PHOSPHOLIPASE C ACTIVITY IS REGULATED BY ARF6 DURING ACROSOMAL EXOCYTOSIS

PELLETAN, LEONARDO E; SUHAIMAN LAILA; VAQUER CINTIA C; MAYORGA LUIS S; BELMONTE, SILVIA A

Buenos Aires

Congreso; XLVIX Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular. SAIB; 2013

SAIB

The acrosome is a large membrane-limited granule that overlies the sperm nucleus. In response to physiological stimuli, sperm undergo exocytosis of this granule. This is a special type of calcium-dependent exocytosis termed the acrosome reaction (AR) which is a prerequisite for fertilization. By using biochemical, exocytosis assays and microscopy techniques we demonstrated that the small GTPase ARF6 is present in, localizes to the acrosomal region of, and is required for calcium and DAG-induced exocytosis in human sperm. Myristoylated and GTPgammaS loaded-ARF6 induces exocytosis in the absence of extracellular calcium and promotes the activation of phosphatidylinositol 4-phosphate 5-kinase leading to phosphatidylinositol 4,5-bisphosphate (PIP2) synthesis. The GTPase-induced AR relies on PLC activity and DAG production. We present direct evidence that active ARF6 increases PLC activity causing PIP2 hydrolysis. Consistent with this result, ARF6 promotes an IP3-dependent intraacrosomal calcium release required for AR. Challenging sperm with calcium or DAG boosts the exchange of GDP for GTP on ARF6, suggesting that both stimuli promote a GEF activity in sperm that activates recombinant ARF6 turning it into an AR inducer. ARF6 has a dual role in human sperm exocytosis inducing both the synthesis and the hydrolysis of PIP2 sitting at a critical point during the exocytotic cascade.