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© The Rockefeller University Press, 0021-9525/2003/8/647 $5.00
The Journal of Cell Biology, Volume 162, Number 4, 647-659

ARF6 regulates a plasma membrane pool of phosphatidylinositol(4,5)bisphosphate required for regulated exocytosis

Department of Biochemistry, University of Wisconsin, Madison, WI 53706

Address correspondence to Thomas F.J. Martin, Dept. of Biochemistry, University of Wisconsin, 433 Babcock Drive, Madison, WI 53706. Tel.: (608) 263-2427. Fax: (608) 262-3453. email: tfmartin{at}facstaff.wisc.edu

ADP-ribosylation factor (ARF) 6 regulates endosomal plasma membrane trafficking in many cell types, but is also suggested to play a role in Ca²⁺-dependent dense-core vesicle (DCV) exocytosis in neuroendocrine cells. In the present work, expression of the constitutively active GTPase-defective ARF6^Q67L mutant in PC12 cells was found to inhibit Ca²⁺-dependent DCV exocytosis. The inhibition of exocytosis was accompanied by accumulation of ARF^Q67L, phosphatidylinositol 4,5-bisphosphate (PIP₂), and the phosphatidylinositol 4-phosphate 5-kinase type I (PIP5KI) on endosomal membranes with their corresponding depletion from the plasma membrane. That the depletion of PIP₂ and PIP5K from the plasma membrane caused the inhibition of DCV exocytosis was demonstrated directly in permeable cell reconstitution studies in which overexpression or addition of PIP5KI restored Ca²⁺-dependent exocytosis. The restoration of exocytosis in ARF6^Q67L-expressing permeable cells unexpectedly exhibited a Ca²⁺ dependence, which was attributed to the dephosphorylation and activation of PIP5K. Increased Ca²⁺ and dephosphorylation stimulated the association of PIP5KI with ARF6. The results reveal a mechanism by which Ca²⁺ influx promotes increased ARF6-dependent synthesis of PIP₂. We conclude that ARF6 plays a role in Ca²⁺-dependent DCV exocytosis by regulating the activity of PIP5K for the synthesis of an essential plasma membrane pool of PIP₂.

Key Words: PIP₂; PIP5K; DCV; ARF; Ca²⁺-dependent exocytosis

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