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Journal of Biochemistry Advance Access originally published online on October 11, 2008
Journal of Biochemistry 2009 145(1):21-30; doi:10.1093/jb/mvn137
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© The Authors 2008. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Internalization of the Receptor for Advanced Glycation End Products (RAGE) is Required to Mediate Intracellular Responses

Natalia Sevillano1, María D. Girón1, Mercedes Salido2, Alberto M. Vargas1, José Vilches2 and Rafael Salto1,*

1Department of Biochemistry and Molecular Biology II, School of Pharmacy, University of Granada, Campus de Cartuja sn, E-18071 Granada, Spain; and 2Department of Histology, School of Medicine, University of Cádiz. Confocal Microscopy and Cell Culture Unit, Central Research Services in Health Sciences, Edificio Policlínico, C/Dr Marañon 3, E-11002 Cádiz, Spain

*To whom correspondence should be addressed. Tel: +34-958-246363, Fax: +34-958-248960, E-mail: rsalto{at}ugr.es

Received September 25, 2008; Accepted October 6, 2008


  Abstract

To dissect the rat receptor for advanced glycation end products (RAGE) subcellular distribution and trafficking in eukaryotic cells, an expression system coding for a fusion protein between the RAGE and an enhanced green fluorescent protein (EGFP) has been used. The RAGE–EGFP protein is expressed at the plasma membrane of CHO-k1 and Neuro-2a (N2a) cells and retains the capacity to bind Texas Red-labelled advanced glycation end products (AGEs). AGEs addition to the cell cultures induced a change in the subcellular distribution of the fluorescent RAGE–EGFP protein compatible with an internalization of the AGEs–RAGE complex. Furthermore, while N2a cells expressing the RAGE–EGFP showed an increase in ERK1/2 phosphorylation and NF-{kappa}B DNA binding in response to AGEs, pre-incubation with dansyl-cadaverine or phenylarsine oxide, inhibitors of receptors internalization, blocked the activation of ERKs and other intracellular responses mediated by AGEs. These results suggest that internalization plays a key role in the signal transduction mediated by RAGE.

Key Words: enhanced green fluorescent protein, ERK1/2, NF-{kappa}B, receptor for advanced glycation end products, receptor internalization

Abbreviations: AGEs, advanced glycation end products; CHO-k1, Chinese hamster ovary cell line; DAPI, 4'-6-Diamidino-2-phenylindole; DC, dansyl-cadaverine; DMEM, Dulbecco's modified Eagle's medium; EGFP, enhanced green fluorescent protein; EMSA, electrophoretic mobility shift assay; ERK, extracellular signal-regulated kinases; FBS, fetal bovine serum; N2a, neuro-2a neuroblastoma cell line; PAO, phenylarsine oxide; PBS, phosphate-buffered saline; RAGE, receptor for advanced glycation end products


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