Structural basis of the transmembrane domain dimerization and rotation in the activation mechanism of the TRKA receptor by nerve growth factor

Abstract

Tropomyosin-receptor kinases (TRKs) are essential for the development of the nervous system. The molecular mechanism of TRKA activation by its ligand nerve growth factor (NGF) is still unsolved. Recent results indicate that at endogenous levels most of TRKA is in a monomer-dimer equilibrium and that the binding of NGF induces an increase of the dimeric and oligomeric forms of this receptor. An unsolved issue is the role of the TRKA transmembrane domain (TMD) in the dimerization of TRKA and the structural details of the TMD in the active dimer receptor. Here, we found that the TRKA-TMD can form dimers, identified the structural determinants of the dimer interface in the active receptor, and validated this interface through site-directed mutagenesis together with functional and cell differentiation studies. Using in vivo cross-linking, we found that the extracellular juxtamembrane region is reordered after ligand binding. Replacement of some residues in the juxtamembrane region with cysteine resulted in ligand-independent active dimers and revealed the preferred dimer interface. Moreover, insertion of leucine residues into the TMD helix induced a ligand-independent TRKA activation, suggesting that a rotation of the TMD dimers underlies NGF-induced TRKA activation. Altogether, our findings indicate that the transmembrane and juxtamembrane regions of TRKA play key roles in its dimerization and activation by NGF.

Keywords: brain derived neurotrophic factor (BDNF); nerve growth factor (NGF); neurotrophin; nuclear magnetic resonance (NMR); p75 neurotrophin receptor; receptor tyrosine kinase; transmembrane domain; tropomyosin receptor kinase A (TRKA); tropomyosin receptor kinase B (TRKB).

Figure 1.

NMR structure of TRKA transmembrane domain dimers in DPC micelles. A, the ¹H/¹⁵N HSQC spectrum of the human TRKA–TMD-wt. The human TRKA–TMD was solubilized in an aqueous suspension of DPC micelles at a DPR of 50:1 and at 45 °C and pH 5.9. ¹H–¹⁵N backbone and side-chain resonance assignments are shown. B, schematic representation of the NMR spatial structure of the human TRKA–TMD dimer in detergent micelles, from different angles. The surface of one TMD helix is represented with the NMR dimer interface in red. The TMD of the other monomer within the dimer is represented as ribbons, and side chains are indicated as sticks. The location of the NMR dimer interface LXXFAXXF on the NMR structure is colored red, and other mutated residues are colored orange. The putative dimerization motif SXXXG is shown in blue. The Protein Data Bank accession code is 2n90. C, alignment of the amino acid sequences of the transmembrane helical domain of TRKA from different species (h, Homo sapiens; r, Rattus norvegicus; m, Mus musculus; c, Gallus gallus; z, Danio rerio; x, Xenopus laevis). The residues participating in the NMR dimer interface are highlighted in red.

Figure 2.

Functional identification of the active dimer interface in the TRKA–TMD. A, amino acid sequence of the transmembrane domain of rat TRKA showing the location of the single cysteine residue substitutions. B, Western blotting of HeLa cells transfected with the indicated TRKA–TMD constructs and analyzed using nonreducing SDS-PAGE, showing the formation of covalent cysteine dimers (d) arising from monomers (m) cross-linked using molecular iodine (I₂) in the presence or absence of NGF. C, quantification of the data in B derived from at least three independent experiments. Bars represent standard error of the mean. Statistics were performed using two-way ANOVA and Dunnett's multiple comparison test using GraphPad software. ****, p < 0.0001; ***, p = 0.0004. D, PC12nnr5 cells were transfected with the indicated constructs and 48 h later analyzed for the presence of neurites. The percentage of cells with at least one neurite longer than the cell body was quantified. At right fluorescence microscopy showing the formation of neurites in the PC12nnr5 cells co-transfected with TRKA and GFP. E, top panel, HeLa cells transfected with the indicated TRKA constructs were stimulated with NGF (10 ng/ml) for 0, 5, or 15 min. The immunoblots show the activation (autophosphorylation) of TRKA as detected using an antibody specific for TRKA P-Tyr^674/675. The levels of total TRKA are shown below each blot. Molecular weight markers are shown at left. Bottom panel, quantification of the data from the top panel derived from at least three independent experiments. Bars represent standard error of the mean. Statistics were performed using two-way ANOVA and Dunnett's multiple comparison test using GraphPad software. p values of conditions significantly different from WT are shown on top of the error bars. F, cytometric analysis of the expression of the TRKA mutants at the plasma membrane (orange bars) and quantification of the percentage of PC12nnr5 differentiated cells (multicolored bars) transfected with the indicated TRKA constructs together with GFP. EV, empty vector. The percentage of the total GFP-positive transfected cells with a neurite twice as long as the length of the cell body was quantified. G, helical wheel model of the TRKA–TMD showing the two dimer interfaces identified by NMR and by functional studies. An arrow shows the putative rotation from the two interfaces. Bars represent the standard error of at least three independent experiments. Statistical analysis was performed using ordinary one-way ANOVA, using Bonferroni's multicomparison test compared with wt. The p values of significant differences are shown. ns, not significant.

Figure 3.

Lys⁴¹⁰ and Lsy⁴¹¹ of eJTM are cross-linked with BS3 upon NGF binding. A, location of the Lys residues in the crystal structure of the rat TRKA-ECD/NGF complex (Protein Data Bank code 2IFG) (16). The Lys residues, shown in green, are located in the extracellular juxtamembrane region of the TRKA–ECD. B, Western immunoblots of lysates of HEK293 cells transfected with the indicated TRKA constructs (see Fig. 3A) and incubated with or without NGF in the presence the cross-linker BS3. Molecular weights are indicated at left. Actin was assayed as a loading control.

Figure 4.

A preferred dimer interface in the TRKA juxtamembrane region. A, amino acid sequence of the rat TRKA cysteine mutant constructs that are mutated in the region of the eJTM closest to the TMD. B, quantification of the ratio of dimer:monomer of the TRKA mutants as determined using nonreducing SDS-PAGE. C, quantification of the activation of TRKA (with and without NGF) by quantifying the signal from the phosphorylation of the Tyr^674/675 signal using Western blotting of the cysteine mutants in the JTM region. D, scatter plot of the dimerization of TRKA cysteine mutants respect to its activation in the absence of NGF. A regression fit using the active (green) and inactive (red) cysteine mutant dimers is shown with the indicated r². E, cytometric analysis of the expression of the TRKA mutants at the plasma membrane of HeLa cells. F, PC12nnr5 cell differentiation assay of TRKA-wt and TRKA-K410C in the presence and absence of NGF. G, quantification of the differentiation of PC12nnr5 cells transfected with the indicated TRKA constructs and incubated in the absence (blue bars) or presence (green bars) of NGF. H, model of the eJTM into an ideal α-helix showing the spatial location of the indicted residues. Error bars represent the standard error of the mean. Statistics were done using two-way ANOVA and Dunnett's multiple comparison test using GraphPad software. The p values of significant differences are shown. ****, p < 0.0001. EV, empty vector.

Figure 5.

Rotation of the TRKA–TMD as a mechanism of TRKA activation by NGF. A, effect of NGF on the activation of overexpressed TRKA. HeLa cells transfected with the indicated constructs were stimulated with NGF (10 ng/ml) for 0, 5, 15, or 30 min. The immunoblots show the activation of TRKA as detected using an antibody specific for the autophosphorylated P-Tyr^674/675, P-Tyr⁴⁹⁰, and P-Tyr⁷⁵¹ residues and for the downstream activated P-ERK. The levels of total TRKA and total ERK are shown below each blot. Actin was blotted as a loading control. A representative blot of at least three independent experiments is shown. B, amino acid sequences of the rat TRKA Leu insertion mutants indicating the location of the inserted Leu residues. C, schematic drawing of the TRKA–TMD-ICD showing how the different numbers of inserted Leu residues (blue) in the TMD induce a different rotation of the intracellular domain (green). D, HeLa cells were transfected with the indicated TRKA–TMD insertion mutants. The immunoblots show the activation of TRKA as detected using an antibody specific for the TRKA autophosphorylation P-Tyr^674/675 residues. The levels of total TRKA are shown below. Actin was assayed as a loading control. E, quantification of the data in D from at least three independent experiments. Bars represent standard error of the mean. Statistical analysis was performed using one-way ANOVA with Dunnett's multicomparison test and TRKA-wt as a control. The p value of the significant difference is shown. F, PC12nnr5 cell differentiation of TRKA–TMD insertion mutant-transfected cells incubated in the absence of NGF. The percentage of the total GFP positive-transfected cells with a neurite twice as long as the length of the cell body was quantified. Bars represent the standard error of at least four independent experiments. Statistical analysis was performed using one-way ANOVA with Dunnett's multicomparison test and TRKA-wt as a control. The p values of significant differences are shown. EV, empty vector.

Figure 6.

Model of the role of TRKA eJTM and TM domains in receptor activation. TRKA is in equilibrium monomer-dimer by TMD interactions. Binding of NGF stabilizes the TRKA dimers and induces a rearrangement of the eJTM that couples ligand binding to rotation of the TMD. For simplicity only the TMD plus the JTM region is shown. The NGF dimer is shown in red.