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The Interaction of Talin with the Cell Membrane Is Essential for Integrin Activation and Focal Adhesion Formation

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The Interaction of Talin with the Cell Membrane Is Essential for Integrin Activation and Focal Adhesion Formation The interaction of talin with the cell membrane is essential for integrin activation and focal adhesion formation Krishna Chinthalapudia, Erumbi S. Rangarajana, and Tina Izarda,1 aCell Adhesion Laboratory, Department of Integrative Structural and Computational Biology, The Scripps Research Institute, Jupiter, FL 33458 Edited by Barry Honig, Howard Hughes Medical Institute and Columbia University, New York, NY, and approved August 23, 2018 (received for review April 11, 2018) Multicellular organisms have well-defined, tightly regulated mech- major phosphorylation sites, T144 and T150) (3), and the talin anisms for cell adhesion. Heterodimeric αβ integrin receptors play preceding F0 subdomain has a ubiquitin-like fold. The talin F3 central roles in this function and regulate processes for normal cell subdomain harbors the primary β-integrin–binding site (4, 5). The functions, including signaling, cell migration, and development, talin rod domain consists of 13 domains, R1–R13, composed of 62 binding to the extracellular matrix, and senescence. They are in- amphipathic α-helices arranged into four-helix (R2, R3, R4, and volved in hemostasis and the immune response, participate in leu- R8) and five-helix (R1, R5, R6, R7, R9, R10, R11, R12, and R13) kocyte function, and have biological implications in angiogenesis bundle domains (6, 7) and a C-terminal dimerization domain (6). and cancer. Proper control of integrin activation for cellular commu- Each domain has unique properties, including binding to other nication with the external environment requires several physiolog- talin domains, to integrin, and to vinculin (8, 9). A secondary ical processes. Perturbation of these equilibria may lead to constitutive integrin-binding site in the rod domain (residues 1,974–2,293) of integrin activation that results in bleeding disorders. Furthermore, uncertain function has also been identified (10). There are three integrins play key roles in cancer progression and metastasis in which actin-binding sites located on the head domain and R4–R8 and certain tumor types exhibit higher levels of various integrins. Thus, the R13 subdomains (6, 11, 12), and 13 vinculin-binding sites that are integrin-associated signaling complex is important for cancer therapy α – single amphipathic -helices (7, 13 20). BIOPHYSICS AND development. During inside-out signaling, the cytoskeletal protein talin The role of lipids in integrin activation remains unclear despite plays a key role in regulating integrin affinity whereby the talin head a large body of literature and the known functional importance of COMPUTATIONAL BIOLOGY domain activates integrin by binding to the cytoplasmic tail of talin attachment to the membrane (8). In the first stages of cell β-integrin and acidic membrane phospholipids. To understand the attachment, the talin F3 FERM domain binds to the NPxY motif mechanism of integrin activation by talin, we determined the crystal of the integrin cytoplasmic β tail, thereby inducing reorganization structure of the talin head domain bound to the acidic phospholipid of the integrin heterodimer and activating integrin (5, 21–24). phosphatidylinositol 4,5-bisphosphate (PIP2), allowing us to design a Talin attachment to the plasma membrane is enhanced by phos- – talin lipid-binding deficient talin mutant. Our confocal microscopy with phatidylinositol 4,5-bisphosphate (PIP2), which induces a confor- knockout cells suggests that the talin–cell membrane interaction seems mational change in talin to expose the integrin-binding site (22, essential for focal adhesion formation and stabilization. Basal integrin 25–28). The role of PIP2 in integrin activation is particularly in- activation in Chinese hamster ovary cells suggests that the lipid- teresting since PIP2 is a major phosphoinositide of the inner binding–deficient talin mutant inhibits integrin activation. Thus, mem- brane attachment of talin seems necessary for integrin activation and Significance focal adhesion formation. Vertebrate cell growth, division, locomotion, morphogenesis, angiogenesis | cell adhesion | integrin activation | phospholipids | and development rely on the dynamic interactions of cells with talin activation extracellular matrix components via cell surface complexes termed focal adhesions that are composed of heterodimeric αβ alin is a key player in integrin activation. Vertebrates express integrin receptors, associated signaling molecules, and the large Ttwo isoforms in which talin1 is ubiquitously expressed, while cytoskeletal protein talin. While it is known that talin activation talin2 is found primarily in striated muscle and in the brain. As a and binding to β-integrin requires interactions with lipids, little multidomain cytoskeletal protein, talin contains discrete binding is known regarding the structure and function of inactive vs. sites for acidic phospholipids, β-integrin, actin, and vinculin, as activated talin, and what is known is often disputed. Here we well as layilin, PIPK1γ90, and synemin. Talin links microfilaments report that talin binding to the cell membrane seems necessary to the cytoplasmic membrane at cell-extracellular matrix adhesion for integrin activation and focal adhesion formation, a finding sites. This process depends critically on talin. Talin consists of a that significantly advances our understanding of integrin acti- polypeptide chain of 2,541 amino acids and is often described as vation and might aid the development of novel integrin having an N-terminal FERM (four-point-one, ezrin, radixin, therapeutic agents. moesin) domain connected by a linker (residues 401–482) that harbors a calpain-II cleavage site to a large “rod” domain (resi- Author contributions: K.C., E.S.R., and T.I. designed research; K.C. and E.S.R. performed dues 483–2,541). The talin head domain is different from all other research; K.C., E.S.R., and T.I. analyzed data; and K.C., E.S.R., and T.I. wrote the paper. FERM domain-containing proteins in that it has four subdomains, The authors declare no conflict of interest. F0–F3 (instead of the typical three, F1–F3), and they adopt an This article is a PNAS Direct Submission. extended structure (1) instead of the canonical cloverleaf con- Published under the PNAS license. formation seen in the ERM family of proteins (2). As seen in Data deposition: The atomic coordinates and structure factors have been deposited in the other FERM domain-containing proteins, the talin FERM sub- Protein Data Bank, www.wwpdb.org (PDB ID code 6mfs). domains contain a ubiquitin-like F1, acyl-CoA–binding protein- 1To whom correspondence should be addressed. Email: [email protected]. like F2 and phosphotyrosine-binding–like F3 subdomain. Unlike This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. all other F1 FERM subdomains, the talin F1 subdomain has an 1073/pnas.1806275115/-/DCSupplemental. unstructured insert (F1 loop, residues 133–165, harboring two www.pnas.org/cgi/doi/10.1073/pnas.1806275115 PNAS Latest Articles | 1of6 Downloaded by guest on September 28, 2021 membrane (29, 30), and because talin regulates the local PIP2 concentration in the membrane by binding and activating PIPK1γ (31, 32). PIP2 regulates important processes, such as vesicular trafficking, platelet activation, cytoskeleton organization (33–35), and focal adhesion turnover (25, 26, 36). This process evolves by targeting proteins to the membrane, often through induction of a conformational change or oligomerization (36, 37). In mammals, the heterodimeric integrin transmembrane re- ceptors are composed of 18 distinct α and β chains (38, 39). By responding to extracellular and intracellular stimuli, integrins connect the extracellular matrix to the cytoskeleton, and trans- duce signals across the plasma membrane in both directions, termed outside-in and inside-out signaling, respectively (39). Integrin activation is important in platelets and leukocytes as well as many tissues in which extracellular matrix remodeling, angiogenesis, and cell migration are involved. These processes require tightly controlled integrin activation mechanisms that involve conformational changes of these receptors. Thus, un- derstanding the molecular mechanisms of how talin activates integrin is fundamental for gaining insight into important path- ological states and recognizing how integrin activation might aid the development of novel integrin antagonists. Here we report the talin1 head/PIP2 complex crystal structure together with biochemical and functional data that answer im- portant questions, including how PIP2 activates talin. Our data provide several surprises and answers to longstanding mecha- nistic questions and suggest a mechanism in which on re- cruitment of cytosolic talin by PIPK1γ to the plasma membrane (32), PIP2 activates talin by severing the head–tail interaction, thereby exposing the integrin-binding site. Remarkably, our in vivo data suggest that the talin–PIP2 interaction is crucial for talin localization to the cell membrane, affects the scaffolding of cells, and thus is likely key for cell spreading and adhesion. We further find that disrupting talin binding to the membrane affects integrin activation, and that this talin–PIP2 interaction seems necessary for focal adhesion formation. Collectively, our study provides a major advance in our understanding of the dynamic control of focal adhesions by talin. Results PIP2 Binding to Talin Allosterically Blocks the Integrin and Talin Tail- Binding Sites. We determined the crystal structures of
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