TY - JOUR
T1 - Genetic analysis suggests a surface of PAT-4 (ILK) that interacts with UNC-112 (kindlin)
AU - Qadota, Hiroshi
AU - McPherson, Annie
AU - Corbitt, Rachel
AU - Dackowski, Evan Kelton
AU - Matsunaga, Yohei
AU - Oberhauser, Andres F.
AU - Benian, Guy M.
N1 - Funding Information:
This study was supported, in part, by a previous grant from the American Heart Association (11GRNT7820000) and a more recent grant from the National Institutes of Health (1R01HL160693).
Funding Information:
We thank Dr. Andrew Fire (Stanford University) for C. elegans expression vectors (pPD49.78 and pPD49.83) and Dr. Kozo Kaibuchi (Nagoya University) for pKS-HA and pBS-myc. We thank Laura Fox-Goharioon of Emory University’s Integrated Cellular Imaging (ICI) Core for help in using the N-SIM microscope. The C. elegans wild-type strain, N2, was provided by the Caenorhabditis Genetics Center, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440).
Publisher Copyright:
© 2022 Genetics Society of America. All rights reserved.
PY - 2022/7
Y1 - 2022/7
N2 - Integrin plays a crucial role in the attachment of cells to the extracellular matrix. Integrin recruits many proteins intracellularly, including a 4-pro-tein 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 (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 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.
AB - Integrin plays a crucial role in the attachment of cells to the extracellular matrix. Integrin recruits many proteins intracellularly, including a 4-pro-tein 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 (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 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.
KW - KIPP complex
KW - integrin
KW - integrin adhesion complex
KW - mutant screening
KW - suppressor
UR - http://www.scopus.com/inward/record.url?scp=85134205496&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85134205496&partnerID=8YFLogxK
U2 - 10.1093/g3journal/jkac117
DO - 10.1093/g3journal/jkac117
M3 - Article
C2 - 35536217
AN - SCOPUS:85134205496
SN - 2160-1836
VL - 12
JO - G3: Genes, Genomes, Genetics
JF - G3: Genes, Genomes, Genetics
IS - 7
M1 - jkac117
ER -