The Extracellular Region of the Receptor for Advanced Glycation End Products Is Composed of Two Independent Structural Units

Abstract

The receptor for advanced glycation end products (RAGE) is an important cell surface receptor being pursued as a therapeutic target because it has been implicated in complications arising from diabetes and chronic inflammatory conditions. RAGE is a single membrane spanning receptor containing a very small ~40 residue cytosolic domain and a large extracellular region comprised of 3 Ig-like domains. In this study, high level bacterial expression systems and purification protocols were generated for the extracellular region of RAGE (sRAGE) and the five permutations of single and tandem domain constructs to enable biophysical and structural characterization of its tertiary and quaternary structure. The structure and stability of each of these six protein constructs was assayed by biochemical methods including limited proteolysis, dynamic light scattering, CD, and NMR. A homology model of sRAGE was constructed to aid in the interpretation of the experimental data. Our results show that the V and C1 domains are not independent domains, but rather form an integrated structural unit. In contrast, C2 is attached to VC1 by a flexible linker and is fully independent. The interaction with a known RAGE ligand, Ca 2+ -S100B, was mapped to VC1, with the major contribution from the V domain but clearly defined secondary effects from the C1 domain. The implications of these results are discussed with respect to models for RAGE signaling.

S100B Protein Stimulates Microglia Migration via RAGE-dependent Up-regulation of Chemokine Expression and Release

“…No effects of the protein at nanomolar concentrations were detected unless the amount of expressed RAGE was increased, in which case nanomolar doses of S100B efficiently stimulated microglia migration. These latter findings might be explained by the observation that RAGE undergoes ligand-induced oligomerization, which appears to be required for RAGE signaling (62,76), or that RAGE ligands stabilize naturally occurring RAGE oligomers, an event also proposed to be necessary for RAGE signaling (78); it is possible that increasing the amount of expressed RAGE might either favor (low) S100B-dependent RAGE oligomerization and signaling or increase the probability that preformed RAGE oligomers become stabilized thereby signaling. Overall, the fact that relatively high concentrations of S100B are required for RAGE-dependent stimulation of microglia migration suggests that the ability of RAGE to activate downstream signaling pathways up-regulating chemokine FIGURE 6.…”

S100B and S100A6 Differentially Modulate Cell Survival by Interacting with Distinct RAGE (Receptor for Advanced Glycation End Products) Immunoglobulin Domains

“…The purified RAGE V, VC 1 , and C 2 domains were expressed in E. coli and purified as described previously (24).…”

“…The V, VC 1 , and C 2 domains were purified from E. coli as described previously (24). sRAGE and the RAGE domains were immobilized onto CM5 Biacore sensor chips according to procedures described previously (34).…”

Multiple levels of regulation determine the role of the receptor for AGE (RAGE) as common soil in inflammation, immune responses and diabetes mellitus and its complications

“…The extracellular region is composed of three Ig-like domains: one V-type domain (V) and two C-type domains (C1, C2). The V and C1 domains seem to form an integrated unit, while C2 is attached to VC1 by a flexible linker and is fully independent [19]. The latter is likely to enable the receptor to bind to a wide spectrum of ligands, explaining its involvement in many pathophysiological settings.…”