Adhesive activity of Lu glycoproteins is regulated by interaction with spectrin

Abstract

The Lutheran (Lu) and Lu(v13) blood group glycoproteins function as receptors for extracellular matrix laminins. Lu and Lu(v13) are linked to the erythrocyte cytoskeleton through a direct interaction with spectrin. However, neither the molecular basis of the interaction nor its functional consequences have previously been delineated. In the present study, we defined the binding motifs of Lu and Lu(v13) on spectrin and identified a functional role for this interaction. We found that the cytoplasmic domains of both Lu and Lu(v13) bound to repeat 4 of the ␣ spectrin chain. The interaction of fulllength spectrin dimer to Lu and Lu(v13) was inhibited by repeat 4 of ␣-spectrin. IntroductionThere is mounting interest in the 2 Lutheran (Lu) red cell transmembrane glycoprotein isoforms that serve as receptors for extracellular matrix laminins. Current evidence indicates that Lu-laminin binding contributes to sickle cell vaso-occlusion. [1][2][3][4][5] Lu-dependent sickle red blood cell adhesion appears to involve epinephrine and cyclic adenosine monophosphate activation, supporting the novel concept that inside-out signaling mechanisms may activate red cell adhesion molecules. 6 Moreover, polycythemia vera red blood cells (RBCs) demonstrate increased adherence to vascular endothelium also mediated by Lu-laminin binding, suggesting that this interaction may contribute to the increased thrombosis observed in this myeloproliferative disorder. 7 Lu first appears on the erythroblast surface late in differentiation, 8 and circulating erythrocytes express 1500 to 4000 copies per cell. 9,10 However, Lu expression is not limited to red cells; the isoforms are also present on vascular endothelial cells and epithelial cells in multiple tissues. 11 Intriguing recent data show that Lu expression is enhanced in various carcinomas and during malignant transformation of epithelial cells, pointing to a possible role in cancer biology. [12][13][14][15] To better understand Lu receptor function(s) in both normal and pathologic states, we are investigating the structural interactions of these transmembrane proteins.The 2 Lu isoforms (85 and 78 kDa) 16 are members of the immunoglobulin superfamily (IgSF). 11 The 85-kDa Lu glycoprotein contains 5 disulfide-bonded extracellular IgSF domains, a single hydrophobic membrane span, and a cytoplasmic domain of 59 residues. 11 Its cytoplasmic tail may function in intracellular signaling and polarization to plasma membrane, as suggested by the presence of the consensus motif for binding of Src homology 3 (SH3) domains, 5 potential phosphorylation sites, 11 and a dileucine motif responsible for regulating basolateral localization of Lu in polarized epithelial cells. 17 The 78-kDa isoform (termed Lu(v13) 18 or B-CAM 13 ), generated by alternative splicing of intron 13, differs from the larger form by having a truncated cytoplasmic tail lacking the proline-rich SH3-binding domain, the dileucine motif, and the 5 phosphorylation sites. 18 Erythrocyte membranes contain 5-to 10-fold more Lu than Lu(v13). 17...

JAK2V617F activates Lu/BCAM-mediated red cell adhesion in polycythemia vera through an EpoR-independent Rap1/Akt pathway

“…38 Lu/BCAM adhesion function could be activated by the phosphorylation of its serine 621 12 or by the dissociation of its cytoplasmic domain from the spectrin-based skeleton. 39,40 In sickle cell disease, RBCs exhibit an abnormal cAMP-dependent Lu/BCAM phosphorylation 12,13 associated with high adhesion to laminin. 13,14 This adhesion seems to involve protein kinase A 14 or Rap1.…”

Novel role for the Lu/BCAM–spectrin interaction in actin cytoskeleton reorganization

“…To date, the Lu/BCAM laminin 511/521 receptor function was known to be activated by two pathways in pathological RBCs: one involving the phosphorylation of its cytoplasmic domain by PKA [2]; and the other mediated by Lu/BCAM cytoplasmic domain interaction with the spectrin-based cytoskeleton [15,18]. Our present study indicates that interaction between Lu/BCAM and spectrin regulates epithelial cell adhesion and spreading to laminin 511/521.…”

“…Lu/BCAM is involved in the abnormal adhesion of red cells to laminin 511/521 and endothelium in pathological situations [2,5,16,17]. We and others have demonstrated that the interaction of Lu/BCAM with erythroid spectrin negatively regulated its adhesive receptor function in normal and spectrin-deficient HS RBCs [15,18]. In non-erythroid adherent cells, partial DII-spectrin depletion was associated with loss of cell spreading, defective adhesion and decrease and irregularity of focal adhesion points [32].…”

“…The Lu/BCAM cytoplasmic domain binds to DI-spectrin D4 repeat (D4R) in RBCs [15]. Sequence analyses revealed that the ĮII-spectrin D4R (residues E361 to E486) shares 57% of identity with its ĮI-spectrin corresponding repeat.…”

“…Alteration of the spectrin-based skeleton as well as disruption of the Lu/BCAM-spectrin interaction resulted in an enhanced Lu/BCAM-mediated adhesion of RBCs and K562 cells to laminin 511/521 [15,18]. To investigate whether the interaction with spectrin could similarly modulate Lu/BCAMmediated adhesion of epithelial cells, we analyzed the spreading and adhesion to laminin 511/521 of MDCK cells expressing either wt-Lu or mt-Lu as described under "Experimental" (Fig.…”