Indeed, the quick phase of alkalinization was totally abolished, confirming the inhibition of HCO3/Clexchange. proteins into the 4.1R complex of the human red cell membrane. Keywords:erythrocyte membrane, macromolecular complex, 4.1R, Kell protein, blood group antigens == Introduction == Protein 4.1R is a major protein of the erythrocyte skeleton and is composed Vinorelbine (Navelbine) of four functional domains: the N-terminal 30 kDa domain name, referred to as the FERM (F for 4.1R protein, E for ezrin, R for radixin and M for moesin) domain, the 16 kDa domain, the 10 kDa spectrin-actin binding (SAB) domain and the C-terminal 24 kDa domain (CTD) (Conboy, et al1986,Takakuwa 2000). The analysis of the crystal structure of the FERM domain led to the identification of three globular lobes. Lobe A corresponds to the first 90 amino acids and includes the binding sites for band 3 and the Na+/H+exchanger (NHE1). Lobe B (amino acids 91190) contains the binding sites for GPC/D, XK and DARC (Duffy antigen receptor for chemokines, also termed ACKR1) proteins. The COOH-terminal lobe (Lobe C, amino acids 191280) contains the binding site for p55 protein. Recently,Gauthier,et al(2011)proposed that this Kell protein is present in the 4.1R complex through its conversation with XK. Indeed, XK protein is covalently linked to the Kell glycoprotein by a single disulfide bond (XK Cys347Kell Cys72) (Russo, et al1998). The Kell glycoprotein (93 kDa) is usually a type II single-span membrane protein that carries the Kell blood group system comprising 28 unique antigens (Ji,et al, 2015) including the K1 (Kell) and K2 (cellano) antigens. The two distinct proteins, Kell and XK are responsible for expressing the Kell blood group antigens (Lee, et al2001). Kell protein exhibits an ectodomain that is composed of two domains: the well-conserved membrane-proximal zinc endopeptidase domain name and a more variable membrane-distal domain name (Lee, et al2003). In addition, the Kell protein shares a consensus sequence with the large family of zinc endopeptidases and has endothelin-3 transforming enzyme activity of type II membrane glycoproteins. Gene disruption in mice provided evidence that cellular divalent cation regulation is functionally coupled to the Kell/XK system in erythrocytes and loss of this complex might contribute to the acanthocytosis seen in McLeod syndrome (Rivera, et al2013). A rare phenotype termed Kellnull(Ko) is usually characterized by the absence of Kell protein and Kell antigens from your reddish cell membrane and diminished amounts of XK protein (Khamlichi, et al1995,Redman, et al1999). The absence of any clinical symptoms in Kellnullindividuals suggest that the Kell enzyme activity is not essential for cell survival or that other metalloproteinase could compensate the lack of this protein (Lee, et al2001). The findings from the present study using 4.1() HE (4.1Rnull) human erythrocytes have enabled us to obtain novel insights into the 4.1R complex organization. Indeed, we describe a detailed novel direct conversation involving the skeletal protein 4.1R and the Kell blood group protein. Furthermore, the functional activities of AQP1, Band 3 and RhAG were measured in the 4.1() HE erythrocyte membrane and we show a decrease in the extent of anion exchange. == Materials and methods == == Materials == Primers used in polymerase chain reaction (PCR) and mutagenesis experiments were provided from Eurogentec (Seraing, Belgium). The QuikChange Site-Directed Mutagenesis Kit was from Stratagene (La Jolla, CA, USA). The Protease Inhibitor Cocktail, the pGEX-3X-5 vector and the glutathione-sepharose 4B beads were purchased from Amersham Vinorelbine (Navelbine) Pharmacia Biotech, (Buckinghamshire, UK). NuPAGE Novex Bis-Tris Gels were purchased from Invitrogen (Carlsbad, CA, USA). The Band 3 inhibitor, DIDS (4,4 2-diisothiocyanatostilbene-2,2 2-disulfonic acid disodium salt), was purchased Vinorelbine (Navelbine) from Sigma-Aldrich (St Louis, MO, USA). == Blood samples == Frozen reddish blood cell (RBC) samples from three healthy donors (normal), one 4.1R() one AQP1nulland one Rhnullindividual were obtained from the Centre National de Rfrence H3FL pour les Groupes Sanguins (CNRGS, Paris, France). The patient with 4.1R()has been previously reported (Tchernia, et al1981). == Antibodies == Murine monoclonal antibodies (MAbs) were as follows: anti-Kell (clone 5A11) directed toward the common, intracellular, amino-terminal region of the protein (Jaber, et al1991); anti-GPC (1F6).