A push-pull mechanism for regulating integrin function (original) (raw)
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Structural Studies of Integrin Activation
2016
Fundamental to cell adhesion and migration, integrins are large heterodimeric membrane proteins that link the extracellular matrix to the actin cytoskeleton. Uniquely, these adhesion receptors mediate inside-out signal transduction, whereby extracellular adhesion is activated from within the cell by talin, a large cytoskeletal protein that binds to the cytoplasmic tail of the β integrin subunit via its PTB-like F3 domain. Features of the interface between talin1 and small β3 fragments only have been described previously. Through NMR studies of full-length integrin β tails, we have found that β tails differ widely in their interactions with different talin isoforms. The muscle-specific β1D/talin2 complex exhibited particularly high affinity, leading to the X-ray crystal structure of the β1D tail/talin2 F2-F3 complex. Further NMR and biological experiments demonstrated that integrin activation is induced by a concerted series of interactions between the talin F3 domain and the β tail and between the talin F2 domain and the cell membrane. Additional studies revealed the structural determinants of tight talin2/β1D binding and the basis of more general differences between β1 and β3 talin binding. NMR studies were also performed on tyrosine-phosphorylated integrin tails binding to the PTB domains of talin1 and Dok1, an inhibitor of integrin activation; these revealed that phosphorylation can inhibit integrin activation by increasing the affinity of the β tail for talin competitors. Key residues governing this switch were identified, and proteins were engineered with reversed affinities, offering potentially useful biological tools. Taken together, these results reveal the remarkable complexity of structural features that enable talin and its competitors to mediate this important form of transmembrane signalling.
Oligomerization of the integrin IIb 3: roles of the transmembrane and cytoplasmic domains
Proceedings of The National Academy of Sciences, 2001
Integrins are a family of ␣͞ heterodimeric membrane proteins, which mediate cell-cell and cell-matrix interactions. The molecular mechanisms by which integrins are activated and cluster are currently poorly understood. One hypothesis posits that the cytoplasmic tails of the ␣ and  subunits interact strongly with one another in a 1:1 interaction, and that this interaction is modulated in the course of the activation of ␣IIb3 [Hughes, P. E., et al. (1996) J. Biol. Chem. 271, 6571-6574]. To examine the structural basis for this interaction, protein fragments encompassing the transmembrane helix plus cytoplasmic tails of the ␣ and  subunits of ␣IIb3 were expressed and studied in phospholipid micelles at physiological salt concentrations. Analyses of these fragments by analytical ultracentrifugation, NMR, circular dichroism, and electrophoresis indicated that they had very little or no tendency to interact with one another. Instead, they formed homomeric interactions, with the ␣and -fragments forming dimers and trimers, respectively. Thus, these regions of the protein structure may contribute to the clustering of integrins that accompanies cellular adhesion. I ntegrins, a family of ␣͞ heterodimers, mediate essential cell-cell and cell-matrix interactions (1). Each subunit of the integrin heterodimer is composed of a large extracellular domain, a transmembrane (TM) helix, and a short cytoplasmic (CYTO) tail. Heterodimer formation results from interactions between sequences located in the extracellular domain of each subunit (2). Many cells actively regulate integrin ligand-binding activity (3). The prototypic example of integrin regulation is the platelet integrin ␣IIb3 (4). In unstimulated platelets, ␣IIb3 is inactive, whereas exposing platelets to agonists such as ADP and thrombin enables ␣IIb3 to bind ligands such as fibrinogen and von Willebrand factor. The integrin is activated in a bidirectional manner, in which intracellular events can trigger a conformational change in the extracellular ligand-binding domains (inside-out signaling) or vice versa. In one proposed mechanism for this process, the CYTO tails of ␣IIb and 3 interact in the inactive state through the formation of a salt bridge (5). This interaction is broken and the CYTO domains separate when the integrin is activated. Evidence for this hypothesis came from mutational studies (5), as well as biochemical studies that seemed to show a weak but divalent cation-dependent interaction between peptides corresponding to the CYTO tails of ␣IIb and 3 (6, 7). Further, elegant protein engineering studies by Springer and coworkers (8, unambiguously demonstrated that when the CYTO domains or the C termini of the extracellular domains were forced to interact, the integrin ␣L2 or ␣51 was inactivated. However, a very recent and carefully executed NMR study indicated that the ␣IIb and 3 CYTO tails were unable to interact, even when tethered in close proximity from the same end of a heterodimeric coiled coil (10). Further, the observation that replacing the CYTO tail of ␣IIb with those of ␣5 or ␣6 results in constitutive ␣IIb3 activity, despite the fact that the membrane-proximal portions of the CYTO tails of these ␣ subunits are nearly identical to ␣IIb, suggests that the putative salt bridge cannot account completely for ␣IIb3 regulation (11). Thus, it is important to conduct biophysical experiments on peptide systems that more closely resemble the integrin proteins, and also to modify the original salt bridge hypothesis to include possible interactions with the cytoskeletal and other intracellular proteins (10).
Involvement of Transmembrane Domain Interactions in Signal Transduction by / Integrins
Journal of Biological Chemistry, 2004
The ␣ and  subunits of ␣/ heterodimeric integrins function together to bind ligands in the extracellular region and transduce signals across cellular membranes. A possible function for the transmembrane regions in integrin signaling has been proposed from structural and computational data. We have analyzed the capacity of the integrin ␣ 2 , ␣ IIb , ␣ 4 ,  1 ,  3 , and  7 transmembrane domains to form homodimers and/or heterodimers. Our data suggest that the integrin transmembrane helices can help to stabilize heterodimeric integrins but that the interactions do not specifically associate particular pairs of ␣ and  subunits; rather, the ␣/ subunit interaction constrains the extramembranous domains, facilitating signal transduction by a promiscuous transmembrane helix-helix association.
On the Activation of Integrin αIIbβ3: Outside-in and Inside-out Pathways
Biophysical Journal, 2013
Integrin aIIbb3 is a member of the integrin family of transmembrane proteins present on the plasma membrane of platelets. Integrin aIIbb3 is widely known to regulate the process of thrombosis via activation at its cytoplasmic side by talin and interaction with the soluble fibrinogen. It is also reported that three groups of interactions restrain integrin family members in the inactive state, including a set of salt bridges on the cytoplasmic side of the transmembrane domain of the integrin aand b-subunits known as the inner membrane clasp, hydrophobic packing of a few transmembrane residues on the extracellular side between the aand b-subunits that is known as the outer membrane clasp, and the key interaction group of the bA domain (located on the b-subunit head domain) with the bTD (proximal to the plasma membrane on the b-subunit). However, molecular details of this key interaction group as well as events that lead to detachment of the bTD and bA domains have remained ambiguous. In this study, we use molecular dynamics models to take a comprehensive outside-in and inside-out approach at exploring how integrin aIIbb3 is activated. First, we show that talin's interaction with the membrane-proximal and membrane-distal regions of integrin cytoplasmic-transmembrane domains significantly loosens the inner membrane clasp. Talin also interacts with an additional salt bridge (R734-E1006), which facilitates integrin activation through the separation of the integrin's aand b-subunits. The second part of our study classifies three types of interactions between RGD peptides and the extracellular domains of integrin aIIbb3. Finally, we show that the interaction of the Arg of the RGD sequence may activate integrin via disrupting the key interaction group between K350 on the bA domain and S673/S674 on the bTD.
Biochemistry, 1992
Integrins are aj3 heterodimers that play a major role in cell-cell contacts and in interactions between cells and extracellular matrices. Identification of structural domains that are critical for the expression of such receptors at the cell surface in a functional conformation is one of the major issues that has not yet been resolved. In the present study, the role of the cytoplasmic and transmembrane domains of each of the subunits has been examined using platelet GPIIb/IIIa as a prototypic integrin. GPIIb/IIIa (aIIb/j3,)
Determination of N- and C-terminal Borders of the Transmembrane Domain of Integrin Subunits
Journal of Biological Chemistry, 2004
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.
Journal of Biological Chemistry, 2005
For several integrins, the existence of multiple conformational states has been studied intensively. For the integrin ␣21, a major collagen receptor on platelets and other cell types, however, no such experimental data were available thus far. Recently, our group has developed a monoclonal antibody IAC-1 sensitive to the molecular conformation of ␣21 because it only binds to the activated state of ␣21 on platelets, induced upon inside-out signaling. By investigating IAC-1 binding in combination with collagen binding after inside-out stimulation and outside manipulation, we demonstrated the existence of three different conformations of ␣21 on platelets and Chinese hamster ovary cells as follows: (i) a nonactivated, resting state with no collagen nor IAC-1 binding; (ii) an intermediate state, induced by outside manipulation, with collagen but no IAC-1 binding; and (iii) a fully activated state, induced after inside-out stimulation, with both collagen and IAC-1 binding. Moreover, these different conformational states of ␣21 are dependent on the cell type where ␣21 is expressed, as IAC-1 binding to peripheral blood mononuclear cells and Jurkat cells could also be induced by outside manipulation, in contrast to platelets and ␣21-expressing Chinese hamster ovary cells. Finally, we revealed a functional relevance for these different conformational states because the conformation of ␣21, induced after outside manipulation, resulted in significantly more cell spreading on coated collagen compared with nonactivated or inside-out stimulated cells.
2005
For several integrins, the existence of multiple conformational states has been studied intensively. For the integrin ␣21, a major collagen receptor on platelets and other cell types, however, no such experimental data were available thus far. Recently, our group has developed a monoclonal antibody IAC-1 sensitive to the molecular conformation of ␣21 because it only binds to the activated state of ␣21 on platelets, induced upon inside-out signaling. By investigating IAC-1 binding in combination with collagen binding after inside-out stimulation and outside manipulation, we demonstrated the existence of three different conformations of ␣21 on platelets and Chinese hamster ovary cells as follows: (i) a nonactivated, resting state with no collagen nor IAC-1 binding; (ii) an intermediate state, induced by outside manipulation, with collagen but no IAC-1 binding; and (iii) a fully activated state, induced after inside-out stimulation, with both collagen and IAC-1 binding. Moreover, these different conformational states of ␣21 are dependent on the cell type where ␣21 is expressed, as IAC-1 binding to peripheral blood mononuclear cells and Jurkat cells could also be induced by outside manipulation, in contrast to platelets and ␣21-expressing Chinese hamster ovary cells. Finally, we revealed a functional relevance for these different conformational states because the conformation of ␣21, induced after outside manipulation, resulted in significantly more cell spreading on coated collagen compared with nonactivated or inside-out stimulated cells.
Regulation of integrin function
Seminars in Cancer Biology, 1996
Cells communicate with their environment through several kinds of cell surface receptor. One of the most important families of cell adhesion receptors are the integrins, which include receptors that mediate cell-cell as well as cell-extracellular matrix interactions. A distinctive feature of integrins is their variable adhesive competence that is reversibly modified depending on the state of cell differentiation and/or activation or in response to environmental signals. The acquisition of adhesive function by integrins may be a consequence of conformational changes in these receptors that result in an increased ligand binding affinity. In addition, cells can control integrin-mediated adhesion through other mechanisms, including receptor clustering and association to cytoskeleton, phenomena that regulate the avidity of integrins for ligand molecules without altering their monovalent affinity. These phenomena have collectively been designated as 'post-receptor occupancy events'. These two interesting aspects of the regulation of integrin function are reviewed.