A balance of opposing signals within the cytoplasmic tail controls the lysosomal targeting of P-selectin.

The 35-amino acid cytoplasmic tail of the adhesion receptor P-selectin is subdivided into stop transfer, C1 and C2 domains. It contains structural signals needed for targeting this protein to specialized secretory organelles and to lysosomes. Recently, using site-directed mutagenesis of horseradish peroxidase-P-selectin chimeras, we have uncovered a novel sequence within the C1 domain, KCPL, that mediates sorting from early, transferrin-positive endosomes to lysosomes and therefore operates as a positive lysosomal targeting signal (Blagoveshchenskaya, A. D., Norcott, J. P. , and Cutler, D. F. (1998) J. Biol. Chem. 273, 2729-2737). In the current study, we examined lysosomal targeting by both subcellular fractionation and an intracellular proteolysis assay and found that a balance of positive and negative signals is required for proper lysosomal sorting of P-selectin. First, we have found that within the sequence KCPL, Cys-766 plays a major role along with Pro-767, whereas Lys-765 and Leu-768 make no contribution to promoting lysosomal targeting. In addition, horseradish peroxidase-P-selectin chimeras were capable of acylation in vivo with [3H]palmitic acid at Cys-766, since no labeling of a chimera in which Cys-766 was replaced with Ala was detected. Second, analysis of mutations within the C2 domain revealed that substitution of two sequences, YGVF and DPSP, causes an increase in both lysosomal targeting and intracellular proteolysis suggesting the presence of lysosomal avoidance signals. The inhibition or promotion of lysosomal targeting resulted from alterations in endosomal sorting since internalization was not changed in parallel with lysosomal delivery. Analysis of the double mutants KCPL/YGVF or KCPL/DPSP revealed that although the positive lysosomal targeting signal operates in the early/sorting transferrin-positive endosomes, the negative lysosomal targeting (lysosomal avoidance) signals act at later stages of the endocytic pathway, most likely in late endosomal compartments.