Innate behaviors are genetically encoded, but their underlying molecular mechanisms remain largely unknown. Predator odor 2,4,5-trimethyl-3-thiazoline (TMT) and its potent analog 2-methyl-2-thiazoline (2MT) are believed to activate specific odorant receptors to elicit innate fear/defensive behaviors in naive mice. Here, we conduct a large-scale recessive genetics screen of ethylnitrosourea (ENU)-mutagenized mice. We find that loss of Trpa1, a pungency/irritancy receptor, diminishes TMT/2MT and snake skin-evoked innate fear/defensive responses. Accordingly, Trpa1−/− mice fail to effectively activate known fear/stress brain centers upon 2MT exposure, despite their apparent ability to smell and learn to fear 2MT. Moreover, Trpa1 acts as a chemosensor for 2MT/TMT and Trpa1-expressing trigeminal ganglion neurons contribute critically to 2MT-evoked freezing. Our results indicate that Trpa1-mediated nociception plays a crucial role in predator odor-evoked innate fear/defensive behaviors. The work establishes the first forward genetics screen to uncover the molecular mechanism of innate fear, a basic emotion and evolutionarily conserved survival mechanism.

Tetraspanins play important roles in normal (e.g. cell adhesion, motility, activation, and proliferation) and pathological conditions (e.g. metastasis and viral infection). Tetraspanins interact with integrins and regulate integrin functions, but the specifics of tetraspanin-integrin interactions are unclear. Using co-immunoprecipitation with integrins as a sole method to detect interaction between integrins and full-length tetraspanins, it has been proposed that the variable region (helices D and E) of the extracellular-2 (EC2) domain of tetraspanins laterally associates with a non-ligand-binding site of integrins. We describe that, using adhesion assays, the EC2 domain of CD81, CD9, and CD151 bound to integrin αvβ3, and this binding was suppressed by cRGDfV, a specific inhibitor of αvβ3, and antibody 7E3, which is mapped to the ligand-binding site of β3. We also present evidence that the specificity loop of β3 directly bound to the EC2 domains. This suggests that the EC2 domains specifically bind to the classical ligand-binding site of αvβ3. αvβ3 was a more effective receptor for the EC2 domains than the previously known tetraspanin receptors α3β1, α4β1, and α6β1. Docking simulation predicted that the helices A and B of CD81 EC2 bind to the RGD-binding site of αvβ3. Substituting Lys residues at positions 116 and 144/148 of CD81 EC2 in the predicted integrin-binding interface reduced the binding of CD81 EC2 to αvβ3, consistent with the docking model. These findings suggest that, in contrast with previous models, the ligand-binding site of integrin αvβ3, a new tetraspanin receptor, binds to the constant region (helices A and B) of the EC2 domain.