Date Published: June 15, 2004
Publisher: Public Library of Science
Author(s): Bing-Hao Luo, Timothy A Springer, Junichi Takagi
Abstract: Conformational communication across the plasma membrane between the extracellular and intracellular domains of integrins is beginning to be defined by structural work on both domains. However, the role of the α and β subunit transmembrane domains and the nature of signal transmission through these domains have been elusive. Disulfide bond scanning of the exofacial portions of the integrin αIIβ and β3 transmembrane domains reveals a specific heterodimerization interface in the resting receptor. This interface is lost rather than rearranged upon activation of the receptor by cytoplasmic mutations of the α subunit that mimic physiologic inside-out activation, demonstrating a link between activation of the extracellular domain and lateral separation of transmembrane helices. Introduction of disulfide bridges to prevent or reverse separation abolishes the activating effect of cytoplasmic mutations, confirming transmembrane domain separation but not hinging or piston-like motions as the mechanism of transmembrane signaling by integrins.
Partial Text: Integrins are major metazoan cell adhesion receptors that have the distinctive property of transducing signals across the plasma membrane in both directions. Intracellular binding of cytoskeletal components to integrin cytoplasmic domains activates the ligand binding competency of the extracellular domain (inside-out signaling). Furthermore, ligand binding to integrin extracellular domains is coupled to alterations in cytoplasmic domains that are linked to downstream signaling (outside-in signaling). The three-dimensional architecture of integrin extracellular domains as well as their rearrangement in activation have been revealed by crystal, nuclear magnetic resonance (NMR), and electron microscopic methods (Xiong et al. 2001, 2002; Adair and Yeager 2002; Beglova et al. 2002; Takagi et al. 2002, 2003). NMR structures of integrin α and β subunit cytoplasmic tails (Vinogradova et al. 2000, 2002; Ulmer et al. 2001; Weljie et al. 2002) and a crystal structure of the β subunit tail in complex with the cytoskeletal protein talin (Garcia-Alvarez et al. 2003) yield structural insights. It is generally accepted that an intersubunit association at the cytoplasmic domain maintains integrins in the low-affinity state (Hughes et al. 1996); however, specific heterodimeric interaction between the isolated cytoplasmic domains in solution is sometimes not observed (R. Li et al. 2001; Ulmer et al. 2001), and when observed the reported structures differ (Vinogradova et al. 2002; Weljie et al. 2002). The dynamic nature of cytoplasmic intersubunit association was revealed using live cell imaging (Kim et al. 2003), which demonstrated upon integrin activation a decrease in fluorescent resonance energy transfer between yellow fluorescent protein and cyan fluorescent protein tags fused to the C-termini of the integrin α and β subunit cytoplasmic domains. This finding demonstrated separation of the cytoplasmic domains; however, whether signal transmission through integrin transmembrane (TM) domains involves hinging or pistoning motions or lateral separation in the plane of the membrane has yet to be definitively established (Hughes et al. 1996; Lu et al. 2001; Takagi et al. 2001, 2002; Gottschalk et al. 2002).
We have obtained for the first time structural information regarding the helix–helix interface between integrin α and β subunit TM domains in the membrane bilayer, and demonstrate that dissociation at this interface occurs upon changes at the cytoplasmic face of the plasma membrane bilayer that activate integrins. Extensive mutagenic cysteine cross-linking experiments revealed the presence of a specific α/β TM helix contact in a resting integrin heterodimer, which is lost upon receptor activation from inside the cell. The data establish the approximate orientation between the integrin TM α helices in the outer leaflet of the membrane bilayer in the resting, low-affinity integrin conformation (see Figure 2C).