Abstract
Receptor-associated protein (RAP) is an endoplasmic reticulum/Golgi protein involved in the processing of receptors of the low density lipoprotein receptor family. A approximately 95-kDa membrane glycoprotein, designated gp95/sortilin, was purified from human brain extracts by RAP affinity chromatography and cloned in a human cDNA library. The gene maps to chromosome 1p and encodes an 833-amino acid type I receptor containing an N-terminal furin cleavage site immediately preceding the N terminus determined in the purified protein. Gp95/sortilin is expressed in several tissues including brain, spinal cord, and testis. Gp95/sortilin is not related to the low density lipoprotein receptor family but shows intriguing homologies to established sorting receptors: a 140-amino acid lumenal segment of sortilin representing a hitherto unrecognized type of extracellular module shows extensive homology to corresponding segments in each of the two lumenal domains of yeast Vps10p, and the extreme C terminus of the cytoplasmic tail of sortilin contains the casein kinase phosphorylation consensus site and an adjacent dileucine sorting motif that mediate assembly protein-1 binding and lysosomal sorting of the mannose-6-phosphate receptors. Expression of a chimeric receptor containing the cytoplasmic tail of gp95/sortilin demonstrates evidence that the tail conveys colocalization with the cation-independent mannose6-phosphate receptor in endosomes and the Golgi compartment.
Highlights
Sorting of newly synthesized lysosomal enzymes from the Golgi compartment to late endosomes in eukaryotic cells is a sophisticated transport process involving specific sorting receptors in the trans-Golgi network
In the present study we have identified, purified, and characterized a ϳ95-kDa protein by receptor-associated protein (RAP) affinity chromatography of membrane protein extracts from human brain
Binding of 125I-RAP was not inhibited by urokinase in complex with plasminogen activator inhibitor type-1 (200 nM), and 125I-labeled urokinaseinhibitor complex did not bind, verifying that the RAP binding protein was different from the very low density lipoprotein receptor
Summary
M6P, mannose-6-phosphate; CD, cationdependent; CHAPS, 3-((cholamidopropyl)D-imethylammoniol)1-propanesulfonate; CI, cation-independent; LDL, low density lipoprotein; TGN, trans-Golgi network; Vps10p, vacuolar protein sorting 10 protein; known sorting receptors that bind to phosphorylated mannose residues in lysosomal hydrolases [1]. A M6P-independent sorting pathway has been demonstrated by identification of the vacuolar protein-sorting 10 protein (Vps10p) [2] and a highly homologous protein encoded by the yeast VTH2 gene [3]. Both are capable of targeting yeast carboxypeptidase Y to lysosomes [2, 3]. Transfection analyses of the novel receptor, which represents the first known RAP binding receptor not related to the LDL receptor family, disclosed that the cytoplasmic tail conveys localization to the Golgi and CI-M6P receptorrich vesicles
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