RESIDUES THROUGHOUT THE CYTOPLASMIC DOMAIN AFFECT THE INTERNALIZATION EFFICIENCY OF P-SELECTIN

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.