Phosphorylation-Dependent Perturbations of the 4.1R-Associated Multiprotein Complex of the Erythrocyte Membrane

Abstract

The bulk of the red blood cell membrane proteins are partitioned between two multiprotein complexes, one associated with ankyrin R, the other with protein 4.1R. Here we examine the effect of phosphorylation of 4.1R on its interactions with its partners in the membrane. We show that activation of protein kinase C in the intact cell leads to phosphorylation of 4.1R at two sites, serine-312 and serine-331. This renders the 4.1R-associated transmembrane proteins GPC, Duffy, XK and Kell readily extractable by nonionic detergent with no effect on the retention of band 3 and Rh, both of which also interact with 4.1R. In solution, phosphorlyation at either serine suppresses the capacity of 4.1R to bind to the cytoplasmic domains of GPC, Duffy and XK. Phosphorylation also exerts an effect on the stability in situ of the ternary spectrin-actin-4.1R complex, which characterizes the junctions of the membrane skeletal network, as measured by the enhanced competitive entry of a β-spectrin peptide possessing both actin- and 4.1R-binding sites. Thus phosphorylation weakens the affinity of the 4.1R for β-spectrin. The two 4.1R phosphorylation sites lie in a domain flanked in the sequence by the spectrin- and actin-binding domain and a domain containing the binding sites for transmembrane proteins. It thus appears that phosphorylation of a regulatory domain in 4.1R results in structural changes transmitted to the functional interaction centers of the protein. We consider possible implications of our findings to altered membrane function of normal reticulocytes and sickle red cells.

Antibody-Mediated Immune Suppression of Erythrocyte Alloimmunization Can Occur Independently from Red Cell Clearance or Epitope Masking in a Murine Model

“…In fact, studies done many years ago showed that AMIS could occur across the Rh Kell boundary (9). Although this was originally thought to be a good indication that red cell clearance was involved, in fact we know today that the RhD and Kell transmembrane proteins are part of the protein 4.1R complex and are therefore in very close proximity to each other (54).…”

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“…Changes in the protein phosphorylation state can modify protein-protein interactions and thereby multiprotein complex formation and/or functions. 37,38 Previous studies documented that the Syk-Lyn signaling pathway working in concert can modify band 3 function in healthy RBCs. 27 It is noteworthy that we found increased association of active Lyn with the ChAc membranes in the absence of Syk.…”

Equilibrium physics breakdown reveals the active nature of red blood cell flickering

“…Biochemists identified several phosphorylation sites directly in the network: at the transmembrane protein complexes 4.1R (refs 23,39,40 ) and ankyrin 41 , which anchor the network in the bilayer, but also at the spectrin chains 22 . These phosphorylations have been associated with a decreased mechanical strength of the membrane 42,23,43,6,15 , but the underlying molecular mechanism remains unclear.…”