This project studies proteins (""""""""complex 1"""""""") that participate in specifying polarity. A membrane protein, junctional adhesion molecule (JAM-1), PAR3, PAR6 (two PDZ proteins), and atypical protein kinase C (aPKC) form complex 1. The complex plays a role in assembly of tight junctions (TJ), even though it is absent from the functional structure. The hypothesis to be tested is that JAM-1 is a membrane anchor that recruits PAR3, a scaffold to which a Cdc42-GTP/PAR6 full length/aPKC sub-complex docks, thus locating the complex apical of """"""""primordial"""""""" adherens junctions. Activated aPKC then phosphorylates proteins that help form the final TJ. Our newly- identified PAR6A-short, which cannot bind aPKC or Cdc42 but retains a semi-PDZ domain, regulates TJ formation and/or stabilizes mature TJs. In vitro models are WlF-B cells, which develop a polarized hepatic phenotype, and intestinal Caco-2 cells, which express proteins in a tetracycline-inducible manner. In vivo experiments extend in vitro results to the regenerating liver, where TJ formation is crucial to recovery of organ function. Approaches are molecular biology, morphology (eg, live cell imaging), quantitative biochemistry (eg, fractionation, in vitro binding, protein identification) and use of recombinant adenoviruses to alter/knock-down complex 1 proteins.
Aim #1 tests JAM's role. In #1A, constructs mutated in the cytoplasmic or ectoplasmic (adhesive) domain of JAM are expressed in WlF-B cells to determine if/when these domains function in progression to an hepatic phenotype. Live-cell imaging of GFP-reporters provides insight into the dynamics of the process. In #1B, mutants expressed in hepatic Fao cells are used in a novel bead assay to identify the proteins JAM recruits. In #1C, mutant JAM or RNAi is expressed in livers of 2/3 hepatectomized rats to determine the effects on TJ formation.
Aim #2 tests the roles of PAR6 and PAR6A-short. In #2A, PAR6A-short is induced in tet-off Caco-2 cells at various stages of maturation to distinguish a role in formation versus maintenance of TJs. A second approach is use of RNAi to silence all PAR6 or just PAR6A-short in WIF-B cells. In #2B, in vitro assays test candidates reported to bind full-length PAR6 for binding to PAR6A-short. In #2C, PAR6A-short or RNAi is expressed in vivo to determine the effects on liver regeneration.
Aim #3 uses pharmacological approaches to test the roles of aPKC and protein phosphatase 2A in the development of WlF-B polarity.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK063096-03
Application #
7110343
Study Section
Hepatobiliary Pathophysiology Study Section (HBPP)
Program Officer
Serrano, Jose
Project Start
2004-09-30
Project End
2009-07-31
Budget Start
2006-08-01
Budget End
2007-07-31
Support Year
3
Fiscal Year
2006
Total Cost
$417,855
Indirect Cost
Name
Johns Hopkins University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
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Dikeman, Dustin A; Rivera Rosado, Leslie A; Horn, Troy A et al. (2008) alpha4 beta1-Integrin regulates directionally persistent cell migration in response to shear flow stimulation. Am J Physiol Cell Physiol 295:C151-9
Braiterman, Lelita T; Heffernan, Sean; Nyasae, Lydia et al. (2008) JAM-A is both essential and inhibitory to development of hepatic polarity in WIF-B cells. Am J Physiol Gastrointest Liver Physiol 294:G576-88
Kallay, Laura M; McNickle, Allison; Brennwald, Patrick J et al. (2006) Scribble associates with two polarity proteins, Lgl2 and Vangl2, via distinct molecular domains. J Cell Biochem 99:647-64