A novel 110-kDa receptor protein is involved in endocytic uptake of decorin by human skin fibroblasts.
The small leucine-rich proteoglycan (SLRP) decorin is efficiently internalized by a variety of cultured cells. A 51-kDa protein has previously been described as a receptor mediating endocytosis of decorin and of the structurally related SLRP biglycan. Recent findings suggest that endocytosis of SLRPs may also be mediated by additional receptors. The class-A scavenger receptor, the endocytic mannose receptor, the epidermal growth factor receptor, and insulin-like growth factor-I receptor have emerged as candidates. We used a combined approach of immunoprecipitation and photoactivated cross-linking to identify endocytosis receptors for decorin in human skin fibroblasts. Decorin was purified by HPLC-DEAE-ion exchange chromatography from the secretions of human skin fibroblasts under nondenaturing conditions. Confocal immunofluorescence microscopy revealed that both biotinylated decorin and decorin conjugated to the heterobifunctional cross-linker sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1-3'-dithiopropionate (SASD) were endocytosed with equal efficiency. SASD-conjugated decorin was added to [35S]-methionine-labeled fibroblasts and cross-linked intracellularly to receptor molecules by photoactivation on endocytic uptake. Cross-linked decorin-receptor complexes were purified from the extracts of trypsin-treated fibroblasts by anion exchange chromatography and immunoprecipitation with a decorin-specific antiserum. Analysis by 2D electrophoresis and autoradiography revealed that decorin was specifically cross-linked to a protein of 110 kDa, which exhibited an isoelectric point of 5.5. In a second approach, unlabeled fibroblasts were subjected to decorin endocytosis and photoactivated cross-linking followed by Western blotting of DEAE-purified cell extracts. A shift of biotinylated decorin immunoreactivity from 165 kDa (decorin-receptor complex) to 54 kDa (SASD-conjugated biotinylated decorin) was noted on reductive cleavage of the cross-linker, representing a difference in molecular weight of approximately 110 kDa. The identification of a 110-kDa protein as a novel endocytosis receptor for decorin provides further support for the emerging concept of a redundancy of receptor molecules in the endocytosis of SLRP.
Sofeu Feugaing
David Denis
DD
Department of Obstetrics and Gynecology, Münster University Hospital, Münster, Germany. dsofeu@yahoo.com
Kresse
Hans
H
Greb
Robert R
RR
Götte
Martin
M
D002478:Cells, Cultured; D058575:Decorin; D004705:Endocytosis / Q000502:physiology*; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D008970:Molecular Weight; D011509:Proteoglycans / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000378:metabolism*
C546878:DCN protein, human; D058575:Decorin; D016326:Extracellular Matrix Proteins; D011509:Proteoglycans; D011956:Receptors, Cell Surface