Genetic analysis of the interaction between the N- and C-terminal halves of UNC-112 (kindlin).
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ABSTRACT:
SUBMITTER: Qadota H
PROVIDER: S-EPMC7747009 | biostudies-literature | 2020 Dec
REPOSITORIES: biostudies-literature
Publications
Genetic analysis of the interaction between the N- and C-terminal halves of UNC-112 (kindlin).
Qadota Hiroshi H Luo Yating Y Oberhauser Andres F AF Benian Guy M GM
microPublication biology 20201217
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Project description:Integrin plays a crucial role in the attachment of cells to the extracellular matrix. Integrin recruits many proteins intracellularly, including a 4-protein complex (kindlin, ILK, PINCH, and parvin). Caenorhabditis elegans muscle provides an excellent model to study integrin adhesion complexes. In Caenorhabditis elegans, UNC-112 (kindlin) binds to the cytoplasmic tail of PAT-3 (β-integrin) and to PAT-4 (ILK). We previously reported that PAT-4 binding to UNC-112 is essential for the binding of UNC-112 to PAT-3. Although there are crystal structures for ILK and a kindlin, there is no co-crystal structure available. To understand the molecular interaction between PAT-4 and UNC-112, we took a genetic approach. First, using a yeast 2-hybrid method, we isolated mutant PAT-4 proteins that cannot bind to UNC-112 and then isolated suppressor mutant UNC-112 proteins that restore interaction with mutant PAT-4 proteins. Second, we demonstrated that these mutant PAT-4 proteins cannot localize to attachment structures in nematode muscle, but upon co-expression of an UNC-112 suppressor mutant protein, mutant PAT-4 proteins could localize to attachment structures. Third, overexpression of a PAT-4 mutant results in the disorganization of adhesion plaques at muscle cell boundaries and co-expression of the UNC-112 suppressor mutant protein alleviates this defect. Thus, we demonstrate that UNC-112 binding to PAT-4 is required for the localization and function of PAT-4 in integrin adhesion complexes in vivo. The missense mutations were mapped onto homology models of PAT-4 and UNC-112, and taking into account previously isolated mutations, we suggest a surface of PAT-4 that binds to UNC-112.
| S-EPMC9258589 | biostudies-literature
Project description:C. elegans UNC-112 (kindlin) is required for muscle sarcomere assembly, and is one component of a conserved four-protein complex that associates with the cytoplasmic tail of integrin at the base of integrin adhesion complexes in muscle. The four-protein complex consists of UNC-112 (kindlin), PAT-4 (integrin linked kinase; ILK), PAT-6 (alpha-parvin), and UNC-97 (PINCH). UNC-112 is comprised of 720 amino acid residues and contains FERM and PH domains. The N-terminal half of UNC-112 (1-396 aa) can bind to the C-terminal half of UNC-112 (397-720 aa), and this interaction is inhibited by the association of PAT-4 (ILK) to the N-terminal half of UNC-112. In support of this model, previously, we reported identification of a D382V mutation that results in lack of binding to PAT-4. However, this residue is not conserved in human Kindlins. Here, we report identification of a novel UNC-112 mutation of a conserved residue that cannot bind to PAT-4. UNC-112 E302G cannot bind to PAT-4 and does not localize to integrin adhesion complexes in muscle.
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Project description:Caenorhabditis elegans striated muscle cells attach to basement membrane and transmit the force of muscle contraction through integrin adhesion complexes. The cytoplasmic tail of β-integrin (PAT-3) is associated with a conserved four-protein complex that includes UNC-112 (kindlin), PAT-4 (integrin-linked kinase), PAT-6 (α-parvin/actopaxin), and UNC-97 (PINCH). The proper localization of UNC-112 to muscle integrin adhesion sites requires PAT-4. A recent report (Qadota, H., Moerman, D. G., and Benian, G. M. (2012) A molecular mechanism for the requirement of PAT-4 (integrin-linked kinase (ILK)) for the localization of UNC-112 (kindlin) to integrin adhesion sites. J. Biol. Chem. 287, 28537-28551) suggests a possible molecular mechanism for this requirement: that UNC-112 exists in closed inactive and open active conformations, and conversion to the open active form is promoted by binding to PAT-4 (ILK). Previously, we also reported identification of a single missense mutation in UNC-112, D382V, which abolishes both binding to PAT-4 and normal localization to integrin adhesion sites in vivo. In this report, we describe isolation and characterization of PAT-4 missense mutations that permit binding with UNC-112 D382V and place nine affected residues on a homology model of PAT-4. These nine residues cluster in two regions on the surface of PAT-4, do not overlap the likely binding surface for PAT-6 (α-parvin), and therefore may reside along the interaction surface of PAT-4 for UNC-112 (kindlin). We also show that one of these PAT-4 mutations restores the ability of UNC-112 D382V to localize to integrin adhesions and participate in complex formation.
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Project description:Kindler syndrome is an autosomal recessive disorder characterized by neonatal blistering, sun sensitivity, atrophy, abnormal pigmentation, and fragility of the skin. Linkage and homozygosity analysis in an isolated Panamanian cohort and in additional inbred families mapped the gene to 20p12.3. Loss-of-function mutations were identified in the FLJ20116 gene (renamed "KIND1" [encoding kindlin-1]). Kindlin-1 is a human homolog of the Caenorhabditis elegans protein UNC-112, a membrane-associated structural/signaling protein that has been implicated in linking the actin cytoskeleton to the extracellular matrix (ECM). Thus, Kindler syndrome is, to our knowledge, the first skin fragility disorder caused by a defect in actin-ECM linkage, rather than keratin-ECM linkage.
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Project description:Caenorhabditis elegans muscle cells attach to basement membrane through adhesion plaques. PAT-3 (β-integrin), UNC-112 (kindlin), and PAT-4 (integrin-linked kinase) are associated with these structures. Genetic analysis indicated that PAT-4 is required for UNC-112 to be properly localized. We investigated the molecular basis of this requirement. We show that the cytoplasmic tail of PAT-3 binds to full-length UNC-112 and that the N- and C-terminal halves of UNC-112 bind to each other. We demonstrate competition between the UNC-112 C-terminal half and PAT-4 for binding to the UNC-112 N-terminal half. The D382V mutation results in lack of binding to PAT-4 and lack of localization to adhesion structures. T346A or E349K mutations, which abolish interaction of the N- and C-terminal halves, permit D382V UNC-112 to localize to adhesion structures. The following model is proposed. UNC-112 exists in closed inactive and open active conformations, and upon binding of PAT-4 to the UNC-112 N-terminal half, UNC-112 is converted into the open state, able to bind to PAT-3.
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Project description:Cells perceive and respond to the extracellular matrix via integrin receptors; their dysregulation has been implicated in inflammation and cancer metastasis. Here we show that a guanine nucleotide-exchange modulator of trimeric-GTPase Gαi, GIV (a.k.a Girdin), directly binds the integrin adaptor Kindlin-2. A non-canonical short linear motif within the C terminus of GIV binds Kindlin-2-FERM3 domain at a site that is distinct from the binding site for the canonical NPxY motif on the -integrin tail. Binding of GIV to Kindlin-2 allosterically enhances Kindlin-2's affinity for β1-integrin. Consequently, integrin activation and clustering are maximized, which augments cell adhesion, spreading, and invasion. Findings elucidate how the GIV•Kindlin-2 complex has a 2-fold impact: it allosterically synergizes integrin activation and enables β1-integrins to indirectly access and modulate trimeric GTPases via the complex. Furthermore, Cox proportional-hazard models on tumor transcriptomics provide trans-scale evidence of synergistic interactions between GIV•Kindlin-2•β1-integrin on time to progression to metastasis.
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