The RGD (Arg-Gly-Asp) is a potential cell-binding motif of UNC-52/PERLECAN.

UNC-52/perlecan is a basement membrane (BM) proteoglycan playing an essential role in the muscle cell attachment of C. elegans. The UNC-52 protein contains two RGD (Arg-Gly-Asp) motifs in domains III and IV, a well-characterized tripeptide known for binding to mammalian ß integrin. To investigate the role of the RGD motif in UNC-52/perlecan, we created two mutations in the 2021RGD2023 motif: one mutation changed the RGD to an RGE, and the other deleted the RGD motif. The RGE2023 caused defective actin filaments and aberrant localization of PAT-3 ß integrin and TLN-1/talin. Additionally, the in-frame deletion of RGD2023 resulted in a paralyzed and arrested at two-fold embryonic stages (Pat) phenotype, which is the identical phenotype of the pat-3 ß integrin null allele. These results indicate that RGD2023 is a potential ligand for cell binding and is essential for development and survival. Furthermore, our analysis reveals that the RGD of an invertebrate BM molecule is a potential cell-binding motif, suggesting that the function of the RGD motif is conserved among species.

Mutations in the cell-binding motif of lam-3/laminin α reveal hypercontraction behavior and defective sensitivity to levamisole in Caenorhabditis elegans.

The amino acid sequence Arg-Gly-Asp (RGD) is a cell-binding motif for extracellular matrix proteins. Initially found in fibronectin, the RGD motif is also found in LAM-3/laminin α chain in C. elegans. Laminin, a heterotrimeric glycoprotein, is a significant component of the basement membrane. Mutations in laminin subunits disrupt the extracellular matrix hence inhibit cell adhesion. This study aims to characterize the function of the RGD motif in lam-3/laminin α. Two mutations, lam-3 RGE and lam-3 ΔRGD, were generated. Our analysis of the mutants revealed that the RGD motif is involved in the motility of animals, suggesting that the cell-laminin interaction plays a role in regulating body contraction.

ZYX-1/Zyxin plays a minor role in oocyte transit through the spermatheca in C. elegans.

In C. elegans, oocytes are ovulated into the spermatheca, where they are fertilized before being pushed into the uterus. Contraction in the C. elegans spermatheca is driven by circumferential acto-myosin fibers. The C. elegans zyxin homolog, zyx-1, is expressed in the body wall muscle, pharynx and spermatheca. To our surprise, a CRISPR-generated zyx-1 deletion allele results in no overt developmental phenotypes, and the spermathecal actin cytoskeleton appears wild type, however, oocyte transit through the spermatheca is slower than in wild type animals. This suggests ZYX-1/Zyxin may regulate spermathecal contraction magnitude or timing of spermathecal bag contraction and/or spermathecal-uterine valve dilation.

Deletion of the RGD motif in LON-2/glypican is associated with morphological abnormalities.

The lon-2 gene in Caenorhabditis elegans encodes a heparan sulfate proteoglycan family glypican that negatively regulates the BMP signaling pathway responsible for controlling body length. LON-2 contains multiple functional domains, including an RGD (Arg-Gly-Asp) motif at amino acid number from 348 to 350. A novel mutant allele of lon-2 was investigated in this study. In this mutant allele, lon-2(kq348ΔRGD), the RGD motif at position 348 was deleted. Another pre-existing mutant allele, lon-2(e678), contains a ~9kb deletion and lacks most of the genomic coding sequence. The lon-2(e678) line was used as a reference allele. The novel mutant line was significantly shorter than wild-type animals, suggesting that removal of the RGD motif in LON-2 may improve its ability to inhibit BMP signaling.

A Novel Mutation in an NPXY Motif of ß Integrin Reveals Phenotypes Similar to him-4/hemicentin.

Integrin, an αß heterodimeric cell surface receptor for the extracellular matrix (ECM), carries two tyrosine phosphorylation motifs in the cytoplasmic tail of the ß subunit. NPXY (Asn-Pro-x-Tyr) is a conserved tyrosine phosphorylation motif that binds to the phospho-tyrosine binding (PTB) domain. We generated a tyrosine to glutamic acid (E) mutation to modify tyrosine (Y) into a negatively charged amino NPXY in the ßpat-3 integrin of Caenorhabditis elegans. The transgenic rescue animal displayed defects in gonad migration and tail morphology. Also, the mutant animals produced a high number of males, suggesting that the Y to E mutation in ßpat-3 integrin causes a phenotype similar to that of Him mutant. Further analyses revealed that males of pat-3(Y804E) and him-4/hemicentin share additional phenotypes such as abnormal gonad and unsuccessful mating. A pat-3 transgenic rescue mutant with a non-polar phenylalanine (F) in NPXY, pat-3(Y792/804F), suppressed the high male number, defective mating, inviable zygote, and the abnormal gonad of him-4 mutants, indicating that Y to F mutation in both NPXY motifs suppressed the him-4 phenotypes. This finding supports the idea that the ECM determines the activation state in integrin NPXY motifs; him-4/hemicentin may directly or indirectly interact with integrins and maintain the NPXY non-charged. Our findings provide new insight into a suppressive role of an ECM molecule in integrin NPXY phosphorylation.