[
International C. elegans Meeting,
2001]
One of the looming mysteries in signal transduction today is the question of how mechanical signals, such as pressure or force delivered to a cell, are interpreted to direct biological responses. Elegant genetic and molecular studies by Chalfie and colleagues have identified several proteins thought to form a touch-transducing complex that mediates the response to body touch. At the core of this complex is a mechanically-gated ion channel made up of MEC-4 and MEC-10 subunits. This channel is homologous to the epithelial amiloride Na+ channel (ENaCs) and the acid sensing channels (ASICS)—the DEG/ENaC superfamily. The C. elegans channel subunits have been called degenerins because specific mutant forms of the channel can induce neurodegeneration. Understanding how the MEC-4/MEC-10 channel functions in touch transduction is a major question in the field of mechanical signaling. These channel subunits are positioned in the plasma membrane with N and C terminal domains inside the cell and a large extracellular domain projecting into a specialized extrcellular matrix. It is thought that specific proteins interact with the intracellular and extracellular domains to exert gating tension on the channel. We are currently focusing on the N-terminal domain to gain insight into mechanisms of channel function. The MEC-4 N-terminal domain includes an 87 amino acid stretch that is not highly conserved and a highly conserved domain close to the first transmembrane domain that has similarity to a half-site of thiol protease active sites. The conserved domain has been implicated in protein interactions and all known N-terminal mutations affect this subdomain. In order to gain greater insight into the nature of the MEC-4 amino terminal domain, we have created a homology model of this domain MEC-4{N}. Our model is based on sequence identity and similarity to the pro domains of proteases of the cathepsin protease family. Our homology model depicts MEC-4 {N} as a globular domain which is predominantly alpha helical in nature aside from an extended span of residues which comprises the hydrophobic core of the model. The model presents several interesting structural features, including small lipophilic surfaces (potential protein binding sites) and exposed tyrosines (potential phosphorylation site). Efforts are currently underway to solve the structure of MEC-4 {N}and MEC-10 {N} via NMR spectroscopy. We are testing highlighted residues for importance in channel function using site-directed mutagenesis. To complement genetic studies that identified candidate proteins of the touch transducing complex, we performed a two-hybrid screen for MEC-4 N-interacting proteins. We used the N-terminal domain of MEC-4 as bait to screen a C. elegans yeast two-hybrid cDNA library. We obtained 81 positive clones from a screen of 3 million primary yeast clones. Among these, four candidate interacting proteins were identified that interact in both two-hybrid and GST pulldown experiments. All the candidate interacting proteins are expressed in the touch receptor neurons and can interact with the non-conserved N-terminal domain of MEC-4. Two interacting proteins are probably involved in channel turnover: 2 isolates encode a AAA ATPase homolog located in F23F1.8 (B70326) and 4 isolates are of a them are SINA (seven-in-absentia) homolog located in Y37E.11 (U89792). The AAA ATPase is most likely a subunit of the proteosome required for MEC-4 degradation. RNAi directed against both genes suggests an essential role very early in embryogenesis. Two other candidates identify novel proteins. We obtained 61 isolates corresponding to an ORF located in C15G7.4 (U08022), and 3 isolates corresponding to ORF Y11D7A.12 (A215876). RNAi against these two proteins induces touch abnormalities and thus these are candidate proteins for regulation or function of the MEC-4 channel.