Ligand-induced dynamics of neurotrophin receptors investigated by single-molecule imaging approaches

Abstract

Neurotrophins are secreted proteins that regulate neuronal development and survival, as well as maintenance and plasticity of the adult nervous system. The biological activity of neurotrophins stems from their binding to two membrane receptor types, the tropomyosin receptor kinase and the p75 neurotrophin receptors (NRs). The intracellular signalling cascades thereby activated have been extensively investigated. Nevertheless, a comprehensive description of the ligand-induced nanoscale details of NRs dynamics and interactions spanning from the initial lateral movements triggered at the plasma membrane to the internalization and transport processes is still missing. Recent advances in high spatio-temporal resolution imaging techniques have yielded new insight on the dynamics of NRs upon ligand binding. Here we discuss requirements, potential and practical implementation of these novel approaches for the study of neurotrophin trafficking and signalling, in the framework of current knowledge available also for other ligand-receptor systems. We shall especially highlight the correlation between the receptor dynamics activated by different neurotrophins and the respective signalling outcome, as recently revealed by single-molecule tracking of NRs in living neuronal cells.

Figure 1

Schematic picture of tropomyosin receptor kinases (Trks) and the p75 neurotrophin receptors (p75NTR). (A) Structure of the two receptors: The intracellular (on top) and extracellular (on bottom) domains are highlighted. The following abbreviations are used: CRD (cystein-rich domain); LRR (leucine-rich domain); IgL-D (immunoglobulin-like domain); TKD (tyrosine-kinase domain); CD (chopper domain); DD (death domain); (B) Modified residues of the Trks (on top) and of p75NTR (on bottom) receptors. The following abbreviations are used: TM (transmembrane domain); JM (juxta-membrane domain); N (N-terminus); C (C-terminus). The following symbols are used: Grey ↓ (N- or O-glycosylation sites); green ↓ (ATP-binding site); red ↓ (site of covalent homo-dimerization due to the disulfide bond formed by Cys257 [32,33]). U (ubiquitination-related lysine residues, as derived from studies mainly performed on the TrkA receptor [34,35,36,37]); Y (phosphorylated tyrosine residues, their numeration and function is described in Figure 2); P (palmitoylated Cys residue [38]).

Figure 2

Schematic picture of four different NTs binding to ACP-TrkA. The ACP-TrkA construct and the four ligands investigated for receptor binding [104] are schematically depicted. NGF, NGF R100E mutant (mutNGF, related to HSANV disease [132]), proNGF and NT-3 all bind to the extracellular domain of TrkA receptor but with different affinity, as quantified by the K_d (dissociation constant) values (see the color-coded arrowheads referring to the corresponding K_d values, which are taken from: ^≠ [133]; * [134]; ^† [135]). The evoked physiological responses are also different among the four ligands and are summarized at the C-terminus of the receptor, highlighted by arrowheads with the same color-code. Intracellular effectors recruited at phosphorylated tyrosine residues and leading to the activation of the MAP kinase, the Akt and PLCγ signaling pathways, upon TrkA-NT binding are also schematically depicted. The numbering of tyrosine residues refers to the rat TrkA cDNA sequence. Note that while Y499 and 794 only have recruitment function, Y683/4 constitute with Y679 (not depicted) the activation loop of tyrosine kinase activity. The figure has been adapted from [104].