Arf6: a key molecule regulating acrosomal exocytosis

PELLETÁN LEONARDO, MAYORGA LUIS AND SILVIA A. BELMONTE

Mar del Plata, Buenos Aires, Argentina

Congreso; XLIII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular. SAIB; 2007

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular, SAIB

Sperm acrosome reaction (AR) is a special type of Ca++-regulated exocytosis. Our previous results suggested that AR needs phosphatidylinositol 4,5-bisphosphate(PIP2) and phosphatidic acid(PA) after the opening of SOC channels. ADP-ribosylation factors (Arfs) are a family of monomeric GTP-binding proteins. In chromaffin cells, Arf6 activates both PI4P5Kinase and PLD1; both enzymes are thought to be coupled during signaling to support simultaneous increases in PIP2 and PA. We demonstrated that anti-Arf6 antibodies abrogated Ca++-triggered AR. This inhibition is reversed by PIP2 remarking that Arf6 and PIP2 are essential molecules in AR. To study the role of Arf6 in AR, we co-transformed E. coli with plasmids encoding 6His-Arf6 and N-myristoyltransferase, to obtain myristoylated Arf6.  Recombinant Arf6 loaded with GDPbS inhibits Ca++-triggered AR confirming its involvement in the calcium pathway. Surprisingly, Arf6 activated with GTPgS induced AR per se in permeabilized cells. No effect was observed when cells were challenged with Arf6GDPbS or non-myristolated Arf6. Arf6GTPgS-induced AR was abrogated by anti-PLD antibodies or the PLCPH domain (specifically binds PIP2) suggesting that Arf6 is upstream PLD1 and PIP2 production probably regulating their function and production, respectively. Our results constitute the first evidence suggesting a key role of Arf6 in regulating AR