Abstract
Protein production within the secretory pathway is accomplished by complex but organized processes. Here, we demonstrate that the growth factor midkine interacts with LDL receptor-related protein 1 (LRP1) at high affinity (K(d) value, 2.7 nm) not only at the cell surface but also within the secretory pathway during biosynthesis. The latter premature ligand-receptor interaction resulted in aggregate formation and consequently suppressed midkine secretion and LRP1 maturation. We utilized an endoplasmic reticulum (ER) retrieval signal and an LRP1 fragment, which strongly bound to midkine and the LRP1-specialized chaperone receptor-associated protein (RAP), to construct an ER trapper. The ER trapper efficiently trapped midkine and RAP and mimicked the premature ligand-receptor interaction, i.e. suppressed maturation of the ligand and receptor. The ER trapper also diminished the inhibitory function of LRP1 on platelet-derived growth factor-mediated cell migration. Complementary to these results, an increased expression of RAP was closely associated with midkine expression in human colorectal carcinomas (33 of 39 cases examined). Our results suggest that the premature ligand-receptor interaction plays a role in protein production within the secretory pathway.
Highlights
Protein production within the secretory pathway is tightly regulated by complex but organized processes, which include folding in the endoplasmic reticulum (ER),2 glycosylation, and vesicular transport to the cell surface through the ER and Golgi apparatus
MK Binds to the Second and Fourth Ligand-binding Domains of LDL receptor-related protein 1 (LRP1) on the Cell Surface—LRP1 consists of two heterodimeric subunits that are produced through furin-mediated processing in the trans-Golgi (Fig. 1A)
These results indicate that the MK-LRP1 autocrine loop was activated, and the enhanced expressions of receptor-associated protein (RAP) and MK are closely associated in human colorectal carcinomas
Summary
DNA Constructs, and Antibodies—CHO K1 cells were cultured in DMEM with 10% fetal bovine serum. For chemical cross-linking, cells were washed with ice-cold phosphate-buffered saline containing calcium and magnesium (PBSϩ) as well as 0.1% bovine serum albumin (PBS-BSA), and supplied with 5 ml of ice-cold DMEM with 10% fetal bovine serum. Cells were scraped in 500 l of detaching buffer, collected by centrifuge, and lysed with 200 l of lysis buffer (20 mM Tris-HCl (pH 7.5), 0.6% CHAPS, 150 mM sodium chloride, 1 mM calcium chloride, 1 mM magnesium chloride, and 1 mM PMSF). For in vitro cross-linking, 10 l of protein solution dialyzed against appropriate buffers was mixed with 2 l of 125I-labeled MK (ϳ10 ng) and was incubated at 20 °C for 1 h. Collagen I-coated chambers were inserted into wells of a 24-well plate, where each well contained 600 l of 0.2% BSA and 0.4% FBS/DMEM with or without 50 ng/ml PDGF-BB, and preincubated at room temperature for 10 min. Viable cells were estimated with a Cell Counting Kit-8 (DOJINDO)
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