RESIDUES THROUGHOUT THE CYTOPLASMIC DOMAIN AFFECT THE INTERNALIZATIONEFFICIENCY OF P-SELECTIN

The cytoplasmic domains of many membrane proteins have short sequences , usually including a tyrosine or a di-leucine, that function as sorti ng 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 secret ory granules, for endocytosis, and for movement from endosomes to lyso somes, The domain has a membrane-distal sequence, YGVFTNAAF, that rese mbles some tyrosine-based signals, We studied the effects of deletions and mutations in the cytoplasmic tail of human P-selectin on its inte rnalization in clathrin-coated pits of transfected Chinese hamster ova ry 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 truncat ed 18-residue cytoplasmic domain with a single substitution (K761A/H77 3Stop) was internalized nearly three times as fast as wild-type P-sele ctin; this construct contained no di-leucine, tyrosine, or other known sorting motif, Substitution of residues throughout the cytoplasmic do main 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 de nsity lipoprotein receptor than when attached to the corresponding dom ains of P-selectin. Thus, we were unable to identify a short internali zation signal in the cytoplasmic tail of P-selectin, Residues througho ut the cytoplasmic domain, and perhaps the transmembrane sequence to w hich the domain is attached, affect the efficiency of internalization.