A conserved LDL-receptor motif regulates corin and CD320 membrane targeting in polarized renal epithelial cells

Abstract

Selective protein distribution on distinct plasma membranes is important for epithelial cell function. To date, how proteins are directed to specific epithelial cell surface is not fully understood. Here we report a conserved DSSDE motif in LDL-receptor (LDLR) modules of corin (a transmembrane serine protease) and CD320 (a receptor for vitamin B12 uptake), which regulates apical membrane targeting in renal epithelial cells. Altering this motif prevents specific apical corin and CD320 expression in polarized Madin–Darby canine kidney (MDCK) cells. Mechanistic studies indicate that this DSSDE motif participates in a Rab11a-dependent mechanism that specifies apical sorting. In MDCK cells, inhibition of Rab11a, but not Rab11b, expression leads to corin and CD320 expression on both apical and basolateral membranes. Together, our results reveal a novel molecular recognition mechanism that regulates LDLR module-containing proteins in their specific apical expression in polarized renal epithelial cells.

A Deep Insight Into Regulatory T Cell Metabolism in Renal Disease: Facts and Perspectives

“…Renal tubule epithelial cells(RTECs) are composed of a single layer of epithelium and have different morphological characteristics and functions according to the position of renal tubules. For example, in the proximal convoluted tubules, the wall is composed of a single layer of cuboidal epithelial cells ( 54 ). The lumen is small and irregular and is an important part of tubular reabsorption.…”

Probing the functional consequence and clinical relevance of CD320 p.E88del, a variant in the transcobalamin receptor gene

“…Madin-Darby Canine Kidney (MDCK) cells (China Center for Type Culture Collection, 3142C0001000000147, authenticated by STR profiling) were grown on glass coverslips in 12-well plates (Corning, 3513) containing Dulbecco's modified Eagle's medium (Corning, 10-0130CVRC) and 10% fetal bovine serum (Gibco, 16000-044) at 37 C with 5% CO 2 . Plasmids expressing human CD320 wild-type (WT) and variants p.E88del and p.S162A with an N-terminal Flag tag were transfected into the cells using PolyJet reagents (SignaGen Laboratories, SL100688), as described previously (Zhang et al, 2020).…”

“…After 24 h, the cells were fixed with methanol at room temperature for 5 min and incubated with 5% (w/v) bovine serum albumin in phosphate-buffered saline at 37 C for 1 h. Immunostaining was conducted with an anti-Flag antibody (Sigma, F1804, 1:500) and an anti-ZO-1 antibody (Cell Signaling, 8193 s, 1:300) and Alexa Fluro-488 or 594-labeled secondary antibodies (Thermo Fisher, A32766 and A32740, 1:500) (Zhang et al, 2020). The stained cells were examined with a confocal laser scanning microscope (Olympus, FV1000).…”

“…The CD320 S162A construct replaces the serine with an alanine in the central residue of the DSSDE motif in the second LDLR-A domain of CD320. It exhibits loss of specific localization to the apical membrane (Zhang et al, 2020) and was included as a control (Figure 5). There was no significant difference in the localization…”

“…When the DSSDE motif is intact, proteins localize specifically to the apical membrane. However, altering this motif in the eighth LDLR-A domain in corin or the second LDLR-A domain in CD320 results in a loss of this specificity (Zhang et al, 2020). Although this DSSDE motif is not perfectly preserved in the first LDLR-A domain in CD320 (DGSDE), the final glutamic acid is the first of three consecutive glutamic acid residues, one of which is deleted in the p.E88del variant of CD320.…”