81, 7825C7832 [PMC free article] [PubMed] [Google Scholar] 20. measured transcytosis of pIgR by biotinylating it Entacapone at the BL surface and quantitating the subsequent release of biotin-SC into the AP medium in the absence or presence of pIgA. AP SC release was reduced from 49.81 1.47% (pIgR-WT) to 37.84 3.79% (pIgR-A580V), = 0.008 in the absence of pIgA (Fig. 2SC in AP and BL media, and pIgR). = 12). = 14). Binding of pIgA to the pIgR is known to increase the rate of Entacapone transcytosis of the pIgR. We therefore determined the effect of the mutation on AP SC release in the presence of pIgA added to the BL medium. Under this condition, AP SC release was also decreased by the mutation, 54.6 1.29% (pIgR-WT) and 46.32 2.79% (pIgR-A580V), = 0.015 (Fig. 213.45 1.47% (A580V), = 0.002) and 80 min (27.71 Entacapone 2.15% (WT) 21.85 2.01% (A580V), = 0.05) (Fig. 2mutant, respectively, was 10.84 0.72% 15.2 1.05% at 5 min, = 0.014; 27.13 3.13% 41.78 3.82% at 10 min, = 0.015; and 38.93 3.18% 64.13 5.61% at 30 min, = 0.002. Open in a separate window Physique 3. A580V mutation in human pIgR CD2 increases pIgA endocytosis in pIgR-expressing MDCK cells. = 4 at 5 min, = 6 at 10 and 30 min). (heavy chain) and (light chain). represent different bands from Entacapone your same gel and exposure. The N-terminal, extracellular region of pIgR consists of five immunoglobulin-like domains, which become SC after proteolytic cleavage. This is connected by a stalk region to the single membrane-spanning segment of pIgR. The exact site(s) of the cleavage that converts pIgR to SC has not been definitively decided (14, 15). The Ala-580 mutation is in the stalk region of pIgR and close to the likely cleavage site(s). It is possible that the pIgR-580V mutation decreases the rate of cleavage of pIgR at the AP surface. Our measurement of pIgR transcytosis scores release of SC into the AP medium, and so a decrease in cleavage would decrease the apparent rate of transcytosis. The same holds true for measurement of transcytosis of pIgA bound to the pIgR. Furthermore, decreased cleavage could increase the amount of pIgR that enters the AP endocytic pathway rather than undergoing cleavage and thereby increase the apparent rate of endocytosis. To measure pIgR cleavage directly, we biotinylated pIgR at the AP surface at 4 C and then warmed the cells to 37 C for 5 min and quantitated the release of biotinylated SC. Indeed, the rate of cleavage of pIgR to SC was decreased by the pIgR-A580V mutation. Cleavage of WT A580V, respectively, was 24.03 0.94% 17.26 0.61%, = 0.03 (Fig. 4). This suggests that the reduction in pIgR-A580V transcytosis is likely due to reduced cleavage (13, 16, 17). Open in a separate window FIGURE 4. A580V mutation in human pIgR decreases pIgR AP cleavage in pIgR-expressing MDCK cells. = 8). is shown. represent different bands from the same gel and exposure. DISCUSSION The pIgR has been studied extensively as a model for membrane trafficking and transcytosis in polarized epithelial cells. Its 103-residue cytoplasmic domain contains sorting signals for.