Dept. of Zoology,

Faculty of Science, AssiutUniversity, Assiut, 71516, Egypt

Role of Ceramide in Transferrin receptor movement in the plane of the plasma membrane

(With 4 Figures)

By

A-B.M. Abdel-Shakor andRana Alawadhi^*

*Dept of Science, Faculty of Basic Education, Kuwait.

(Received at 15/10/2009)

دور السيراميد في حرکة مستقبل الترانسفرين علي سطح غشاء البلازما

أبوبکر محمد الطيب عبد الشکور ، رنا العوضي

الترانسفرين هو بروتين موجود في بلازما الدم ويفرزة الکبد ووظيفته الأساسية هي نقل الحديد الي الخلايا. عندما يرتبط الترانسفرين بمستقبله علي سطح  الغشاء الخلوى فان المستقبل يتحرک علي غشاء البلازما ثم يتجمع في تجاويف الکلاثرين الموجودة في غشاء الخلية. بعد ذلک يدخل الترانسفرين مع مستقبلة الي داخل الخلية خلال هذه التجاويف. الهدف من هذا البحث هو التعرف علي الميکانيکية التي يتبعها مستقبل الترانسفرين في الحرکة علي غشاء البلازما والتي تقودة الي تجاويف الکلاثرين. أدت نتائج هذا البحث الي التعرف علي الوسيط الليبيدي المعروف بأسم السيراميد (ceramide) علي أنه العامل الأساسي في ميکانيکية حرکة مستقبل الترانسفرين. لقد وجد أن مستوي السيراميد يزداد خلال أول دقيقة بعد اتحاد الترانسفرين بمستقبلة کما هو مستنتج من طريقة  DGK لقياس مستوي السيراميد والکشف المناعى الخلوي للسيراميد . أوضحت النتائج أن نشاط أنزيمASMase  يزداد ونشاط إنزيم SMS  ينخفض خلال الدقيقة الأولي وبذلک يزداد مستوي السيراميد خلال هذه الفترة. وللتأکيد من أن السيراميد له دور في حرکة المستقبل وتعرفه علي تجاويف الکلاثرين تم تثبيط نشاط ASMase  بواسطة عقار Imiprarmine وأيضا تم معادلة السيراميد النتائج بواسطة الأجسام المضادة للسيراميد. لقد وجد أن الخلايا التي تم تثبيط السيراميد بها يحدث لها عدم إعادة الترانسفرين الي الخارج وتبقي هذه الخلايا محتفظة بالترانسفرين داخلها. تم أيضا استخدام طريقة التجزئة الخلوية(Cell Fractionation)  في تتبع حرکة مستقبل الترانسفرين علي سطح غشاء البلازما. لقد وجد أن تثبيط السيراميد يؤدي الي إنضمام اعداد کبيرة من المستقبل الي مناطق في غشاء الخلية بعيدة عن تجويف الکلاثرين والتي تعرف بأسم الرافت (Rafts). تعد الخلايا السرطانية أکثر الخلايا تمثيلا لمستقبل الترانسفرين علي أغشيتها الخلوية لذلک فإن نتائج هذا البحث تلقى بعض الضوء علي فهم ميکانيکية عمل مستقبل الترانسفرين والتي يمکن الاستفادة منها في مقاومة الخلايا السرطانية.

SUMMARY

After ligation with transferrin (Tf), transferrin receptor (TfR) is aggregated on cell surface in clathrin coated pits and then internalized into the cell. In the present work, the mechanism of (Tf/TfR) and the role of Ceramide in this mechanism were studied. After Tf/TfR ligation, Ceramide level on plasma membrane outer leaflet was temporarily elevated as clarified by immunocytochemical detection of cell surface Ceramide in human "jurkat" T lymphoma cell line. Ceramide elevation was confirmed by Diacylgelycerol kinase assay for biochemical Ceramide measurement. The fast and brief Cer elevation was the result of acid sphingomyelinase activity, and no detection of neutral sphingomyelinase activity was noticed. Sphingomyelin synthase activity was obviously decreased in the same time frame of Ceramide generation, to maintain the elevated level of Ceramide. Inhibition of Ceramide generation by Imipramine, an acid sphingomyelinase inhibitor, or neutralization of the generated Ceramide by anti-Ceramide mAb lead to miss-internalization of transferrin, instead of being clathrin mediated it becomes, most probably, raft mediated as indicated by altered Tf recycling and cell fractionation studies. Miss-internalization of transferrin leads to abrogation of cell growth and finally apoptotic cell death.

Abbreviations: ASMase – Acid Sphingomyelinase; Cer – Ceramide; DGK - Diacylgelycerol kinase; NSMase – Neutral Sphingomyelinase; SM – Sphingomyelin; SMS – Sphingomyelin synthase; Tf – Transferrin; TfR – Transferrin receptor.

Key words: Transferrin, ceramide, sphingomyelin, acid sphingomyelinase, clathrin, jurkat cells.

Introduction

Transferrin (Tf) is an 80 kDa plasma glycoprotein secreted by the liver, function as iron carrier. When Tf is loaded with iron its binding capacity towards its membrane receptor, TfR is increased, then Tf/TfR complex is aggregated into clathrin coated pits. The receptor and the empty Tf is recycled back to the cell surface after discharge of iron, ready for another round of iron uptake (Huebers and Finch, 1987). The internalization and trafficking events of different receptors and agents via clathrin pathway are complex molecular machinery. Intensive studies were carried out to clarify the protein molecules chairing in this machinery. Recently lipid molecules attract the attention of the researchers as functioning molecules in clathrin endocytosis pathway (De Camilli et al., 1996; Roth, 1999; Di Paolo et al., 2004; Padron et al., 2006). The role of lipid molecules in this process was additionally clarified by inhibition or mutation in the lipid modifying enzymes leading to blocking of endocytosis events (Hughes and Parker, 2001; Du et al., 2003, 2004). Cells have the ability to direct different molecules to be internalized through different pathways and to different intracellular targets. For example TfR and T cell receptor (TCR) are in close and physical association to each others (Salmeron et al., 1995). Upon activation, TCR is aggregated into lipid rafts (Boniface et al, 1998; Monks et al., 1998) whereas; TfR is aggregated into clathrin coated pits. How cells control the movement of specific receptor or agent towards specific membrane domain, however still need to be clarified.

Hydrolysis of Sphingomyelin (SM) results in Cer releasing, which is catalyzed by three sphingomyelinases, acid, neutral and alkaline with the respective pH optimum activity (Kolesnick et al., 2000; Hannun and Obeid, 2008). Another pathway for Cer generation is by de novo synthesis, which involving serine-palmitoyl-CoA transferase as the key enzyme. Among sphingomyelinases, ASMase is the best characterized one; it was shown to be critically involved in many cellular activities (Gulbins and Kolesnick, 2003). ASMase is found in two forms, lysosomal and secretory that are both derived from the same gene but differ in their glycosylation pattern (Schissel et al., 1998). Cer is a fast moving lipid molecules has the tendency to spontaneous fusing with each others forming Cer platforms. Cer molecules in such platforms seem to be tightly backed with each others forming rather stable membrane domains (Veiga et al., 1999; Grassmé et al., 2001 a, b; Megha and London, 2004). This help in trapping and clustering receptor molecules in Cer rich platforms resulting in a high receptor density within a small area of cell membrane (Veiga et al., 1999; Megha and London, 2004). This high receptor density helps them to be in close association with their signaling molecule or to be excluded from undesirable molecule as phosphatases. Another study (Abdel Shakor et al., 2004) clarified that Cer acts as a driving force for Fc gamma RII to be trapped into SM and cholesterol rich membrane domain (membrane rafts). The immediate and fast generation of Cer is the character of ASMase activity whereas other sphingomyelinases seem to act slower (Gulbins and Kolesnick, 2003 and Dumitru and Gulbins, 2006). In the present study, it is shown that Cer is generated within seconds of Tf/TfR interaction, the generated Cer drive the movement of the receptor in the plane of the plasma membrane towards clathrin coated pits and away from raft trapping. The current work was designed to study the mechanism of TfR movement in the plane of the plasma membrane and the role of the lipid mediator Cer in this process.

Materials and methods

Materials

Transferrin Alexa fluor 488 conjugated and goat anti-mouse IgM Alexa fluor 546 were from Iinvitrogen. Anti lyn rabbit IgG from Santa Cruz Biotechnology, anti Cer IgM monoclonal MID15B4 from Alexis, SuperSignal West Pico chemiluminescent substrate (Pierce). C6-NBD-Cer and C6-NBD-SM from Avanti. All other chemicals, if not mentioned, were purchased from Sigma.

Cell culture, Tf treatment and measurement.

Human T lymphocytes, (Jurkat cell line) was routinely cultured in RPMI 1640 supplemented with 10% calf serum in 100% humidified atmosphere with 5% CO₂ at 37°C. After 48 h, inoculated cells were harvested and 2×10⁶ cells were kept in serum free medium for 30 min at 37°C for elimination of endogenous Tf. Cells were left as control or treated with 30 μM Imipramine, 10 μg/ml anti-Cer IgM MID15B4 mAb and 10 μg/ml irrelevant mouse IgM for 1 hr before Tf uptaking. Cells were treated for different time points at 37°C with 5 μg/ml of human Tf alexa fluor 488 conjugated. To stopping Tf/TfR movement and internalization, cells were washed trice with ice cold PBS (pH 7.2). Cells were fixed with 2% formaldehyde for 30 min at ambient temperature. For measurement of Tf uptaking, 2×10⁶ cells were processed for uptake of alexa fluor 488 conjugated Tf for 5 and 15 min. After fixation, cells washed once with acidic PBS (pH 4.5) to remove the membrane attached Tf. Immediately, cells were washed twice with normal PBS, lysed by 1% NP-40, in Tris buffer pH 7.2. After centrifugation at 5,000 rpm for 5 min, aliquots of supernatant were collected and measured for fluorescence intensity at excitation/emission 495/519 nm. Fluorescence intensity was presented as percentage of the highest mean value.

Cell surface Ceramide immunocytochemistry

Cells were processed for Tf uptaking for different time points as described above, cytospinned onto glass slides. Blocking of the active sites was done by 0.1% BSA for 30 min at room temperature. For visualization of cell surface Cer, cells were treated with 1 μg/ml anti-Cer IgM MID15B4 mAb for 1 h at room temperature followed by Goat anti mouse IgM Alexa fluor 546, conjugated. Cells were examined with a fluorescent microscopy (BX60-34FFB1; Olympus).

Measurements of ASMase and NSMase activities

Sphingomyelinases activities were assayed based on a previously described method (Okazaki et al., 1989 and Tanaka et al., 1996). Breively, cells (2.5x 10⁶) were homogenized in 1 ml lysis buffer containing 10 mM Tris-HCL (pH 7.5), 1 mM EDTA, 1% Triton X-100, 1 mM phenylmethyl-sulfonyl fluoride, 10 μg/ml pepstatin A and 2.5 μg/ml aprotenin. A breive sonication was done for ASMase activity determination. Cell lysates was centrifuged at 12,000 x g for 10 min at    4 °C, supernatant was used as enzyme source. The protein concentration was determined using a protein assay kit (Bio-Rad). 20 μg of protein was mixed with the reaction buffer for NSMase activity, including 100 mM Tris-HCL (pH 7.5), 10 mM MgCl₂, 5 mM dithiothreitol, 54 μM C6-NBD-SM and 0.1% Triton X-100 ( total volume 100μl). For ASMase activity, the protein was mixed with the reaction buffer including 100 mM acetate buffer (pH 5.0), 54 μM C6-NBD-SM  and 0.1 % Triton X-100 (total volume 100 μl). The mixure was incubated for 30 min at 37 °C, the reaction was stopped by adding 2 ml chloroform/methanol (2:1, v/v) and then 900 μl of H₂O and centrifuged at 3,000 x g for 5 min to separate the organic and aquas phases. The lower phase was collected and the solvent was evaporated. Aliquots were then applied to TLC plates (Whatman, LK6DF Silica gel) and NBD-lipids were resolved in a solvent system containing chloroform/methanol/12 mM MgCl₂ (65/25/4, v/v/v), the fluorescence was detected and quantified by image analyser, (LAS-2000, FujiFilm, Japan).

Measurement of SMS activity

SMS activity was measured as described previously (Yamaoka  et al., 2004) with minor modifications. 5 x 10⁶ cells were homogenized in lysis buffer containing 20 mM Tris-HCL (pH 7.5), 2 mM EDTA, 10 mM EGTA, 1 mM phenylmethyl-sulfonyl fluoride, 2.5 μg/ml leupeptin and 2.5 μg/ml aprotenin. Cells were left on ice for 10 min and then homogenised by passing 10 times through  27 gauge needle. The homogenates were centrifuged (2500 rpm, 5 min, 4 °C), the supernatants used as enzyme source. Supernatants (100 μg protein) were incubated with (20 μg) C6-NBD-Cer/phosphatidylcholine (1:10) liposomes in buffer containing 10 mM Tris-HCL (pH 7.5),1mM EDTA (in total volume 100 μl) for 60 min at 37 °C. Reaction was stopped by adding     2 ml chloroform: methanol (2:1, v/v) and then 900 μl of H₂O and centrifuged at 3000 rpm for 5 min to separate the two phases. The lower phase was collected and the solvent was evaporated. Aliquots were then applied to TLC plates and NBD-lipids were resolved in a solvent system containing chloroform/methanol/12 mM MgCl₂ (65/25/4, v/v/v), the fluorescence was detected and quantified, (LAS-2000, Fuji, Japan).

Measurment of Cer

Cer was quantified using diacylglycerol kinase assay (DGK) as described elswere (Bielawska et al., 2001). Briefly human holo Tf 5 μg/ml was added to 2.5 x 10⁶ jurkat cells in serum free rpmi 1640 medium for different time points. Total cellular lipids were extracted (Bligh and Dayer, 1959) and dried, then solubilized in 7.5 % octyl-β-D-glucose, 25 mM dioleolphosphatidylglycerol solution. Lipid micelles were mixed with the reaction buffer containing 50 mM Imidazol (pH 6.6), 5 mM LiCl, 12.5 mM MgCl₂, 1 mM EGTA (pH 6.6), 19.36 μM DTPA, 3.8 μg/μl DGK membrane from DGK-overexpressed E. coli,100 μM ATP and 4 μ Ci [γ-³²P] ATP (Amersham) in total 100μl volume.The mixture was incubated  at 37 °C for 30 min. Aliquots of extracted lipids were spotted on TLC plates and lipids were separated in a solvent system containing chloroform/acetone/methanol/acetic acid/water (10:4:3:2:1, v/v/v/v/v), radioactive lipids, were visualized and quantified by BAS2000 Image Analyzer (Fuji, Japan). Lipid spots corresponding to Cer-phosphate were identified compared with the known standards. Lipid phosphate level was measured compared with the phosphate level of linear concentrations of NaH₂PO₄ and then Cer levels were normalized to lipid phosphate levels and expressed as (pmol/nmol phosphate).

DAPI staining and apoptotic percentage

Subconfluent cells were washed twice with serum free medium, treated or not with Imipramine, anti-Cer mouse IgM mAb and irrelevant mouse IgM, reseeded at 2×10⁵ cells/ml and incubated at the same medium supplemented with 5 μg/ml human holo transferrin in 5% CO₂ at 37°C. Cells were processed for cellular nuclear chromatin changes detection by DAPI staining method, where cells were washed and fixed with 1% glutraldehyde for 30 min, then labelled with 2 μg/ml DAPI. Apoptotic percentage were caculated by counting 200 cells at least for each treatment for three independent expriments.

Cell fractionation

Cell fractionation was carried out using Triton X-100 cold lysis of cells, essentially as described (Korzeniowski et al., 2003). Cells (12.5 × 10⁶ per sample) treated or not with Imipramine as previously described were treated with 5 mg/ml human holo Tf for one min at 37 °C. Cells were lysed for 30 min at 0 °C in 200 ml of buffer containing 0.2% Triton X-100, 100 mM NaCl, 2 mM EDTA, 2 mM EGTA, 30 mM Hepes     (pH 7.5), phosphatase inhibitors (1 mM Na₃VO₄, 50 mM phenylarsine oxide, 30 mM p-nitrophenylphosphate) and a cocktail of protease inhibitors. Cells were further disrupted by repeated passages through an 18-Guage needle. After clarification (1.5 minute, 480 x g, 4°C), cell lysates (200 μl) were supplemented with 40% OptiPrep and 10% sucrose     (600 ml total volume) and the mixtures was transferred to centrifuge tubes. The samples were overlaid with 30%, 25%, 20%  (400 ml of each) and 0% OptiPrep (300 ml) in 0.2% TX-100 lysis buffer containing 10% sucrose, and centrifuged for 3 hours at 170,000 x g, 4°C (RCM 100 ultracentrifuge, Sorvall). Seven fractions of 300 ml were collected from the top of the gradient. Aliquots were subjected to SDS-page and immuno-detection of TfR and Lyn kinase were carried out.

Results

Tf enhances temporary Cer generation in the plasma membrane.

In the present study, early time points of Tf uptaking resulted in cell surface Cer generation. Cer generation was early and temporarily, it was obviously increased after one min of Tf addition and then Cer rendered to its normal level. Cer level tends to increase slightly again after ten min of Tf addition (Fig. 1 A, B). The elevation of Cer level upon Tf uptaking was accessed by immunocytochemical detection of cell surface Cer (Fig. 1A) and was confirmed biochemically by Diacylgelycerol kinase (DGK) assay (Fig. 1B).The kinetics of Tf uptaking and recycling was studied to determine at which stage Cer is produced. jurkat cells started to internalize Tf after two min of Tf addition, and Tf was stayed intracellular till the minute 5 and then the recycling back to the extracellular milieu after the minute 5 (Fig. 1C). Depending on these data, it is clear that Tf attached to cell surface for 1 min and during this time Cer is generated on cell surface before Tf internalization. Upon internalization of Tf, Cer was rapidly disappeared or metabolized. Theses data indicate that Cer might play a role in the movement of TfR in the plane of the plasma membrane.

Fig. 1: Temporary and breif Cer elevation upon Tf treatment as shown by immunocytochemical detection of cell surface Cer (A) and by DGK assay (B).Tf uptaking and recycling kinetics in jurkat cells (C).

Activation of ASMase and inhibition of SMS upon Tf uptaking

Looking for the enzymes responsible for Cer generation, ASMase and NSMase activities were investigated during time course of Tf uptaking. ASMase was found to be activated in the early time points of Tf addition to cells (Fig. 2A), reaching its maximum activity at one min and then gradually return almost to its basic level of activity. It seems that ASMase is the only SMase that responsible for Cer generation; hence NSMase had almost no change of its activity (Fig. 2A). SMS activity was decreased dramatically in the same time frame and then returns gradually to the basic level of activity (Fig. 2B). It is clear that Cer generation is occurred by ASMase early and brief activation together with inhibition of SMS activity at the same time to maintain Cer level without metabolizing by SMS. After this time point, Cer seems to be metabolized by the basic activity level of SMS giving SM.

Fig. 2:  Activation of ASMase but not NSMase (A) and downregulation of SMS activity upon Tf treatment (B).

Cer is important for Tf/TfR internalization through clathrin and avoiding rafts.

Tf was internalized and remains for relatively short time, almost 5 minutes (at least in jurkat cells) inside cells and then starts to be recycled back to the plasma membrane and then to the extracellular space after the minute 5 as indicated from the Tf uptaking and recycling kinetic curve (Fig. 1C). To investigate the role of Cer in Tf uptaking and/or recycling, abrogation of Cer generation by inhibition of ASMase activity with Imipramine, and neutralization of cell surface Cer by anti-Cer mAb was carried out. The amount of intracellular Tf was estimated at 5 min and 15 min (the uptaking and recycling times, respectively). The percentage of intracellular fluorescent Tf at 15 min was 89 and 100 of the highest mean fluorescence in anti-Cer and Imipramine treated cells, respectively compared with 18 and 31 in control and irrelevant Ab treated cells, respectively (Fig. 3A). Whereas, Tf internalization (5 min) was not affected under such treatment. This observation lent the attention to the probability that Tf missed its way to the recycling endosomes. For more analysis of Tf recycling alteration under Cer abrogation, TfR redistribution on cell surface following Tf ligation was studied.

It is well known that TfR is one of the receptors that did not trap into membrane microdomains, known as rafts; it is frequently used as non-rafts marker. TfR avoids rafts to be collected and internalized through clathrin machinery. After 1 min of Tf treatment, cell fractionation study of cold Triton, TX-100 cell lysis revealed that, in control cells, TfR mainly concentrated in non-rafts fractions number 4-7, and no detection of TfR could be found in lighter fractions “rafts fractions” number 1 and 2 (Fig. 3B). Inhibition of Cer generation by Imipramine leads to a clear shift of the main bands of TfR towards lighter fractions number 1 and 2 (Fig. 3B), these fractions represents raft fractions as indicated by Lyn tyrosine kinase, one of the raft markers. It seems that Cer generation is so important for TfR to avoid raft trapping and direction to clathrin-coated pits, its proper site of collection and internalization. Taking together, inhibition of Cer generation upon Tf/TfR interaction, leads to trapping of TfR into rafts and then most probably internalized through raft machinery. This result confirms the assumed role of Cer in controlling TfR movement in the plane of the plasma membrane, away from rafts and towards clathrin coated pits.

Fig. 3: Fluorescencet intinisty measurments of the intracellular Tf at 5 and 15 min of Tf treatment in control and after Cer abrogation, represented as the percentage of the highest mean fluorescence (A). Cell fractionation study showing the redistribution of TfR on cell surface after 1 min of Tf addition in control and in 30 μM Imipramine treated cells, raft fractions are indicated by Lyn, Tyrosine Kinase (B).

Internalization of Tf through non-clathrin machinery inhibits cell growth.

Tf is an important growth factor for cell growth and division, the importance of Tf as a growth factor is not restricted to its function as iron carrier, but Tf\TfR interaction has its own signaling pathway, which enhances cell growth and division. Hence TfR has its specific signaling and its specific entry pathways, it will be a matter of interest to investigate the role of Tf internalization pathway on cell growth, by other words, is clathrin an obligatory pathway for Tf to be a powerful growth factor? To address this question, DAPI staining (Fig. 4A) revealed that apoptotic cell death after 24 hr reached almost 50% in cells treated with Imipramine or anti-Cer Ab, compared with almost 6% of the control and irrelevant Ab treated cells (Fig. 4B). This result indicated that Tf internalization through clathrin machinery is important for its mitogenic effect; changing the entry pathway “as internalization through rafts” leads to the loss of Tf mitogenic effect and finally apoptotic cell death.

Fig. 4: Fluorescent microscopy of chromatin changes after 24 hrs  of cell inoculation using DAPI staining in control and in 30 μM Imipramine treated cells (A). Percentage of apoptotic cell death as determined from DAPI staining after 24 hrs inoculation of cells under different treatments of Cer inhibition, cell with unhomogenous nucleus staining was considered as apoptotic, 200 cells at least were counted for each treatment for three independent experiments (B).

Discussion

There are several mechanisms for internalizing different molecules from cell surface to various intracellular destinations. In general these mechanisms are clathrin coated-pits, non-clathrin coated pits, phagocytosis (for uptake of relatively large particles), caveolae/rafts, membrane ruffling and STEM formation (Surface-connected Tubules Entering Macrophages) (Maxfield and McGraw, 2004). It is still an open question in the field of membrane trafficking is how distinct ligands follow a specific internalization pathway to different intracellular targets.

 The current study through the light on a mechanism that may help in understanding the receptor movement on cell membrane and internalization through specific site of plasma membrane. The present data indicates that, when Tf attaches to its cell surface receptor, TfR, it takes about 1 min before internalization. Within the frame of this time, ASMase is briefly activated hydrolyzing cell surface SM, which is enriched in the membrane outer leaflet (Pomorski and Menon, 2006), giving rise to Cer. To maintain the level of Cer without metabolizing, SMS activity was decreased and then returns to its basic level of activation, returning Cer back to SM. The brief Cer generation upon TfR activation is so important to direct TfR towards clathrin coated pits and avoiding trapping into rafts.

ASMase is the best-characterized sphingomyelinase; it was shown to be involved in many forms of cellular activities. In addition, ASMase was shown to bind to the extracellular leaflet of the plasma membrane, however, it is still unknown whether this form of ASMase is a secretory, lysosomal or a third type of ASMase. Cer is a rapid moving lipid molecule; its release alters the biophysical characters of the membrane (Huang et al., 1996). Cer molecules are spontaneously associated and tightly bind to each others resulting in the formation of Cer-enriched membrane microdomains. These microdomains have the tendency to fuse again with each others forming Cer-enriched macrodomains (Grassmé et al., 2001a, b). Indeed, these Cer-enriched platforms are almost different from rafts (Megha, and London, 2004). Cer-enriched membrane platforms were shown to trap and cluster many receptors leading to sorting of specific proteins in a high density level for perfect signaling (Grassmé et al., 2003, Nurminen  et al., 2002 and Cremesti et al., 2001). The role of cell surface Cer generation in receptor movement and sorting seems to be used by many receptors and agents (Bionda et al., 2007, Grassmé et al., 2002, Göggel et al., 2004 and Pfeiffer et al, 2001). The mechanism followed by these receptors to be sorted and clustered is poorly understood. One study mentioned to the importance of the transmembrane region of the receptor that determine trapping or excluding from membrane domains (Bock and Gulbins, 2003). Another study clarified that cell surface Cer is generated in two different membrane domains; one near the receptor and the other is inside rafts. By the spontaneous moving and tight binding of Cer molecules, the two domains are bind to each other leading to trapping of receptor into rafts (Abdel Shakor et al., 2004). SM found in different subpools on plasma membrane (Linardic and Hannun, 1994).

TfR has been shown in direct and physical association with        T cell receptor and important for its activation (Batista et al., 2004). It is well known that TCR is clustered into rafts upon activation, but TfR is aggregated in clathrin coated pits and avoiding rafts. This two different compartmentalization of two receptors what are in close and physical association with each others gives the impression of the presence of specific mechanism that differentiates between the two receptor clustering pathways. In this study, Cer is suggested to be the driving lipid molecule that differentiates between raft and clathrin trapping.          

There is direct evidence that SM utilizes clathrin machinery upon cell entry (Koval and Pagano, 1990), but it is still unknown whether clathrin machinery differentiates between different SM pools. In other words, clathrin machinery uptake rafts or non-rafts SM or both. In recent studies (Masuyama et al., 2009 and Deinhardt et al., 2006), some cell surface proteins or attached agents already found in lipid rafts but its internalization is performed through clathrin machinery. this means that rafts or non-rafts SM can be internalized through clathrin machinery depending on the protein or agent to be internalized.

In the current study, the part of TfR that trapped into rafts upon Cer inhibition is most probably not internalized through clathrin coated pits because there was altered Tf recycling. The molecular mechanism followed by Cer to direct TfR towards clathrin pits and to avoid rafts however needs to be clarified. There was a detectable slight increasing of Cer level at 10 min after Tf addition to cells; this increasing may be according to a second round of Tf internalization after the fast recycling that occurs at 8-10 minutes. Tf should be internalized through clathrin machinery to recycle back to extracellular space for another round of internalization. Miss-internalization of Tf/TfR leads to accumulation of Tf inside cells without recycling as shown in the present study.

This accumulation of Tf inside cells leads to loss of mitogenic capacity of Tf and growth arrest and finally death. The loss of Tf mitogenic function is more probably not related to iron transportation, because cells can take up iron by different ways as low molecular weight iron chelates, non-receptor mediated Tf internalization and intercellular stores as ferritin, but however cells are in need of Tf/TfR for proliferation (Brock, 1981). It seems that Tf has an additional role unrelated to its iron-donating properties (Brock and Mainou – Fowler, 1983 and Bomford et al., 1983). Taken together, Tf/TfR interaction and their proper internalization pathway through clathrin machinery might itself provide some intracellular signaling that enables cell growth and division.

The present data clarify the role of early and brief Cer elevation in the direction of TfR towards clathrin coated pits and avoiding raft trapping, this role may be applicable for other receptors and agents that internalize via clathrin machinery.

Acknowledgment

Great appreciation to the group of cell biology, Faculty of Science, ZoologyDept.AlexandriaUniversity, Damanhour branch, Damanhour, Egypt for supplementation of DGK membranes.

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