Structural characterization of the integrin alphaIIbbeta3 transmembrane and cytosolic domains.

摘要

Integrin's are the principal cell surface receptors that link the cytoskeleton to the extracellular matrix. They exist in active conformations that can bind extracellular ligands and resting conformations that cannot. The platelet integrin alphaIIbbeta3 is a prototypical regulated integrin that is resting on a circulating platelet and becomes activated to adhere the platelet to the vascular endothelium or subendothelial matrix.;The integrin is composed of alpha and beta subunits and each subunit contains a single transmembrane helix that form an alpha/beta heterodimer in the resting state. Additionally, each subunit contains a cytosolic domain that binds signaling proteins that affect the resting-active equilibrium. Activation signals are transduced across the membrane by separating the transmembrane heterodimer.;The structure of the resting integrin alphaIIbbeta3's transmembrane and cytosolic domains was characterized by molecular modeling and NMR spectroscopy. First, software was developed to model transmembrane helix dimers using experimental mutagenesis results as a modeling restraint. Next, the alphaIIb/beta3 transmembrane heterodimer was modeled and the model was compared to published experimental data and other published models. The model correlated well with experimental findings and converged on the same structure as other top performing models, suggesting this conformation approximates the native interface. The model's interface includes alphaIIb residue Met987 and beta3 residue Leu712. These residues were mutated to cysteine to crosslink peptides corresponding to the alphaIIb and beta3 cytosolic tails, and the disulfide-linked construct was probed by NMR spectroscopy.;NMR revealed that the alphaIIb and beta3 cytosolic tails have a dynamic interface. The alphaIIb subunit is natively unstructured and the beta3 subunit consists of a hydrophobic helix followed by two amphiphilic helices. The amphiphilic portions of beta3 include domains that interact with cytosolic proteins, but the membrane embedding of its hydrophobic faces sequesters some of the interacting residues. This result suggests that the integrin's resting-active equilibrium is coupled to an equilibrium between membrane embedded and solvent exposed conformations of the beta3 cytosolic tail, providing new insight into integrin activation.