Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Aug 15;305(4):C457-67.
doi: 10.1152/ajpcell.00141.2013. Epub 2013 Jun 19.

Carbachol-induced MUC17 endocytosis is concomitant with NHE3 internalization and CFTR membrane recruitment in enterocytes

Thaher Pelaseyed  1 , Jenny K Gustafsson, Ida J Gustafsson, Anna Ermund, Gunnar C Hansson
Affiliations

Carbachol-induced MUC17 endocytosis is concomitant with NHE3 internalization and CFTR membrane recruitment in enterocytes

Thaher Pelaseyed et al. Am J Physiol Cell Physiol. .

Abstract

We have reported that transmembrane mucin MUC17 binds PDZ protein PDZK1, which retains MUC17 apically in enterocytes. MUC17 and transmembrane mucins MUC3 and MUC12 are suggested to build the enterocyte apical glycocalyx. Carbachol (CCh) stimulation of the small intestine results in gel-forming mucin secretion from goblet cells, something that requires adjacent enterocytes to secrete chloride and bicarbonate for proper mucin formation. Surface labeling and confocal imaging demonstrated that apically expressed MUC17 in Caco-2 cells and Muc3(17) in murine enterocytes were endocytosed upon stimulation with CCh. Relocation of MUC17 in response to CCh was specific as MUC3 and MUC12 did not relocate following CCh stimulation. MUC17 colocalized with PDZK1 under basal conditions, while MUC17 relocated to the terminal web and into early endosomes after CCh stimulation. CCh stimulation concomitantly internalized the Na(+/)H(+) exchanger 3 (NHE3) and recruited cystic fibrosis transmembrane conductance regulator (CFTR) to the apical membranes, a process that was important for CFTR-mediated bicarbonate secretion necessary for proper gel-forming mucin unfolding. The reason for the specific internalization of MUC17 is not understood, but it could limit the diffusion barrier for ion secretion caused by the apical enterocyte glycocalyx or alternatively act to sample luminal bacteria. Our results reveal well-orchestrated mucus secretion and trafficking of ion channels and the MUC17 mucin.

Keywords: CFTR; MUC17; Muc3(17), PDZK1; NHE3.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
The transmembrane mucin MUC17 is a component of the glycocalyx covering apical membranes of Caco-2 cells. A: schematic view of the domain organization of MUC17. SS, signal sequence; PTS, proline-, threonine-, and serine-rich region called the mucin domain when O-glycosylated; SEA, SEA domain; TM, transmembrane domain. G↓SVVV represents the consensus sequence of the native SEA cleavage site. S1 and S2 indicate the position of epitopes recognized by anti-MUC17S1 and anti-MUC17S2 pAbs. TTSF represents the class I PDZ binding motif that allows binding to the scaffolding protein PDZK1. B: transmission electron microscopy of microvilli distributed on the apical surface of human duodenal enterocytes (top) and polarized Caco-2 cells (bottom). Insets: magnifications of the larger micrographs. Arrows point at the glycocalyx. Bars in large micrographs = 1 μm. Bars in insets = 100 nm. C: expression of surface glycoproteins on apical surface of Caco-2 cells assessed by surface biotinylation using biotin hydrazide. Biotinylated high-molecular glycoproteins were detected using streptavidin HRP. Arrows indicate separated biotinylated glycoproteins. IB, immunoblot. D: staining of MUC17 on apical surface of polarized Caco-2 cells (left). The specificity of anti-MUC17S1 pAb against MUC17 was assessed by neutralizing the antibody with its corresponding immunizing peptide (P) (right). Arrows point towards the apical plasma membrane.
Fig. 2.
Fig. 2.
MUC17 is endocytosed and redistributes to F-actin-rich terminal web upon stimulation with carbachol (CCh). A: nonstimulated polarized Caco-2 cells (Ctrl) and cells stimulated with CCh (CCh) or a combination of CCh and Dynasore (CCh/Dyn) were analyzed for apical MUC17 expression. Cell surface glycoproteins were biotinylated and MUC17 was detected by Western blot using anti-MUC17S2 pAb. Left: representative Western blot showing input controls, unbound material after affinity precipitation and the precipitated material. Right: bar graph summarizing means ± SE of 4 independent experiments (n = 4; *P < 0.05 is compared with control; au, arbitrary units). B: nonstimulated Caco-2 cells (Ctrl) and CCh-stimulated cells stained for MUC17 (green), F-actin (gray), and DNA (blue). Dashed white lines indicate F-actin intensity maximum. Scale bar = 2 μm. C: normalized intensity profiles obtained from 3 independent experiments showing distribution of MUC17 (green line) in relation to F-actin (gray line) in nonstimulated (left) and cells stimulated with CCh (right). D: bar graph summarizing means ± SE of 3 independent experiments. Solid bars represent amount of MUC17 in nonstimulated cells (Ctrl), whereas open bars represent amount of MUC17 in cells treated with CCh (n = 3 in each group, ***P < 0.001). E, left: normalized intensity profile of MUC3 (blue lines) and MUC12 (red lines) in relation to F-actin (gray line) in nonstimulated cells and CCh-stimulated cells. Right: bar graph summering means ± SE from 3 independent experiments. Solid bars represent amount of MUC3 (blue) and MUC12 (red) in nonstimulated cells, and open bars represent amount of MUC3 (blue) and MUC12 (red) in CCh-treated cells. T, terminal web; M, microvilli.
Fig. 3.
Fig. 3.
Subcellular localization of MUC17 in relation to PDZK1 and EEA1-positive early endosomes. A: nonstimulated and CCh-stimulated Caco-2 cells stained for MUC17 (green), PDZK1 (red), and F-actin (gray). Dashed white lines indicate F-actin intensity maximum. Scale bar = 2 μm. B: normalized intensity profiles for MUC17 (green line) and PDZK1 (red line) plotted in relation to F-actin (gray line). C: 2-dimensional (2D) scatter plots showing correlation between MUC17 (green channel) and PDZK1 (red channel) in nonstimulated (left panel) and CCh-stimulated cells (right). D, left: bar graph summarizing thresholded PCC for colocalization of MUC17 with PDZK1 in nonstimulated and CCh-stimulated cells. Right: MCC M1 for MUC17 and M2 for PDZK1 in nonstimulated cells (n = 3 in each group; ***P < 0.001). E: Caco-2 cells stimulated with CCh and stained for MUC17 (green), EEA1 (red) and F-actin (gray). Top: MUC17 and EEA1 and the merge of the 2 channels in nonstimulated cells. Bottom: staining of MUC17 and EEA1 and the merge of the 2 channels in CCh-stimulated cells. Magnifications show colocalization of MUC17 with EEA1 in yellow. Scale bar = 2 μm. F: 2D scatter plots showing correlation between MUC17 (green channel) and EEA1 (red channel) in nonstimulated cells (left) and CCh-treated cells (right). G: bar graph summarizing thresholded Pearson's correlation coefficient (PCC) obtained from colocalization of MUC17 with EEA1 in nonstimulated and CCh-stimulated cells (left). Mander's correlation coefficients (MCC) M1 for MUC17 and M2 for EEA1 in CCh-stimulated cells are displayed at right (n = 3 in each group; ***P < 0.001).
Fig. 4.
Fig. 4.
MUC17 endocytosis occurs simultaneously with Na+/H+ exchanger 3 (NHE3) internalization and CFTR membrane recruitment. A and D: nonstimulated (left) and CCh-stimulated Caco-2 cells (right) stained for F-actin (gray) as well as MUC17 (green) and NHE3 (red) or MUC17 (green) and CFTR (red). Dashed white lines indicate F-actin intensity maximum. Scale bar = 2 μm. B and E: normalized intensity profiles for nonstimulated (left) and CCh-treated cells (right) from 3 independent experiments showing the distribution of MUC17 (green line) and NHE3 (red line) or MUC17 (green line) and CFTR (red line) in relation to F-actin (gray line). C and F: bar graphs summarizing means ± SE of 3 independent experiments. Solid bars represent distribution of MUC17 (green) and NHE3 (red) or MUC17 (green line) and CFTR (red line) in nonstimulated cells (Ctrl), while open bars show distribution of each protein after CCh stimulation (n = 3 in each group; *P < 0.05, ***P < 0.001).
Fig. 5.
Fig. 5.
Murine Muc3(17) redistributes to the F-actin-rich terminal web of enterocytes upon stimulation with carbachol. Sections from nonstimulated (A) and CCh-stimulated mouse duodenum (B) were stained for Muc3(17) (green), F-actin (red), and DNA (blue). Dashed white lines indicate F-actin intensity maximum. Scale bar = 5 μm. C: normalized intensity profile for Muc3(17) (green line) in relation to F-actin (gray line) in nonstimulated (Ctrl) and tissue stimulated with CCh. D: bar graphs summarizing means ± SE for amount of Muc3(17) in nonstimulated (Ctrl) and CCh-stimulated tissue. (n = 3; ***P < 0.001). E: mucus thickness in mouse duodenal explants, mounted in a horizontal chamber at time point −10 min. ●, Mucus was removed and 100 μM CCh were added at 0 min. Mucus expansion and thickness were measured during 40 min. ○, Nonstimulated tissue. ▲, Villus height (n = 5).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ambort D, Johansson MEV, Gustafsson JK, Nilsson HE, Ermund A, Johansson BR, Koeck PJB, Hebert H, Hansson GC. Calcium and pH-dependent packing and release of the gel-forming MUC2 mucin. Proc Natl Acad Sci USA 109: 5645–5650, 2012 - PMC - PubMed
    1. Axelsson MA, Karlsson NG, Steel DM, Ouwendijk J, Nilsson T, Hansson GC. Neutralization of pH in the Golgi apparatus causes redistribution of glycosyltransferases and changes in the O-glycosylation of mucins. Glycobiology 11: 633–644, 2001 - PubMed
    1. Barlow AL, MacLeod A, Noppen S, Sanderson J, Guérin CJ. Colocalization analysis in fluorescence micrographs: verification of a more accurate calculation of Pearson's correlation coefficient. Microsc Microanal 16: 710–724, 2010 - PubMed
    1. Bustamante C, Marko JF, Siggia ED, Smith S. Entropic elasticity of lambda-phage DNA. Science 265: 1599–1600, 1994 - PubMed
    1. Carlstedt I, Herrmann A, Karlsson H, Sheehan J, Fransson LA, Hansson GC. Characterization of two different glycosylated domains from the insoluble mucin complex of rat small intestine. J Biol Chem 268: 18771–18781, 1993 - PubMed

Publication types

MeSH terms

LinkOut - more resources