Previous studies on the membrane-cytoplasm interphase of human integrin subunits have shown that a conserved lysine in subunits ␣ 2 , ␣ 5 , ␤ 1 , and ␤ 2 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 ␣ 10 and ␤ 8 , 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 ␣ 2 , ␣ 5 , ␣ 10 , ␤ 1 , and ␤ 8 subunits. The TM domain of subunit ␤ 8 is found to be 22 amino acids long, with a second basic residue (Arg 684 ) positioned just inside the membrane at the exoplasmic side, whereas the lipidembedded domains of the other subunits are longer, varying from 25 (␣ 2 ) to 29 amino acids (␣ 10 ). These numbers implicate that the TM region of the analyzed integrins (except ␤ 8 ) 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 ␣ 5 and ␤ 1 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 ␣-helix.Integrins are heterodimeric receptors composed of an ␣ subunit noncovalently associated with a ␤ subunit. Each subunit has an N-terminal extracellular domain, a transmembrane (TM) 1 region and a cytoplasmic domain. The human ␣-and ␤ subunits constitute two unrelated protein families of 18 and 8 members, respectively (1, 2).Integrins mediate cell adhesion to the pericellular matrix and to neighboring cells (1). In addition to the anchoring function, ligand binding to integrins generates intracellular signals required for several cellular processes, including cell migration and proliferation. The ability to bind ligands is regulated by mechanisms acting on the cytoplasmic part of the protein, an unusual receptor feature. Integrin activation by cytoplasmic signals has been shown to involve transmembrane conformational changes (3, 4). Subsequent ligand binding induces further structural rearrangements, as monitored by exposure of new epitopes, in the extracellular as well as in the intracellular domains (5, 6).Recently, significant progress has been made in the elucidation of the mechanisms controlling integrin activation ("inside-out signaling") and ligand-induced signaling ("outside-in signalin...

Adhesion by means of b1-integrins induces the phosphorylation of Akt, an event strictly dependent on the activity of the phosphatidylinositol 3-kinase (PI3K). Binding of the p85 regulatory subunit of PI3K to phosphorylated tyrosine 397 in focal adhesion kinase (FAK) is considered to be the mechanism of cell adhesion-induced activation of class Ia PI3K. Here we show that PI3K-dependent phosphorylation of Akt in response to ligation of b1-integrins occurs efficiently in the absence of FAK tyrosine phosphorylation. Akt S473 phosphorylation was strongly promoted both in cells expressing the integrin subunit splice variant b1B, which is unable to activate FAK, and in FAK knockout cells. In addition, we found this phosphorylation to be independent of the Src family kinases Src, Fyn and Yes. These results indicate that a major pathway for adhesion-dependent activation of PI3K/Akt is triggered by the membrane proximal part of the b1 subunit in a FAK and Src-independent manner.

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