The composition and function of the plasma membrane is maintained by a complex intracellular traffic moving cell surface glycoproteins between organelles. This requires the recognition and sorting of different classes of proteins, not only during biosynthesis, but also during redistributive processes, such as the internalization and recycling of receptors during receptor-mediated endocytosis, or during the down-regulation of hormone receptors. Disease can result form the failure of any one of these processes, or from its subversion for use by a pathogen such as a virus. Experiments described in this application are designed to reveal molecular details of the cellular mechanisms that control the intracellular transport of a major class of cell surface glycoproteins. Through genetic engineering, gain-of-function mutants have been produced that are sorted into pathways from which they were previously excluded. This allows features of proteins important for intracellular sorting events to be distinguished from those important for the structure of the protein as a whole. Six experiments are proposed (1) to determine the number and relationship of independent mechanisms for recognition of proteins by coated pits during endocytosis; (2) to identify the proteins controlling sorting in the endocytic pathway; (3) to identify the proteins responsible for sorting transmembrane glycoproteins in polarized epithelial cells; (4) to determine the role of the transmembrane domain in the intracellular transport of glycoproteins. Mutants made previously will be used to establish in vitro assays for internalization and recycling for the purpose of identifying essential cellular components of these events. Permanent cell lines expressing these mutants will be used to determine which proteins compete for recognition by components of coated pits. Genetic engineering will be used to introduce progressively smaller mutations into glycoproteins to identify the amino acid sequences responsible for interesting changes in their intracellular traffic. The secondary, tertiary and quarternary structure of features important for sorting will be investigated. Synthetic peptides that mimic transport signals will be used to identify cellular proteins that interact with them in experiments employing chemical cross-linking, direct affinity chromatography, or anti-idiotype antibodies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM037547-05
Application #
3292867
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1986-12-01
Project End
1994-11-30
Budget Start
1990-12-01
Budget End
1991-11-30
Support Year
5
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Type
Schools of Medicine
DUNS #
City
Dallas
State
TX
Country
United States
Zip Code
75390
Siu, Ka Yu; Yu, Mei Kuen; Wu, Xinggang et al. (2011) The non-catalytic carboxyl-terminal domain of ARFGAP1 regulates actin cytoskeleton reorganization by antagonizing the activation of Rac1. PLoS One 6:e18458
Shvartsman, Dmitry E; Kotler, Mariana; Tall, Renee D et al. (2003) Differently anchored influenza hemagglutinin mutants display distinct interaction dynamics with mutual rafts. J Cell Biol 163:879-88
Padron, David; Wang, Ying Jie; Yamamoto, Masaya et al. (2003) Phosphatidylinositol phosphate 5-kinase Ibeta recruits AP-2 to the plasma membrane and regulates rates of constitutive endocytosis. J Cell Biol 162:693-701
Tall, Renee D; Alonso, Miguel A; Roth, Michael G (2003) Features of influenza HA required for apical sorting differ from those required for association with DRMs or MAL. Traffic 4:838-49
Wang, Ying Jie; Wang, Jing; Sun, Hui Qiao et al. (2003) Phosphatidylinositol 4 phosphate regulates targeting of clathrin adaptor AP-1 complexes to the Golgi. Cell 114:299-310
Yu, Sidney; Roth, Michael G (2002) Casein kinase I regulates membrane binding by ARF GAP1. Mol Biol Cell 13:2559-70
Keren, T; Roth, M G; Henis, Y I (2001) Internalization-competent influenza hemagglutinin mutants form complexes with clathrin-deficient multivalent AP-2 oligomers in live cells. J Biol Chem 276:28356-63
Lewis, C M; Latham, K; Roth, M G (2000) A screen of random sequences for those that alter the trafficking of the influenza virus hemagglutinin in vivo. Traffic 1:282-90
Melikyan, G B; Markosyan, R M; Roth, M G et al. (2000) A point mutation in the transmembrane domain of the hemagglutinin of influenza virus stabilizes a hemifusion intermediate that can transit to fusion. Mol Biol Cell 11:3765-75
Brown, C M; Roth, M G; Henis, Y I et al. (1999) An internalization-competent influenza hemagglutinin mutant causes the redistribution of AP-2 to existing coated pits and is colocalized with AP-2 in clathrin free clusters. Biochemistry 38:15166-73

Showing the most recent 10 out of 40 publications