Determination of N- and C-terminal Borders of the Transmembrane Domain of Integrin Subunits

Previous studies on the membrane-cytoplasm interphase of human integrin subunits have shown that a conserved lysine in subunits α2, α5, β1, and β2 is embedded in the plasma membrane in the absence of interacting proteins (Armulik, A., Nilsson, I., von Heijne, G., and Johansson, S. (1999) in J. Biol. Chem. 274, 37030–37034). Using a glycosylation mapping technique, we here show that α10 and β8, two subunits that deviate significantly from the integrin consensus sequences in the membrane-proximal region, were found to have the conserved lysine at a similar position in the lipid bilayer. Thus, this organization at the C-terminal end of the transmembrane (TM) domain seems likely to be general for all 24 integrin subunits. Furthermore, we have determined the N-terminal border of the TM domains of the α2, α5, α10, β1, and β8 subunits. The TM domain of subunit β8 is found to be 22 amino acids long, with a second basic residue (Arg684) positioned just inside the membrane at the exoplasmic side, whereas the lipidembedded domains of the other subunits are longer, varying from 25 (α2) to 29 amino acids (α10). These numbers implicate that the TM region of the analyzed integrins (except β8) would be tilted or bent in the membrane. Integrin signaling by transmembrane conformational change may involve alteration of the position of the segment adjacent to the conserved lysine. To test the proposed “piston” model for signaling, we forced this region at the C-terminal end of the α5 and β1 TM domains out of the membrane into the cytosol by replacing Lys-Leu with Lys-Lys. The mutation was found to not alter the position of the N-terminal end of the TM domain in the membrane, indicating that the TM domain is not moving as a piston. Instead the shift results in a shorter and therefore less tilted or bent TM α-helix.