title: Residues throughout the cytoplasmic domain affect the internalization efficiency of P-selectin.
abstract: 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 clathrin-coated 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.
authors.affiliation: W. K. Warren Medical Research Institute, Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, USA.
authors.forename: H
authors.initials: H
authors.lastname: Setiadi
authors.forename: M
authors.initials: M
authors.lastname: Disdier
authors.forename: S A
authors.initials: SA
authors.lastname: Green
authors.forename: W M
authors.initials: WM
authors.lastname: Canfield
authors.forename: R P
authors.initials: RP
authors.lastname: McEver
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D004705:Endocytosis; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019007:P-Selectin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid
chemical_list: D019007:P-Selectin; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins