Structural Basis and Mechanism of TrkA Activation by NGF through Ligand-Induced Rotation of Transmembrane Domain Dimers.

The molecular mechanism of TrkA activation have been poorly studied in comparison with those of other RTK family members. TrkA, like most RTKs, is in a monomer- dimer equilibrium in the absence of their ligand, and an unsolved issue is how ligand- binding can activate a pre-formed inactive dimer. Here we show that the transmembrane domain (TMD) of TrkA can form dimers using two different interfaces. We characterized the structural determinants of TrkA-TMD dimerization and identified the dimer interface of the active receptor. We validated this interface using site-directed mutagenesis together with functional and cell differentiation studies with the full-length TrkA receptor expressed in mammalian cells. Using in vivo crosslinking we identified a reordering of the extracellular juxtamembrane region after ligand binding. This conformational change could be mimicked by replacement of some residues with cysteine and the spontaneous formation of ligand-independent active dimers. In addition the cysteine-scanning mutagenesis of the juxtamembrane region revealed that the position of the mutation relative to the TMD was more important for activation than the dimerization propensity of the mutant, suggesting a preferred interface for the active receptor. Insertion of leucine residues into the TMD helix induces a ligand-independent TrkA activation depending on the number of residues inserted suggesting a specific orientation of the kinase domains with respect to each other. Altogether our data suggests that the transmembrane and juxtamembrane regions of the receptor mediate switching between the active and inactive states of the TrkA dimer.