  Integrins are transmembrane proteins involved in hemostasis, wound healing,
immunity and cancer. In response to intracellular signals and ligand binding,
integrins adopt different conformations: the bent (resting) form; the
intermediate extended form; and the ligand-occupied active form. An integrin
undergoing such conformational dynamics is the heterodimeric platelet receptor
alphaIIbbeta3. Although the dramatic rearrangement of the overall structure of
alphaIIbbeta3 during the activation process is potentially related to changes
in the protein secondary structure, this has not been investigated so far in a
membrane environment. Here we examine the Mn2+- and drug-induced activation of
alphaIIbbeta3 and the impact on the structure of this protein reconstituted
into liposomes. By quartz crystal microbalance with dissipation monitoring and
activation assays we show that Mn2+ induces binding of the
conformation-specific antibody PAC-1, which only recognizes the extended,
active integrin. Circular dichroism spectroscopy reveals, however, that
Mn2+-treatment does not induce major secondary structural changes of
alphaIIbbeta3. Similarly, we found that treatment with clinically relevant
drugs (e.g. quinine) led to the activation of alphaIIbbeta3 without significant
changes in protein secondary structure. Molecular dynamics simulation studies
revealed minor local changes in the beta-sheet probability of several
extracellular domains of the integrin. Our experimental setup represents a new
approach to study transmembrane proteins, especially integrins, in a membrane
environment and opens a new way for testing drug binding to integrins under
clinically relevant conditions.
