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 stru ctural signals needed for targeting this protein to specialized secret ory organelles and to lysosomes. Recently, using site-directed mutagen esis of horseradish peroxidase-P-selectin chimeras, we have uncovered a novel sequence within the C1 domain, KCPL, that mediates sorting fro m early, transferrin-positive endosomes to lysosomes and therefore ope rates 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 f ound that a balance of positive and negative signals is required for p roper lysosomal sorting of P-selectin. First, we have found that withi n the sequence KCPL, Cys-766 plays a major role along with Pro-767, wh ereas Lys-765 and Leu-768 make no contribution to promoting lysosomal targeting. In addition, horseradish peroxidase-P-selectin chimeras wer e capable of acylation in vivo with [H-3]palmitic acid at Cys-766, sin ce 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 promoti on of lysosomal targeting resulted from alterations in endosomal sorti ng since internalization was not changed in parallel with lysosomal de livery. 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 end ocytic pathway, most likely in late endosomal compartments.