Signal transduction in receptor for advanced glycation end products (RAGE): solution structure of C-terminal rage (ctRAGE) and its binding to mDia1

Abstract

The receptor for advanced glycation end products (RAGE) is a multiligand cell surface macromolecule that plays a central role in the etiology of diabetes complications, inflammation, and neurodegeneration. The cytoplasmic domain of RAGE (C-terminal RAGE; ctRAGE) is critical for RAGE-dependent signal transduction. As the most membrane-proximal event, mDia1 binds to ctRAGE, and it is essential for RAGE ligand-stimulated phosphorylation of AKT and cell proliferation/migration. We show that ctRAGE contains an unusual α-turn that mediates the mDia1-ctRAGE interaction and is required for RAGE-dependent signaling. The results establish a novel mechanism through which an extracellular signal initiated by RAGE ligands regulates RAGE signaling in a manner requiring mDia1.

FIGURE 1.

ctRAGE contains a folded segment. A, domain structure of human RAGE (NCBI accession code NP_001127) and sequence alignment of the ctRAGE construct used in this study. NCBI accession codes for rat RAGE, mouse RAGE, and dog RAGE are NP_445788, NP_031451, and NP_01104546, respectively. Trp-363 of human full-length RAGE corresponds to Trp-2 of ctRAGE. Conserved residues are in red. Residues mutated to Ala are in blue. exRAGE, extracellular immunoglobulin domain of RAGE; TMH, transmembrane helix. B, ¹⁵N HSQC NMR spectrum of ctRAGE. The assignments of the backbone amide protons and nitrogens are shown by the residue sequence number. Amide proton peaks show limited chemical shift dispersion, from 7.5 to 8.8 ppm, which is characteristic of predominantly unstructured proteins. C, steady state ¹⁵N NOEs indicate that the N terminus of ctRAGE, residues 2–15, is folded. Residues 16–42 exhibit low or even negative values of steady state ¹⁵N NOE, which indicate an unstructured C-terminal tail. D, hydrogen-deuterium exchange experiment. The ¹⁵N HSQC spectrum shows residual amide proton and nitrogen peaks from [U-¹⁵N]ctRAGE remaining after 5 min at 6 °C. The ¹⁵N HSQC experiment was conducted at 6 °C to slow the hydrogen-deuterium exchange rate. Amide protons that form hydrogen bonds, and also amide protons of charged amino acids, exhibit impeded exchange rates.

FIGURE 2.

Solution structure of the ctRAGE fragment (amino acids 2–15). A, cluster of 20 solution backbone traces of ctRAGE. The closeness of the traces to each other reflects the overall quality of the solution structure. The r.m.s. deviation of the ctRAGE cluster is 0.9 Å. B, ribbon diagram of ctRAGE. Hydrogen bonds between Arg-5 and Glu-11 and between Gly-9 and Arg-5 are indicated by black lines. The hydrogen bond pattern indicates that ctRAGE folds into an α-turn. C, electrostatic surface map of ctRAGE. Positive and negative surfaces are colored blue and red, respectively.

FIGURE 3.

ctRAGE interacts with mDia1 FH1. A, sequence alignment of the human mDia1 FH1 construct used in this study (NCBI accession code NP_005210) and mouse mDia1 FH1 (NCBI accession code NP_031884). Conserved residues are in red. The boundaries of mDia1 FH1 small fragments, FH1-pep1, FH1-pep2-del1, FH1-pep2-del2, and FH1-pep2-del3, respectively, are indicated by solid lines above the sequence. B, overlay of ¹⁵N HSQC NMR spectra of free [U-¹⁵N]ctRAGE (black) and the mDia1 FH1-[U-¹⁵N]ctRAGE complex (red). To form the mDia1FH1-ctRAGE complex, 0.5 mm unlabeled mDia1 FH1, in NMR buffer (10 mm potassium phosphate (pH 6.5), 100 mm NaCl, 0.02% (w/v) NaN₃, in 90%/10% H₂O/D₂O) was added into 100 μm [U-¹⁵N]ctRAGE to yield a mDia1 FH1/ctRAGE molar ratios of 1:1. Due to ¹⁵N editing of the experiment, only backbone amide protons and nitrogens of ctRAGE are present in the spectrum. Most peaks do not change their positions, reflecting the fact that only a subset of ctRAGE residues interact with FH1. ctRAGE peaks that are substantially or completely broadened are labeled. C, mDia1 FH1-ctRAGE interaction map. Residues broadened during the NMR titration experiment are indicated in red.

FIGURE 4.

S100B stimulates AKT activation in vascular smooth muscles cells via ctRAGE-mDia1 FH1 interaction through Arg-5 and Gln-6. A, wild type, vector-transfected, or full-length double mutant R366A/Q367A human RAGE-transfected SMCs were stimulated with 10 μg/ml S100B for the indicated times. Total lysates were subjected to Western blotting with antibodies against phospho-AKT or total AKT. Quantified levels of phosphorylated AKT to total AKT in the wild type-transfected SMCs at different times following stimulation with S100B are shown. -Fold changes are relative to control. Error bars, S.D. (*, p < 0.05). B, RAGE expression levels in the indicated transfected wild type SMCs were assessed by Western blots probed with primary anti-RAGE antibody followed by anti-GAPDH antibody as a loading control. C, wild type, vector-transfected, or full-length double mutant R366A/Q367A human RAGE-transfected SMCs were stimulated with 10 μg/ml S100B for 5 min. Where indicated, cells were pretreated with 10 μm PI3K inhibitor LY294002 or with vehicle, DMSO. Total lysates were subjected to Western blotting with antibodies against phospho-AKT or total AKT. Quantified levels of phosphorylated AKT to total AKT in the wild type-transfected SMCs following stimulation with S100B are shown. -Fold changes are relative to control. Error bars, S.D. (*, p < 0.05). Assays were performed in triplicate, and the results shown are representative of three independent experiments. Arg-366 and Gln-367 of full-length RAGE correspond to Arg-5 and Gln-6 of ctRAGE, respectively. NS, nonstimulated.

FIGURE 5.

S100B stimulates migration (A) and proliferation (B) in vascular SMCs mainly via ctRAGE-mDia1 FH1 interaction through Arg-5 and Gln-6. Wild type, vector-transfected, full-length double mutant R366A/Q367A human RAGE-transfected, Dia-1-transfected, or Dia-1 and full-length double mutant R366A/Q367A human RAGE-transfected SMCs were treated with 10 μg/ml S100B or 10 ng/ml PDGF for 5 or 48 h. At the end of that time, migration (A) and proliferation (B), respectively, were assessed. Assays were performed in triplicate, and the results shown represent three independent experiments. Error bars, S.D. (*, p < 0.005). Arg-366 and Gln-367 of full-length RAGE correspond to Arg-5 and Gln-6 of ctRAGE, respectively.

FIGURE 6.

Model of RAGE-induced activation of mDia1. The FH1 and FH2 domains of mDia1 are required for mDia1 activity. DID and DAD, N-terminal diaphanous inhibitory domain and C-terminal diaphanous autoregulatory domain of mDia1, respectively. mDia1 is autoinhibited due to the interaction between the regulatory diaphanous inhibitory and diaphanous autoregulatory domains, DID and DAD, respectively. RAGE can constitutively bind to mDia1 by using the ctRAGE-FH1 interaction. Clustering of mDia1 molecules due to extracellular RAGE (exRAGE) binding to a RAGE ligand results in intermolecular domain swapping and partial restoration of mDia1 activity.