The majority of receptors responsible for activation of distinct cell types within the immune system assemble with dimeric signaling modules through interaction of a basic transmembrane residue with a pair of acidic residues of the signaling dimer. Because assembly of other membrane proteins requires specific interactions along extended stretches of transmembrane helices, we examined how transmembrane sequences flanking the polar interaction site contribute to assembly for three receptors that associate with different signaling modules-the natural killer cell receptors KIR and NKG2D and the Fc receptor for IgA, FcalphaRI. The KIR and NKG2D receptors assembled with the DAP12 and DAP10 dimers, respectively, even when the entire KIR or NKG2D transmembrane domains were replaced by polyleucine sequences with a properly positioned basic residue. In contrast, a high degree of specificity for the basic side chain could be observed because the KIR-DAP12 and FcalphaRI-Fcgamma interactions favored lysine or arginine, respectively. Steric hindrance among incompatible extra-membranous domains and competition for signaling modules also contributed to specificity of assembly. These results demonstrate that these interactions are focused on the polar site created by three ionizable transmembrane residues, and explain how the DAP12 and Fcgamma signaling modules can assemble with large, non-overlapping sets of receptors that have highly divergent transmembrane sequences.