Evidence for a granule targeting sequence within platelet factor 4

Abstract

Platelets achieve bleeding arrest at sites of vascular injury via secretion of secretory proteins from their storage granules, termed α-granules. We have recently analyzed granule targeting of platelet factor 4 (PF4), a secretory α-granule chemokine, and demonstrated that PF4 α-granule storage relied upon determinants within PF4 mature sequence. To define these determinants, PF4 mutants fused to the fluorescent reporter protein green fluorescent protein were generated by progressive deletions and site-directed mutagenesis. They were then transfected in AtT20 cells and assessed for granule targeting by colocalization with ACTH-containing granules, using laser scanning confocal microscopy. This strategy identified the amino acid 41-50 (LIATLKNGRK) sequence as most critical for PF4 granule targeting and/or storage; its deletion from PF4 induced a marked decrease in granule storage (from 81 ± 2% to 17 ± 3%, p ≤ 0.0001). Ala-scanning mutagenesis of LIATLKNGRK narrowed down the targeting motif to LKNG. A direct role for LKNG in α-granule targeting -was confirmed in the thrombopoietin-induced human megakaryocytic Dami cells, in which the LKNG-green fluorescent protein chimera exhibited an 82.5 ± 1.8% colocalization with the α-granule proteins von Willebrand factor and P-selectin. LKNG is poorly conserved within the chemokine family. However three-dimensional alignments of the human α-granule chemokines Nap-2 (neutrophil-activating peptide) and RANTES (Regulated upon Activation Normal T Cell Expressed and Secreted) with PF4 revealed that LKNG, a surface-exposed hydrophilic turn/loop, matched Nap-2 (LKDG) and RANTES (TRKN) peptides with similar features. Moreover Nap-2 and RANTES peptides exhibited the same α-granule targeting efficiency than LKNG. We therefore postulate that the three-dimensional and physicochemical characteristics of PF4 LKNG are of general relevance to α-granule targeting of chemokines and possibly of other α-granule proteins. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.