A Pleckstrin Homology Domain Specific for Phosphatidylinositol 4,5-Bisphosphate (PtdIns-4,5-P2) and Fused to Green Fluorescent Protein Identifies Plasma Membrane PtdIns-4,5-P2 as Being Important in Exocytosis

Ronald W Holz,Michael D Hlubek,Scott D Sorensen,Stephen K Fisher,Tamas Balla,Shoichiro Ozaki,Glenn D Prestwich,Edward L Stuenkel,Mary A Bittner

doi:10.1074/jbc.m000925200

Abstract

Kinetically distinct steps can be distinguished in the secretory response from neuroendocrine cells with slow ATP-dependent priming steps preceding the triggering of exocytosis by Ca(2+). One of these priming steps involves the maintenance of phosphatidylinositol 4, 5-bisphosphate (PtdIns-4,5-P(2)) through lipid kinases and is responsible for at least 70% of the ATP-dependent secretion observed in digitonin-permeabilized chromaffin cells. PtdIns-4,5-P(2) is usually thought to reside on the plasma membrane. However, because phosphatidylinositol 4-kinase is an integral chromaffin granule membrane protein, PtdIns-4,5-P(2) important in exocytosis may reside on the chromaffin granule membrane. In the present study we have investigated the localization of PtdIns-4,5-P(2) that is involved in exocytosis by transiently expressing in chromaffin cells a pleckstrin homology (PH) domain that specifically binds PtdIns-4, 5-P(2) and is fused to green fluorescent protein (GFP). The PH-GFP protein predominantly associated with the plasma membrane in chromaffin cells without any detectable association with chromaffin granules. Rhodamine-neomycin, which also binds to PtdIns-4,5-P(2), showed a similar subcellular localization. The transiently expressed PH-GFP inhibited exocytosis as measured by both biochemical and electrophysiological techniques. The results indicate that the inhibition was at a step after Ca(2+) entry and suggest that plasma membrane PtdIns-4,5-P(2) is important for exocytosis. Expression of PH-GFP also reduced calcium currents, raising the possibility that PtdIns-4,5-P(2) in some manner alters calcium channel function in chromaffin cells.

Highlights

From the Departments of ‡Pharmacology and ¶¶Physiology, and the **Mental Health Research Institute, University of Michigan, Ann Arbor, Michigan 48109, the ‡‡Endocrinology and Reproduction Research Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892-4510, and the §§Department of Medicinal Chemistry, University of Utah, Salt Lake City, Utah 84112
Distinct steps can be distinguished in the secretory response from neuroendocrine cells with slow ATP-dependent priming steps preceding the triggering of exocytosis by Ca2؉
In the present study we have investigated the localization of PtdIns-4,5-P2 that is involved in exocytosis by transiently expressing in chromaffin cells a pleckstrin homology (PH) domain that binds PtdIns4,5-P2 and is fused to green fluorescent protein (GFP)

Summary

Introduction

From the Departments of ‡Pharmacology and ¶¶Physiology, and the **Mental Health Research Institute, University of Michigan, Ann Arbor, Michigan 48109, the ‡‡Endocrinology and Reproduction Research Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892-4510, and the §§Department of Medicinal Chemistry, University of Utah, Salt Lake City, Utah 84112. Distinct steps can be distinguished in the secretory response from neuroendocrine cells with slow ATP-dependent priming steps preceding the triggering of exocytosis by Ca2؉. One of these priming steps involves the maintenance of phosphatidylinositol 4,5bisphosphate (PtdIns-4,5-P2) through lipid kinases and is responsible for at least 70% of the ATP-dependent secretion observed in digitonin-permeabilized chromaffin cells. Two cytosolic factors that were necessary for ATP-dependent priming of exocytosis were identified as a phosphatidylinositol transfer protein [15] and phosphatidylinositol 4-phosphate kinase [16]. It has long been known that PtdIns 4-kinase, one of the key enzymes in PtdIns-4,5-P2 synthesis, is an integral

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Conclusion