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Mechanism of talin recruitment to integrin adhesion sites
Mechanism of talin recruitment to integrin adhesion sites
Integrins are heterodimeric transmembrane receptors that facilitate the adhesion of cells to their surrounding extracellular matrix and to other cells. Integrins can adopt an inactive, thermodynamically favored or an active conformation with much higher ligand binding affinity. The latter is induced and stabilized by direct association with the intracellular adapter molecules talin and kindlin. The cellular signaling processes that trigger the formation of the high affinity conformation are named integrin inside-out signaling. Active integrins recruit a large complex of scaffold and signaling molecules that mediate integrin clustering, provide a link to the actin cytoskeleton and form signaling hubs regulating processes such as cell spreading, migration, adhesion stability, differentiation, proliferation and gene expression (outside-in signaling). A series of cell biological studies raised the hypothesis that integrin inside-out activation requires a so-called trimeric “integrin activation complex” consisting of talin, the adapter protein RIAM and the small membrane-bound GTPase Rap1. However, in vivo studies revealed that this complex is only involved in regulating leukocyte β2 integrins. Based on these observations, the main aim of my doctoral thesis was to decipher an alternative, RIAM independent pathway of talin membrane recruitment and integrin activation. Following up a previously detected, but due to its very weak nature, not further studied direct interaction between Rap1 and the N-terminal talin F0 domain, a detailed biochemical and cell biological characterization confirmed specific GTP-dependent binding of Rap1 to talin F0 and revealed that this direct interaction is important to recruit talin to adhesion sites and to regulate adhesion and spreading of fibroblasts in vitro (paper 1). To assess the in vivo relevance of the talin F0/Rap1 interaction a Rap1-binding deficient talin 1 knockin mouse model carrying specific mutations in the talin 1 F0 domain was generated. A detailed characterization of these mice revealed impaired hemostasis and leukocyte adhesion and extravasation defects due to reduced integrin activity in platelets and neutrophils, respectively (paper 2). Since blocking the talin F0/Rap1 interaction only partially impaired integrin activity in vitro and in vivo, the contribution of other known talin interacting factors, such as membrane lipids and RIAM as well as the role of a second novel Rap1-binding site in the talin F1 domain for integrin activity regulation were investigated. These studies showed that binding of talin F0 and F1 domains to Rap1 and talin F2 domain to membrane lipids act synergistically on talin recruitment to adhesion sites and promote integrin activation, cell adhesion and spreading (paper 4). In addition, I contributed to a project that identified and characterized the interaction of kindlin-3 and the paxillin family protein leupaxin. This study showed that kindlin-3 dependent recruitment of leupaxin into podosomes increases their lifetime by regulating PTP-PEST-mediated paxillin dephosphorylation (paper 3).
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Bromberger, Thomas
2020
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Bromberger, Thomas (2020): Mechanism of talin recruitment to integrin adhesion sites. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

Integrins are heterodimeric transmembrane receptors that facilitate the adhesion of cells to their surrounding extracellular matrix and to other cells. Integrins can adopt an inactive, thermodynamically favored or an active conformation with much higher ligand binding affinity. The latter is induced and stabilized by direct association with the intracellular adapter molecules talin and kindlin. The cellular signaling processes that trigger the formation of the high affinity conformation are named integrin inside-out signaling. Active integrins recruit a large complex of scaffold and signaling molecules that mediate integrin clustering, provide a link to the actin cytoskeleton and form signaling hubs regulating processes such as cell spreading, migration, adhesion stability, differentiation, proliferation and gene expression (outside-in signaling). A series of cell biological studies raised the hypothesis that integrin inside-out activation requires a so-called trimeric “integrin activation complex” consisting of talin, the adapter protein RIAM and the small membrane-bound GTPase Rap1. However, in vivo studies revealed that this complex is only involved in regulating leukocyte β2 integrins. Based on these observations, the main aim of my doctoral thesis was to decipher an alternative, RIAM independent pathway of talin membrane recruitment and integrin activation. Following up a previously detected, but due to its very weak nature, not further studied direct interaction between Rap1 and the N-terminal talin F0 domain, a detailed biochemical and cell biological characterization confirmed specific GTP-dependent binding of Rap1 to talin F0 and revealed that this direct interaction is important to recruit talin to adhesion sites and to regulate adhesion and spreading of fibroblasts in vitro (paper 1). To assess the in vivo relevance of the talin F0/Rap1 interaction a Rap1-binding deficient talin 1 knockin mouse model carrying specific mutations in the talin 1 F0 domain was generated. A detailed characterization of these mice revealed impaired hemostasis and leukocyte adhesion and extravasation defects due to reduced integrin activity in platelets and neutrophils, respectively (paper 2). Since blocking the talin F0/Rap1 interaction only partially impaired integrin activity in vitro and in vivo, the contribution of other known talin interacting factors, such as membrane lipids and RIAM as well as the role of a second novel Rap1-binding site in the talin F1 domain for integrin activity regulation were investigated. These studies showed that binding of talin F0 and F1 domains to Rap1 and talin F2 domain to membrane lipids act synergistically on talin recruitment to adhesion sites and promote integrin activation, cell adhesion and spreading (paper 4). In addition, I contributed to a project that identified and characterized the interaction of kindlin-3 and the paxillin family protein leupaxin. This study showed that kindlin-3 dependent recruitment of leupaxin into podosomes increases their lifetime by regulating PTP-PEST-mediated paxillin dephosphorylation (paper 3).