Tags

Type your tag names separated by a space and hit enter

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.
J Biol Chem. 2000 Jun 09; 275(23):17878-85.JB

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.

Authors+Show Affiliations

Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109, USA. holz@umich.eduNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

10747966

Citation

Holz, R W., et al. "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." The Journal of Biological Chemistry, vol. 275, no. 23, 2000, pp. 17878-85.
Holz RW, Hlubek MD, Sorensen SD, et al. 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. J Biol Chem. 2000;275(23):17878-85.
Holz, R. W., Hlubek, M. D., Sorensen, S. D., Fisher, S. K., Balla, T., Ozaki, S., Prestwich, G. D., Stuenkel, E. L., & Bittner, M. A. (2000). 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. The Journal of Biological Chemistry, 275(23), 17878-85.
Holz RW, et al. 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. J Biol Chem. 2000 Jun 9;275(23):17878-85. PubMed PMID: 10747966.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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. AU - Holz,R W, AU - Hlubek,M D, AU - Sorensen,S D, AU - Fisher,S K, AU - Balla,T, AU - Ozaki,S, AU - Prestwich,G D, AU - Stuenkel,E L, AU - Bittner,M A, PY - 2000/4/5/pubmed PY - 2000/7/25/medline PY - 2000/4/5/entrez SP - 17878 EP - 85 JF - The Journal of biological chemistry JO - J. Biol. Chem. VL - 275 IS - 23 N2 - 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. SN - 0021-9258 UR - https://www.unboundmedicine.com/medline/citation/10747966/A_pleckstrin_homology_domain_specific_for_phosphatidylinositol_4_5_bisphosphate__PtdIns_45_P2__and_fused_to_green_fluorescent_protein_identifies_plasma_membrane_PtdIns_45_P2_as_being_important_in_exocytosis_ L2 - http://www.jbc.org/cgi/pmidlookup?view=long&pmid=10747966 DB - PRIME DP - Unbound Medicine ER -