TrkA COOH-terminal tailits relevance on receptor stability and signaling for cell differentiation and survival

Resumen

The nerve growth factor receptor TrkA contributes to the survival and differentiation of several neuronal populations. Although main signaling pathways and adaptor molecules activated after ligand binding to the receptor are well studied, recently a growing number of TrkA interacting proteins have been described as novel mechanisms of receptor regulation. Receptor trans-autophosphorylation, additionally to activating signaling cascades relevant for cell differentiation and survival, such as MAPKs and PI3K-Akt, also induces a transient increase in intracellular calcium concentration. Therefore, the Ca2+ sensor calmodulin (CaM) also participates in TrkA signaling. Our group was interested in elucidating the relationship between CaM and TrkA, and discovered that CaM interacts directly with the C-terminal tail of TrkA in a Ca2+ dependent manner. Consequently, we sought to find the exact position of the calmodulin binding site on TrkA sequence. Since available prediction software was unable to find a putative CaM binding site in TrkA intracellular domain, we focused on the C-terminal tail, a domain rich in hydrophobic amino acids in conserved positions. We introduced point mutations on Leu784 and Val790 by switching them to Ala, located on our predicted CaM binding site. However, TrkA-L784A and TrkA-L784A/V790A mutants did not lose CaM binding, therefore they were not involved on CaM binding. Nonetheless, we demonstrated that these hydrophobic aminoacids are important for the binding and regulation of Nedd4-2 Ub ligase. We observed a stronger interaction of both C-terminal mutant receptors with Nedd4-2, corroborated with a higher colocalization of both proteins by immunofluorescence. As a consequence, we observed an enhanced basal multimonoubiquitination of mutant receptors by Nedd4-2. These results correlated with a decrease in TrkA abundance due to a faster late endosome/lysosome targeting, leading to a higher degradation rate of mutant receptors. Despite the reduction in the amount of membrane receptor caused by the C-terminal changes, TrkA mutants were able to activate signaling cascades and were even more efficient in promoting neurite outgrowth than the wild-type receptor. Our results demonstrate that the C-terminal tail conformation of TrkA regulates Nedd4-2 binding and activity. Moreover, TrkA ubiquitination by Nedd4-2 promotes TrkA endosomal trafficking to late endosome/lysosome leading to receptor degradation. In addition, TrkA multimonoubiquitination does not interfere with the activation of signaling cascades, but rather potentiates receptor signaling leading to differentiation.