Amyloid-beta peptide is central to the pathology of Alzheimer's disease, because it is neurotoxic--directly by inducing oxidant stress, and indirectly by activating microglia. A specific cell-surface acceptor site that could focus its effects on target cells has been postulated but not identified. Here we present evidence that the 'receptor for advanced glycation end products' (RAGE) is such a receptor, and that it mediates effects of the peptide on neurons and microglia. Increased expressing of RAGE in Alzheimer's disease brain indicates that it is relevant to the pathogenesis of neuronal dysfunction and death.

The receptor for advanced glycation end products (RAGE), a newly-identified member of the immunoglobulin superfamily, mediates interactions of advanced glycation end product (AGE)-modified proteins with endothelium and other cell types. Survey of normal tissues demonstrated RAGE expression in situations in which accumulation of AGEs would be unexpected, leading to the hypothesis that under physiologic circumstances, RAGE might mediate interaction with ligands distinct from AGEs. Sequential chromatography of bovine lung extract identified polypeptides with M r values of Ϸ12,000 (p12) and Ϸ23,000 (p23) which bound RAGE. NH 2 -terminal and internal protein sequence data for p23 matched that reported previously for amphoterin. Amphoterin purified from rat brain or recombinant rat amphoterin bound to purified sRAGE in a saturable and dose-dependent manner, blocked by anti-RAGE IgG or a soluble form of RAGE (sRAGE). Cultured embryonic rat neurons, which express RAGE, displayed dose-dependent binding of 125 I-amphoterin which was prevented by blockade of RAGE using antibody to the receptor or excess soluble receptor (sRAGE). A functional correlate of RAGE-amphoterin interaction was inhibition by anti-RAGE F(ab) 2 and sRAGE of neurite formation by cortical neurons specifically on amphoterin-coated substrates. Consistent with a potential role for RAGEamphoterin interaction in development, amphoterin and RAGE mRNA/antigen were co-localized in developing rat brain. These data indicate that RAGE has physiologically relevant ligands distinct from AGEs which are likely, via their interaction with the receptor, to participate in physiologic processes outside of the context of diabetes and accumulation of AGEs.Incubation of proteins or lipids with aldose sugars results in nonenzymatic glycation and oxidation (1-7). Following formation of the reversible early glycation products, Schiff bases and Amadori products, further complex molecular rearrangements result in irreversible advanced glycation end products (AGEs). 1Factors favoring nonenzymatic glycation include delayed protein turnover, as in amyloidoses, accumulation of macromolecules with high lysine content, and situations with elevated glucose levels, as in diabetes. AGE formation occurs during normal aging, and at an accelerated rate in diabetes, in which their accumulation in the plasma and vessel wall has been speculated to underlie the pathogenesis of vasculopathy (1, 2, 4).One of the principal means through which AGEs impact on cellular elements is through interaction with cellular binding proteins. Although there are several possible cell-associated polypeptides with which AGEs might interact (8, 9), our work has focussed on the receptor for AGEs (RAGE), as its expression in endothelium, vascular smooth muscle, mononuclear phagocytes, and the central nervous system suggests strategic loci for interaction with the glycated ligands (10, 11). The potential pathophysiologic relevance of AGE-RAGE interaction was emphasized by studies demonstrating that blockade of RAGE pr...

S100/calgranulin polypeptides are present at sites of inflammation, likely released by inflammatory cells targeted to such loci by a range of environmental cues. We report here that receptor for AGE (RAGE) is a central cell surface receptor for EN-RAGE (extracellular newly identified RAGE-binding protein) and related members of the S100/calgranulin superfamily. Interaction of EN-RAGEs with cellular RAGE on endothelium, mononuclear phagocytes, and lymphocytes triggers cellular activation, with generation of key proinflammatory mediators. Blockade of EN-RAGE/RAGE quenches delayed-type hypersensitivity and inflammatory colitis in murine models by arresting activation of central signaling pathways and expression of inflammatory gene mediators. These data highlight a novel paradigm in inflammation and identify roles for EN-RAGEs and RAGE in chronic cellular activation and tissue injury.

The cholecystokinin (CCK) family of peptides and their receptors are widely distributed throughout the gastrointestinal and central nervous systems where they regulate secretion, motility, growth, anxiety, and satiety. The CCK receptors can be subdivided into at least two subtypes, CCKA and CCKB on the basis of pharmacological studies. We report here the purification of the CCKA receptor to homogeneity from rat pancreas by using ion-exchange and multiple affinity chromatographic separations. This allowed partial peptide sequencing after chemical/enzymatic ceavage and synthesis of degenerate oligonucleotide primers. These primers were used for initial cloning of the cDNA from rat pancreas by PCR. The predicted protein sequence ofthe cDNA clone contained the five partial peptide sequences obtained from the purified protein.Seven putative transmembrane domains suggest its membership in the guanine nucleotide-binding regulatory proteincoupled receptor superfamily. In vitro transcripts of the cDNA clone were functionally expressed in Xenopus oocytes and displayed the expected agonist and antagonist specificity.The cholecystokinin (CCK) family of peptides was originally isolated from the mammalian gastrointestinal tract (1) and was one of the first gastrointestinal peptides to be discovered in the brain (2). Their receptors appear throughout the gastrointestinal and nervous systems and can be pharmacologically classified into two subtypes, CCKA and CCKB (3). CCKA receptors mediate physiologic gallbladder contraction, pancreatic growth and enzyme secretion, delayed gastric emptying, relaxation of the sphincter of Oddi, and potentiation of insulin secretion (3). The CCKA type receptor also appears in the anterior pituitary, the myenteric plexus, and areas of the central nervous system (midbrain) where CCK-containing dopaminergic neurons have been implicated in the pathogenesis of schizophrenia, Parkinson disease, drug addiction, and feeding disorders (4). Experimental rat pancreatic carcinogenesis is promoted by CCK through the CCKA type receptor (5). CCK acting at peripheral CCKA receptors and at central CCKA and CCKB gastrin receptors plays a significant role in the nervous system control of appetite (6).Recently, antagonists highly selective and potent for each of the CCK receptor subtypes have been developed. The two most potent and selective antagonists are L-364,718 (7) and PD134308 (8) for CCKA and CCKB receptors, respectively.In pancreatic acinar cells, CCKA receptors are coupled to a guanine nucleotide-binding regulatory protein (G protein), which activates phospholipase C, breakdown of inositol phospholipids, mobilization of intracellular calcium, and activation of protein kinase C (3). Activation of this pathway in Xenopus oocytes by CCK receptors that have been functionally expressed in the plasma membrane after injection of either rat brain total RNA (9) or the rat pancreatic acinar carcinoma cell line, AR42J (10), mRNA (11) results in a depolarizing current due to Ca2+-dependent chloride channels o...

The mammalian bombesin-like peptides gastrin-releasing peptide (GRP) and neuromedin B regulate numerous and varied cell physiologic processes in various cell types and have also been implicated as autocrine growth factors influencing the pathogenesis and progression of human small cell lung carcinomas. We report here the molecular characterization of the bombesin/GRP receptor. Structural analysis of cDNA clones isolated from Swiss 3T3 murine embryonal fibroblasts shows that the GRP receptor is a member of the guanine nucleotide binding protein-coupled receptor superfamily with seven predicted hydrophobic transmembrane domains. In vitro transcripts from cloned cDNA templates encompassing the predicted protein coding domain, when injected into Xenopus oocytes, resulted in expression of functional GRP receptors. The predicted amino acid sequence of the open reading frame in cDNA clones matches the aminoterminal sequence as well as the sequence of four tryptic fragments isolated from the purified protein. Expression of the GRP receptor cDNA in model systems potentially provides a powerful assay for the development of subtype-specific receptor antagonists that may prove to be of therapeutic importance in human small cell lung carcinoma.The mammalian bombesin-like peptides gastrin-releasing peptide (GRP) and neuromedin B (NMB) are regulatory peptides of importance in a wide variety of cell physiologic processes including secretion, smooth muscle contraction, and modulation of neuron firing rate (for review, see refs. 1 and 2). In addition, bombesin-like peptides can function as growth factors in Swiss 3T3 murine embryonal fibroblasts (3) and have been implicated as autocrine growth factors in the pathogenesis of some human small cell lung carcinomas (4). The bioactivities associated with the mammalian bombesinlike peptides are mediated by high-afflinity binding to cell surface receptors present on many target cells (for review, see ref. 1). The properties of high-affinity bombesin/GRP receptors found in relatively high numbers (=100,000 receptors per cell) on Swiss 3T3 fibroblasts have been extensively studied (5), with subsequent purification of the protein to near homogeneity (6). Molecular cloning ofthe gene encoding the Swiss 3T3 bombesin/GRP receptor is the next step to understanding the diversity and function of mammalian bombesin-like peptides and their receptors in physiologic and pathologic processes. In this paper, we report the isolation and characterization of cDNA clones § encoding the Swiss 3T3 bombesin/GRP receptor, defining the structure of the receptor polypeptide. The clones obtained should provide a basis for further molecular analysis ofthe structure, function, and expression of receptors in Swiss 3T3 cells. In addition, these clones provide a means for a similar analysis of this receptor and other related bombesin peptide receptors expressed in the various cell types known to respond to this family of peptides. MATERIALS AND METHODSReceptor Protein Purification and Peptide Sequencing. The ...