Genetic analysis of the interaction between the N- and C-terminal halves of UNC-112 (kindlin)

Qadota, Hiroshi; Luo, Yating; Oberhauser, Andrés F.; Benian, Guy M.

doi:10.17912/micropub.biology.000342

Cited by 2 publications

(7 citation statements)

References 13 publications

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“…Furthermore, all four mutation sets (F301S, N37S, A96V, and Q27R plus F117S) can bind to both PAT-4 Q308H and PAT-4 I432F. Previously (Qadota et al 2020), we reported generating a homology model of UNC-112 based on the crystal structure of human kindlin-3 (Bu et al 2020). We placed our suppressor mutations on this model.…”

Section: Resultsmentioning

confidence: 99%

“…UNC-112 N-terminal half with F301S, N37S, A96V, or Q27Q/F117S can bind to PAT-4 with Q308H and I432F. B, Structure of UNC-112 (Qadota et al 2020) based on the human kindlin-3 3D structure (PDB: 7C3M) (Bu et al 2020) modelled with Swiss-Model (Waterhouse et al 2018), and showing corresponding mutated residues. The color scheme matches the colors of the domains in the schematic of part A. Residues that were affected by single amino acid changes appearing in part A (N37, A96, and F301) are shown in enlarged windows.…”

Section: Resultsmentioning

confidence: 99%

“…Figure 6 summarizes our results to date using a random mutagenesis approach (Qadota and Benian 2014) to isolate non-binding mutants and their suppressor mutants (Qadota et al 2012; Qadota et al 2014, Qadota et al 2020; Qadota et al 2021; this study). Other than deletion and non-sense mutations, there are reports about two unc-112 alleles (no reports for pat-4 ) : First, the Unc allele, unc-112(r367) , is a missense mutation, T85I (Rogalski et al 2000).…”

Section: Discussionmentioning

confidence: 99%

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Genetic Analysis Suggests a Surface of PAT-4 (ILK) that Interacts with UNC-112 (kindlin)

Benian

Qadota

McPherson

et al. 2022

Preprint

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The transmembrane protein integrin plays a crucial role in the attachment of cells to the extracellular matrix. Integrin recruits many proteins intracellularly, including a four protein complex (Kindlin, Integrin linked kinase (ILK), PINCH, and parvin). C. elegans muscle provides an excellent model to study integrin adhesion complexes. In C. elegans, UNC-112 (Kindlin) binds to the cytoplasmic tail of PAT-3 (b-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 (ILK) and UNC-112 (Kindlin), we took a genetic approach. First, we isolated mutant PAT-4 proteins that cannot bind to UNC-112. Then, we isolated suppressor mutant UNC-112 proteins that restore interaction with mutant PAT-4 proteins. Second, these mutant PAT-4 proteins cannot localize to attachment structures in nematode muscle, but upon co-expression of an UNC-112 suppressor mutant protein, the mutant PAT-4 protein could localize to attachment structures. Third, overexpression of a mutant PAT-4 protein results in disorganization of the adhesion plaques at muscle cell boundaries, and co-expression of the UNC-112 supressor 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.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Genetic Analysis Suggests a Surface of PAT-4 (ILK) that Interacts with UNC-112 (kindlin)

Benian

Qadota

McPherson

et al. 2022

Preprint

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“…WT denotes wild type. b) Structure of UNC-112 ( Qadota et al 2020 ) based on the human kindlin-3 3D structure (PDB: 7C3M) ( Bu et al 2020 ) modelled with Swiss-Model ( Waterhouse et al 2018 ), and showing corresponding mutated residues. The color scheme matches the colors of the domains in the schematic of part a. Residues that were affected by amino acid changes appearing in part a (Q27, N37, A96, F117, and F301) are shown in enlarged windows.…”

Section: Resultsmentioning

confidence: 99%

“…Furthermore, all 4 mutation sets (F301S, N37S, A96V, and Q27R plus F117S) can bind to both PAT-4 Q308H and PAT-4 I432F. Previously ( Qadota et al 2020 ), we reported generating a homology model of UNC-112 based on the crystal structure of human kindlin-3 ( Bu et al 2020 ). We placed our single amino acid suppressor mutations on this model.…”

Section: Resultsmentioning

confidence: 99%

Genetic analysis suggests a surface of PAT-4 (ILK) that interacts with UNC-112 (kindlin)

Qadota

McPherson

Corbitt

et al. 2022

G3 Genes|Genomes|Genetics

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Integrin plays a crucial role in the attachment of cells to the extracellular matrix. Integrin recruits many proteins intracellularly, including a four protein complex (Kindlin, Integrin linked kinase (ILK), PINCH, and parvin). C. elegans muscle provides an excellent model to study integrin adhesion complexes. In C. 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 two-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 disorganization of adhesion plaques at muscle cell boundaries, and co-expression of the UNC-112 supressor 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.

show abstract

Genetic analysis of the interaction between the N- and C-terminal halves of UNC-112 (kindlin)

Cited by 2 publications

References 13 publications

Genetic Analysis Suggests a Surface of PAT-4 (ILK) that Interacts with UNC-112 (kindlin)

Genetic Analysis Suggests a Surface of PAT-4 (ILK) that Interacts with UNC-112 (kindlin)

Genetic analysis suggests a surface of PAT-4 (ILK) that interacts with UNC-112 (kindlin)

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