase activity and the PX domain of SNX17 are needed for endosomal localization, but function is only partially dependent on its PX domain. (A) Schematic diagram of domain structure of SNX17^*15.03 encoding His-tagged sorting nexin 17 lacking a PX domain. Known domains (PX, FERM) are boxed and amino acid length are indicated underneath. The site at which pDHH-SNX17^*15.03 begins is indicated with an arrow above.(B) Wortmannin treatment or loss of the PX domain relocate SNX17 to the cytosol. CHO or HEK293 (unpublished data) cells nucleofected with SNX17-GFP 1 day postnucleofection were incubated with medium (A) or medium containing 50 nM wortmannin (B) for 15 min and examined by epifluorescence microscopy. HEK-293 cells nucleofected with pSNX17-HH (C) and pDHH-SNX17^*15.03 (D) were fixed 1 day postnucleofection and examined by confocal microscopy. Bars, 10 μm. (C) HEK-293 cells were conucleofected with ssHRP^P-selectin and pSNX17-HH or pDHH-SNX17^*15.03. After 24 h His-tagged complexes were isolated using Probond and eluted from the beads, and the HRP activity was determined as in MATERIALS AND METHODS. Data are expressed as a percentage of the binding ssHRP^P-selectin and pSNX17-HH. Each bar represents the mean ± SE of at least five independent determinations. (D) HEK-293 cells were conucleofected with ssHRP^P-selectin and pSNX17-HH or pDHH-SNX17^*15.03 and after 24 h were subjected to TX114 partitioning. HRP activity in aqueous and the detergent phases were measured, and the clipping activity was determined. Data are expressed as a percentage of clipping that occurs with ssHRP^P-selectin and pSNX17-HH obtained from five independent experiments.

SNX17 expression enhances the internalization of P-selectin from the cell surface. HEK-293 cells were transfected with pPEX, encoding for P-selectin, and an inducible SNX17-GFP variant (pTRE-bsd-SNX17-GFP). SNX17-GFP expression not induced (control cells) or induced with 2 μg/ml doxycycline for 12 h. Cells were labeled with anti-P-selectin antibody on ice, warmed for indicated times at 37°C to allow internalization then fixed, stained with Alexa 647-labeled secondary antibody and analyzed by flow cytometry as described in materials and methods. Data points shown are means (±SE) from three independent experiments are shown from control cells (□), cells gated as expressing low SNX17-GFP (▪), and cells gated as expressing high levels of SNX-GFP (▴).