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Trophoblast giant-cell differentiation is well-characterized at the molecular level, yet very little is known about how molecular changes affect the cellular functions of trophoblast in embryo implantation. We have found, using both explanted E7.5 mouse embryo ectoplacental cone and the rat choriocarcinoma (Rcho-1) cell line, that trophoblast differentiation is distinguished by dramatic changes in cytoarchitecture and cell behavior. Undifferentiated trophoblast cells contain little organized actin and few small, peripheral focal complexes and exhibit high membrane protrusive activity, while differentiated trophoblast giant cells contain prominent stress fibers, large internal as well as peripheral focal adhesions, and become immotile. The dramatic changes in cell behavior and cytoskeletal organization of giant cells correlate with changes in the activities of the Rho family of small GTPases and a decrease in tyrosine phosphorylation of focal adhesion kinase. Together, these data provide detailed insight into the cellular properties of trophoblast giant cells and suggest that giant-cell differentiation is characterized by a transition from a motile to a specialized epithelial phenotype. Furthermore, our data support a phagocytic erosion, rather than a migratory infiltration, mechanism for trophoblast giant-cell invasion of the uterine stroma.

We previously identified the 11 amino acid C1 region of the cytoplasmic domain of P-selectin as essential for an endosomal sorting event that confers rapid turnover on P-selectin. The amino acid sequence of this region has no obvious similarity to other known sorting motifs. We have analyzed the sequence requirements for endosomal sorting by measuring the effects of site-specific mutations on the turnover of P-selectin and of the chimeric protein LLP, containing the lumenal and transmembrane domains of the low density lipoprotein receptor and the cytoplasmic domain of P-selectin. Endosomal sorting activity was remarkably tolerant of alanine substitutions within the C1 region. The activity was eliminated by alanine substitution of only one amino acid residue, leucine 768, where substitution with several other large side chains, hydrophobic and polar, maintained the sorting activity. The results indicate that the endosomal sorting determinant is not structurally related to previously reported sorting determinants. Rather, the results suggest that the structure of the sorting determinant is dependent on the tertiary structure of the cytoplasmic domain. INTRODUCTIONThe function of membrane-bounded organelles requires correct targeting of resident membrane proteins to each organelle and in many cases selective cycling of membrane proteins between organelles. Selective localization and targeting of membrane proteins to their appropriate destinations depend on structural features of these proteins, termed sorting determinants. Sorting determinants are recognized by sorting machinery that functions to concentrate proteins bearing the appropriate sorting determinants into specific transport vesicles, which can then carry their cargo vectorially to the correct destination. A number of sorting determinants have been characterized, including those that mediate localization to clathrin-coated pits in the trans-Golgi network (TGN) 11 or at the cell surface, sorting to the basolateral cell surface in polarized epithelial cells, and selective transport from endosomes to lysosomes (Sandoval and Bakke, 1994;Mellman, 1996;Kirchhausen et al., 1997;Marks et al., 1997).Many sorting determinants are contained in short segments of amino acid sequence in the cytoplasmic domains of transmembrane proteins (Sandoval and Bakke, 1994;Marks et al., 1997). These short sequences have been defined by mutagenesis experiments in which systematic substitution of amino acid residues identifies only a few mutations that disrupt sorting activity. To date, the most prevalent and most extensively studied determinants that operate in post-Golgi trafficking pathways are those that require a tyrosine residue and additional residues in specific contexts * Present address: Department of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville, VA 22908. † Corresponding author: Department of Cell Biology, Box 439 HSC, University of Virginia, Charlottesville VA 22908. E-mail address: sag4y@virginia.edu. 1 Abbreviations used: CHO, Ch...

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