Naslov
Internalization, Recycling and Degradation of CD44

Autori
Karleuša, Ljerka ; Mahmutefendić, Hana ; Blagojević Zagorac, Gordana ; Ilić Tomaš, Maja ; Lučin, Pero

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Skup
2012 Annual Meeting Of The Croatian Immunological Society

Mjesto i datum
Marija Bistrica, Hrvatska, 05.10.2012. - 06.10.2012

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
CD44; internalization; recycling

Sažetak
Introduction: CD44 molecule, a receptor for hyaluronic acid, is present at the cell surface in lipid-ordered and lipid-disordered plasma membrane (PM) environment. It has been shown that CD44 associate with non-clathrin Arf6-associated endosomal carriers, similar to Major Histocompatibility Class I (MHC-I) proteins. However, very little is known about its membrane turnover, internalization route and endosomal trafficking. Objectives: The objective of this research was to investigate the internalization, recycling and degradation pathways of CD44. Materials and Methods: In our experiments we used Balb 3T3 fibroblasts and L cells transfected with murine H-2 Ld allele (L-Ld cells). CD44 molecule was followed by monoclonal antibody (mAb) IM-7 and MHC-I molecules were followed by mAbs MA-215 (Kd), 34-5-8S (Dd), 30-5-7 (fully conformed Ld) and 64-3-7 (open forms of Ld). Internalization rates of cell surface proteins were determined by flow cytometry using the internalization assay based on mAb labeling at the cell surface and their intracellular routes by immunofluorescence and confocal analysis using various modifications of the pulse-chase internalization assay. In order to clarify the endocytic pathways of CD44 we used a variety of chemical inhibitors of endocytosis, vesicular transport and cell signaling and colocalization of internalized proteins with markers of endosomal compartments and pathways (EEA-1 and transferrin receptor for erly endosomes, Lamp1/2, LBPA, EGF-EGFR, manose-6-phosphate receptor and dextran for late endosomes). Results: CD44 molecules localized in plasma membrane (PM) tubular invaginations that were resistant to Triton X-100 extraction. The internalization rate of CD44 was low and the cell surface half-life, indicating its high recycling rate that takes place via long PM tubular invaginations since very little of internalized CD44 colocalized with internalized TfR and EEA1 and almost no CD44 was detected in the juxtanuclear recycling compartment. Consequently, very little of internalized CD44 colocalized with late endosomal markers indicating that very little CD44 internalize into late endosomes in spite of a large mambrane turnover, as determined by NBD-C6 ceramide. Aluminum fluoride (AlF4+) increased the internalization rate of CD44, same as to the MHC-I molecules, confirming our conclusion that peripheral recycling maintains the low internalization rate. In the presence of chemical compound U18666a, which accumulate cholesterol in endosomes and disrupts late endosomal mobility, a significant fraction of cell surface CD44 was retained in vesicular endosomal carriers suggesting that peripheral cholesterol membrane load determines the recycling capacity. Methyl-β-cyclodextrin, a drug that disrupts membrane cholesterol load, hindered CD44 internalization. Finally, the pH disrupting drug Concanamycin A did not change the internalization rate of CD44, whereas increased internalization of MHC-I molecules, suggesting that CD44 is retains in the pH neutral environment and internalized via neutral endosomal carriers, Conclusion: Long cell surface half-life of the CD44 molecules is maintained by high rate of peripheral recycling via long plasma membrane tubular invaginations. The endocytic route of CD44 and MHC-I molecules is distinct although they are all collected in the same vacuolar early endosomes at later stages of the early endosomal route.

Izvorni jezik
Engleski

Znanstvena područja
Temeljne medicinske znanosti

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