Abstract
Lutheran (Lu) blood group and basal cell adhesion molecule (B-CAM) antigens reside on two glycoprotein (gp) isoforms Lu and Lu(v13) that belong to the Ig superfamily and differ only by the size of their cytoplasmic tail. Lu/B-CAM gps have been recognized as laminin alpha5 receptors on red blood cells and epithelial cells in multiple tissues. It has been shown that sickle red cells exhibit enhanced adhesion to laminin alpha5 when intracellular cAMP is up-regulated by physiological stimuli such as epinephrine and that this signaling pathway is protein kinase A- and Lu/B-CAM-dependent. In this study, we analyzed the relationship between the phosphorylation status of Lu/B-CAM gps and their adhesion function to laminin alpha5. We showed that Lu isoform was phosphorylated in sickle red cells as well as in erythroleukemic K562 and epithelial Madin-Darby canine kidney cells and that this phosphorylation is enhanced by different stimuli of the PKA pathway. Lu gp is phosphorylated by glycogen synthase kinase 3 beta, casein kinase II, and PKA at serines 596, 598, and 621, respectively. Alanine substitutions of serines 596 and 598 abolished phosphorylation by glycogen synthase kinase 3 beta and casein kinase II, respectively, but had no effect on adhesion of K562 cells to laminin under flow conditions. Conversely, mutation of serine 621 prevented phosphorylation by PKA and dramatically reduced cell adhesion. Furthermore, stimulation of K562 cells by epinephrine increased Lu gp phosphorylation by PKA and enhanced adhesion to laminin. It is postulated that modulation of the phosphorylation state of Lu gp might be a critical factor for the sickle red cells adhesiveness to laminin alpha5 in sickle cell disease.
Highlights
Lutheran (Lu)1 blood group and basal cell adhesion molecule (B-CAM) antigens are both carried by two glycoprotein isoforms, Lu and Lu(v13), that belong to the Ig superfamily and differ only by the size of their cytoplasmic tail (59 versus 19 amino acids)
We demonstrated that Lu gp but not Lu(v13) isoform is phosphorylated by glycogen synthase kinase 3  (GSK-3), casein kinase II (CKII), and PKA, at serines 596, 598, and 621, respectively
Epinephrine Stimulates Adhesion of K562 recombinant cells expressing Lu gp (K562-Lu) but Not K562Lu(v13) to Laminin ␣5 under Flow Conditions—Since Lu gp exhibited enhanced phosphorylation in K562 cells when treated with br-cAMP, forskolin, or epinephrine, we examined the effects of epinephrine on K562 cell adhesion to laminin ␣5 as it is the major mediator of the physiologic stress response known to elevate cAMP levels [33]
Summary
Lutheran; B-CAM, basal cell adhesion molecule; gp, glycoprotein; PKA, protein kinase A; CKII, casein kinase II; GSK-3, glycogen synthase kinase 3 ; MDCK, Madin-Darby canine kidney; mAb, monoclonal antibody; br-cAMP, 8-bromo-cAMP; WT, wild-type; GST, glutathione S-transferase. The Lu/B-CAM gps represent the unique red cell receptors for laminin ␣5 in normal and in sickle red blood cells [3,4,5]. It was recently shown that the physiologic stress mediator epinephrine, acting through the 2-adrenergic receptor, increased the adhesion of sickle red blood cells to laminin ␣5 via a cAMP and PKA-dependent signaling pathway [25]. Lu/B-CAM gps were identified as receptors that mediate the stimulated adhesion of sickle red cells to laminin ␣5 under continuous flow conditions [26]. This signaling pathway may participate in vaso-occlusion events. We demonstrated that PKA-mediated phosphorylation of the Lu gp at Ser-621 positively regulates the adhesion function of Lu gp under flow conditions
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