Interestingly, when blots of normal erythroid cells were reacted with the G/14C2 antibody only the 66?kDa band was observed, whereas no TFR2 band (95?kDa) was detected using the G/14E8 antibody in cell components derived from these cells (Number ?(Figure44A)

Interestingly, when blots of normal erythroid cells were reacted with the G/14C2 antibody only the 66?kDa band was observed, whereas no TFR2 band (95?kDa) was detected using the G/14E8 antibody in cell components derived from these cells (Number ?(Figure44A). Open in a separate window Figure 4 Western blotting analysis of TFR2 protein in cell extracts of normal erythroblasts, K562 and HepG2?cellsCellular lysates were electrophoresed, transferred to a PVDF membrane and probed with the anti-TFR2 antibodies G/14C2 and G/14E8. erythropoiesis, haemochromatosis, iron, liver, transferrin, Kira8 Hydrochloride transferrin receptor Abbreviations: AML, acute myeloid leukaemia; BFU-E, burst-forming unit-erythroid; HFE, haemochromatosis gene product; mAb, monoclonal antibody; PNGase F, peptide N-glycanase F; RTCPCR, reverse transcriptaseCPCR; TFR(1/2), transferrin receptor 1/2; TRITC, tetramethylrhodamine -isothiocyanate Intro TFR2 (transferrin receptor 2) is definitely a new member of the TFR family fortuitously cloned in 1999 during an attempt to identify fresh transcription factors [1]. Two transcripts of these genes were indicated: and . The expected amino acid sequence of the TFR2- protein revealed that this is a type II membrane protein, sharing 45% identity and 66% similarity in its extracellular website with TFR1 [1]. The TFR2- protein lacked the N-terminal portion of the TFR2- protein including the transmembrane website [1]. Studies possess demonstrated the expressed TFR2 protein mediates transferrin-iron uptake into the cells, but has an affinity for transferrin that is 30-fold lower than TFR1 [2]. Unlike TFR1, TFR2 mRNA lacks iron regulatory elements, and TFR2 manifestation may be controlled from the cell cycle rather than by intracellular iron status [3]. Additional variations between TFR1 and TFR2 concern mRNA cells distribution, as evaluated both by SACS Northern blot analysis and by RTCPCR (reverse transcriptaseCpolymerase chain reaction). Using these techniques, it was demonstrated the TFR2 mRNA is definitely recognized mainly in the liver and, among a large panel of cell lines, only in the K562 erythroleukaemia cell collection and HepG2 hepatoblastoma cells [1]. Additional studies have shown high levels of TFR2 manifestation in early erythroid cells and in main leukaemic blasts, mostly derived from the FAB M6 erythroleukaemia subtype [4,5]. Finally, TFR2 manifestation was also observed in the small intestine, although only at the level of crypt cells [6]. The function of TFR2 appears to be different from that of TFR1. In fact, TFR1 knockout mice did not survive beyond embryonic day time 12.5 because of severe anaemia and neurological abnormalities, which clearly indicates that murine TFR2 cannot fully compensate for the functions of TFR1 [7]. Moreover, mice with only one practical TFR1 allele show a phenotype associated with slight tissue-iron depletion, whereas disabling mutations of the TFR2 gene result in haemochromatosis type-3, a genetic form of iron overload exhibiting a medical picture much like HFE (haemochromatosis gene product)-connected hereditary haemochromatosis, including hepatic iron loading [8C10]. Furthermore, target mutagenesis of the murine TFR2 gene generates haemochromatosis, characterized by periportal hepatic iron loading [11]. These observations clearly show that TFR2 is definitely involved in iron homoeostasis under physiological conditions. This conclusion is also supported by a recent study showing a co-localization of TFR2 and HFE in crypt duodenal cells [6]. Accordingly, it was suggested that TFR2 may function as an iron-sensor mechanism. Studies of the TFR2 protein have been limited by the lack of specific reagents. Recently, antibodies specifically reacting with the human being TFR2 have been reported [12,13]. These reagents were clearly useful in terms of determining more precisely Kira8 Hydrochloride the pattern of cells distribution of this receptor, which seems to be limited to hepatocytes, crypt duodenal cells and erythroleukaemia cells [12], and its subcellular Kira8 Hydrochloride localization, showing the receptor is definitely localized to the cell membrane and to some punctate perinuclear subcellular compartments, seemingly related to endocytic vesicles [12,13]. In the present study we have characterized the pattern of manifestation of the TFR2 protein in normal erythroid cells. To perform these studies, we took advantage of the availability of a large panel of anti-TFR2 mAbs (monoclonal antibodies) developed by some of the present authors [12]. Our results have shown that, in normal erythroid cells, TFR2 is definitely indicated at low levels at.