AP-3 adaptor functions in targeting P-selectin to secretory granules in endothelial cells.
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 alpha-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.
Daugherty
B L
BL
Department of Cell Biology, UVa Health System, School of Medicine, PO Box 800732, Charlottesville, VA 22908-0732, USA.
Straley
K S
KS
Sanders
J M
JM
Phillips
J W
JW
Disdier
M
M
McEver
R P
RP
Green
S A
SA
D033965:Adaptor Protein Complex alpha Subunits; D000595:Amino Acid Sequence; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D011992:Endosomes / Q000737:chemistry / Q000378:metabolism; D004727:Endothelium / Q000166:cytology* / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D005434:Flow Cytometry; D005982:Glutathione Transferase / Q000378:metabolism; D006579:Heterozygote; D008099:Liver / Q000378:metabolism; D008168:Lung / Q000166:cytology; D008565:Membrane Proteins / Q000502:physiology*; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D019007:P-Selectin / Q000378:metabolism*; D016716:PC12 Cells; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D022142:Secretory Vesicles / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
D033965:Adaptor Protein Complex alpha Subunits; D008565:Membrane Proteins; D019007:P-Selectin; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase