Wild Type and Transmembrane Domain Mutants were Expressed at Similar Levels on the Surface of Stably Transfected 253T-NT Cells Sequence analysis showed that the CEACAM1-4S transmembrane domain contained multiple GXXXG dimerization motifs and two C-terminal tyrosine residues which in other transmembrane receptors were involved in transmembrane domain dependent signaling events [30, 31, 32] . To determine if these transmembrane domain elements played a part in the anchorage independent phenotype induced by CEACAM1-4S, nine expression vectors encoding CEACAM1-4S with transmembrane domain mutations in the GXXXG motifs and/or tyrosine residues were constructed as described under Methods and shown in Table 1 . These vectors were used to produce stably transfected sublines of 253-NT. Single G424L or G432L mutations or double G424L/G432L mutations were introduced to disrupt two or more GXXXG motifs. Substituting leucine for glycine introduced a large hydrophobic amino acid that maintained the hydrophobic character of the transmembrane domain but disrupted the conformation of the transmembrane domain helix [28] . Transmembrane domain tyrosine residues were mutated to valine, a substitution that replaced tyrosine with a non-polar hydrophobic amino acid lacking a hydroxyl group in its side chain. In other systems, proper spacing of the tyrosine hydroxyl group had been shown to be necessary for signaling events dependent upon protein-protein interactions [41] .