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Thomas Jefferson University - James H. Keen, Ph.D.
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James H. Keen, Ph.D.
Biochemistry and Molecular Biology
Thomas Jefferson University
Jefferson Medical College
Professor, Department of Biochemistry and Molecular Biology
Dean, Jefferson College of Graduate Studies
Member, Cancer Cell Biology and Signaling Program
Kimmel Cancer Center
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Mailing Address
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233 S 10th St; BLSB/915
Philadelphia, Pennsylvania 19107
United States
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Expertise and Research Interests
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Coordinate control of exocytosis and endocytosis is known to exist, ascells maintain a relatively constant surface area despite large variationin the rates of vesicular membrane addition tot he plasma membrane. Thepathways of against activation of G-proteins that result inphosphatidylinositol-4,5-bisphosphate PIP2 cleavage and calciummobilization, and the latter's involvement in stimulated secretion arewell document. However little is known about the mechanism by whichendocytosis is tightly coupled to the rate of secretion. Retrieval ofmembrane is carried out by receptor-mediated endocytosis through coatedpits and vesicles. The structural proteins of the plasma membrane coatstructure include clathrin and AP-2, an assembly adaptor or associatedprotein. Studies in this laboratory have recently identified a discretehigh affinity Kd-10-8 M binding site on the alpha subunit of AP-2 thatrecognized polyphosphoinositols PPIs in the context of a lipid bilayer. Occupancy of this site inhibits clathrin binding and coat assembly by AP- 2, indicating that this is likely to be a regulatory site of majorphysiological significance. This finding, and observations in theliterature which indicate that increases in coated membrane assembly atthe plasma membrane are accompanied by increases in PIP2 cleavage, leadto two working hypotheses: 1 that PIP2 located predominantly orexclusively in the cytoplasmic leaflet of the plasma membranecontributes to the recruitment or retention of AP-2 at the plasmamembrane; 2 that PIP2 regulates the clathrin coat assembly activity ofAP-2 at the plasma membrane. The experiments proposed here are designedto localize the site of PPI binding on the AP-2 a subunit usingradiolabeled photoaffinity probes, peptide fractionation and anti-peptideantibodies. Subsequent site-specific mutagenesis and in vitrotranscriptiontranslation experiments will identify mutant forms of aAthat retain clathrin binding activity but lack functional PPI sites. Transient expression of mutant and wild type proteins in mammalian cellswill then be used to determine the role of the PPI site in recruitmentof AP-2 to the plasma membrane, in coated pit assembly and in receptor- mediated endocytosis. To evaluate the linkage in cells between PIP2cleavage and plasma membrane coated pit formation, quantitative time- course and dose-response changes in the levels of assembled clathrincoated membranes and of PIP2 and PIP3 will be measured in mouseperitoneal macrophages stimulated by an immune complex surface and byplatelet activating factor. Similar measurements will be performed onintact and permeabilized rat mast cells under conditions that willselectively activate exocytosis, PIP2 cleavage, or both. These studieshave broad implications for our understanding of basic processesregulating membrane dynamics in eukaryotic cells, and for development ofpotential therapeutic measurements that depend on receptor-mediatedendocytosis for delivery of agents to the cell interior. The long-term goal of this research program is an understanding of howclathrin coated membranes contribute to intracellular organization andmembrane dynamics. Our approach to these issues has been to study thestructure, activities and interrelationships of the various componentsof the clathrin coated membrane at the molecular level. Clathrin, themajor protein component of the coated membrane, interacts with AP-1 orAP-2, which have been termed assembly, adaptor or associated proteins, to form the completed lattice structure that is found within manydifferent cell types. Another AP-like protein, AP-3, is a majorneuronal protein which is highly conserved but little is known about itsinteractions with clathrin and there are no compelling hypotheses forits intracellular funct
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Keywords
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Biochemistry; Biophysics; Cell Biology; Developmental Biology; Genetics; Human Physiology; Pharmacology; neuron; antibody; clathrin; mast cell; exocytosis; macrophage; radiotracer; calcium flux; cytoskeleton; cell membrane; stoichiometry; immune complex; molecular site; phosphorylation; laboratory mouse; light microscopy; membrane protein; western blotting; laboratory rabbit; membrane activity; membrane structure; electron microscopy; membrane biogenesis; tissue cell culture; phosphatidylinositol; protein biosynthesis; nucleic acid sequence; fluorescence microscopy; site directed mutagenesis; platelet activating factor; protein structure function; confocal scanning microscopy; receptor mediated endocytosis
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Publications
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- Gaidarov I; Keen JH. Phosphoinositide-AP-2 interactions required for targeting to plasma membrane clathrin-coated pits. Journal of Cell Biology. 146(4): 755-64, 23 Aug 1999
- Gaidarov I; Krupnick JG; Falck JR; Benovic JL; Keen JH. Arrestin function in G protein-coupled receptor endocytosis requires phosphoinositide binding. EMBO Journal. 18(4): 871-81, 15 Feb 1999
- Santini F; Marks MS; Keen JH. Endocytic clathrin-coated pit formation is independent of receptor internalization signal levels. Molecular Biology of The Cell. 9(5): 1177-94, 1998
- Santini F; Keen JH. Endocytosis of activated receptors and clathrin-coated pit formation: deciphering the chicken or egg relationship. Journal of Cell Biology. 132(6): 1025-36, 1996
- Goodman OB Jr; Keen JH. The alpha chain of the AP-2 adaptor is a clathrin binding subunit. Journal of Biological Chemistry. 270(40): 23768-73, 6 Oct 1995
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Individual Expertise profile of
James H. Keen, Ph.D., Copyright © James H. Keen, Ph.D..
Last Updated
by James Keen, Ph.D. : Wednesday, September 2, 2009 12:54:20 PM
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