Similar reasoning can be applied to the C-terminus where 5 of 12 transmembrane prediction algorithms placed Y448 outside the transmembrane domain. However, mutation of either Y445 or Y448 led to the soft agar growth enhanced phenotype, indicating first, that Y448 was functionally equivalent to Y445 and thus likely to be located within the transmembrane domain and second, that both tyrosines were required to sustain anchorage independent growth at the level produced by wild type CEACAM1-4S. However, whether Y448 was or was not in the transmembrane was moot since single Y445V or Y448V mutations produced the growth-enhanced phenotype in soft agar. Put another way, the presence of both tyrosines appeared to be necessary to suppress anchorage independent growth, a suppressive effect that apparently involved more than interactions between aromatic side chains since in both single tyrosine to valine mutants (VFLY or YFLV) the remaining tyrosine was paired with F446. Whether the two tyrosines without phenylalanine would be able maintain the wild type CEACAM1-4S phenotype is an open question that is beyond the scope of the present investigation.