| Summary: | The plasma membrane of sperm contains highly dynamic lipid microdomains (rafts), which house signaling proteins with a role in regulating capacitation. We reported that ATP1A4, the testis-specific isoform of Na/K-ATPase, interacted with caveolin-1, Src, epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinases 1/2 (ERK1/2) in raft and non-raft domains of the plasma membrane of bovine sperm during capacitation. The objective of the present study was to use a proteomic approach to characterize the ATP1A4 interactome in rafts and non-rafts from capacitated bovine sperm. The non-raft interactome included hexokinase 1, plakophilin 1, desmoglein 1, 14-3-3 protein ζ/δ, cathepsin D and heat shock protein beta1 proteins exclusively, whereas glutathione <i>S</i>-transferase and annexin A2 were unique to raft interactome. However, a disintegrin and metalloprotease 32 (ADAM 32), histone H4, actin, acrosin, serum albumin and plakoglobin were identified in both raft and non-raft fractions of capacitated sperm. Based on gene ontology studies, these differentially interacted proteins were implicated in cell−cell adhesion, signal transduction, fertilization, metabolism, proteolysis and DNA replication, in addition to acting as transport/carrier and cytoskeletal proteins. Overall, we identified proteins not previously reported to interact with ATP1A4; furthermore, we inferred that ATP1A4 may have a role in sperm capacitation.
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