Abstract
Flotillins are the major structural proteins in erythroid raft domains. We have shown previously that the dynamic nanoscale organization of raft domains in erythroid cells may depend on flotillin-MPP1 interactions. Here, by using molecular dynamic simulations and a surface plasmon resonance-based approach we determined that high-affinity complexes of MPP1 and flotillins are formed via a so far unidentified region within the D5 domain of MPP1. Significantly, this particular “flotillin binding motif” is of key physiological importance, as overexpression of peptides containing this motif inhibited endogenous MPP1-flotillin interaction in erythroid precursor cells, thereby causing lateral disorganization of raft domains. This was reflected by both reduction in the plasma membrane order and markedly decreased activation of signal transduction via the raft-dependent insulin receptor pathway. Our data highlight new molecular details concerning the mechanism whereby MPP1 functionally links flotillins to exert their physiological role in raft domain formation.
Highlights
Flotillins are the major structural proteins in erythroid raft domains
Using MPP1 knockdown erythroid precursor HEL cells and giant plasma membrane vesicles (GPMVs) derived from them, we demonstrated that the marked decrease of MPP1 protein expression is directly associated with significant changes in physicochemical properties of the plasma membrane monitored as an increase in membrane fluidity parameters and phase-separation properties[23,24]
Our previous study showed that direct interaction between MPP1 and flotillins exists in the plasma membrane of red blood cells (RBCs) and functionally contributes to the organization of membrane rafts in living erythroid cells[23,25]
Summary
Flotillins are the major structural proteins in erythroid raft domains. We have shown previously that the dynamic nanoscale organization of raft domains in erythroid cells may depend on flotillin-MPP1 interactions. By using molecular dynamic simulations and a surface plasmon resonance-based approach we determined that high-affinity complexes of MPP1 and flotillins are formed via a so far unidentified region within the D5 domain of MPP1 This particular “flotillin binding motif” is of key physiological importance, as overexpression of peptides containing this motif inhibited endogenous MPP1-flotillin interaction in erythroid precursor cells, thereby causing lateral disorganization of raft domains. The membrane-organizing capacity assigned to flotillins is due to their ability to form o ligomers[28] that serve as active assembly sites controlling numerous different cellular processes such as s ignaling29–32, endocytosis[33,34], and cell a dhesion[35] These features emphasized flotillins as preferable molecular candidates for interacting with MPP1 in the context of plasma membrane lateral organization. A recombinant protein corresponding to this domain via inhibiting this interaction reduced membrane order and markedly decreased activation of signal transduction via the raft-dependent insulin receptor pathway
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