While the mechanism behind the gain-of-function produced by the Y mutations is less clear, there is increasing evidence supporting the functional importance of interactions between transmembrane aromatic amino acids [41] . Although aromatic residues make up only a small percentage of the amino acids in any given protein, they are generally the most highly conserved residues. Interactions between the aromatic rings of phenylalanine, tryptophan (W) or tyrosine are thought to involve pi-stacking, a process that creates an attractive force when aromatic rings assume energetically favored stacking geometries. Accumulating evidence suggests that pi-stacking plays an important role in molecular recognition and self assembly either by contributing energy for driving self assembly or by providing directionality and orientation through stacking geometries [52, 53] . When a bacterial transmembrane database was statistically analyzed by Sal-Man et al, these investigators found that aromatic pairs (WXXW or YXXY) were significantly over-represented compared to their predicted frequency, suggesting a functional role for these sequences [52] . The stabilization of transmembrane domain selfassociation following substitution of tyrosine for glutamine (Q) and serine (S) in the known dimerization motif, QXXS, provided support for this idea and led these authors to conclude that stabilization of transmembrane associations by aromatic residues may be a general mechanism for generating specificity in transmembrane-transmembrane interactions. In considering which amino acid to substitute for the transmembrane tyrosine residues, phenylalanine would intuitively seem to be the logical When separated on native gels, wild type CEACAM1-4S and the single G mutants migrated with an apparent molecular mass that was approximately 100 kDa higher than the double glycine mutant. doi:10.1371/journal.pone.0029606.g010 choice. However, based on recent reports, tyrosine to phenylalanine mutations may or may not disrupt function. Stevens et al [54] reported that a YS/FA mutation of the IgM transmembrane domain had no effect on anti-Ig induced signaling as measured by the activation of tyrosine phosphorylation and did not disrupt the association between IgM and its signaling partners, Ig-alpha/Igbeta. In contrast, a YS/VV mutation diminished both signaling and association with Ig-alpha/Ig-beta. In cases where phenylalanine substitutions fail to mimic tyrosine, the lack of a properly spaced hydroxy group may be the reason [41] . Based on these considerations, it was decided to introduce tyrosine to valine mutations.