During the past 12 years the applicants laboratory has made many observations which helped to define the functional anatomy of the intercellular junctions of intestinal epithelial. It was shown in these studies that, both in health and disease, intestinal epithelial tight junctions could regulate epithelial permeability in a highly dynamic fashion. Given the importance of this barrier to intestinal function, it is now appropriate to focus on the molecular structure of the tight junction and on the molecular basis for the regulation of tight junction assembly and permeability. Such new approaches should include molecular structure/function work focused on molecules which contribute to tight junction structure. The host lab (Professor Daniel Louvard, Director of Cell and Molecular Biology, Pasteur Institute, Paris) not only excels in such molecularly based structure/function work in intestinal epithelia, but this lab has recently made the serendipitous discovery of a tight- junction specific small GAP binding protein of the ra family. This novel protein has been cloned and sequenced by the host laboratory. Specific members of this large protein family are generally located at different and highly specific sites within cells. Moreover, members of this family typically behave as functional switches to control membrane interactions/targeting and transmembrane signaling at such specific sites. Accordingly, this fellowship application proposes to initiate molecular structure/function studies of this tight junction-specific ra (J-ra) protein which could play a role in tight junction regulation and/or assembly. During the first visit (August 1, 1993 - January 30, 19994), under the supervision of Dr. Louvard and in association with molecular biologists in his laboratory (including Dr. Arnold Zahraoui who was one of the first to clone human ra proteins), the applicant will prepare plasmids which will express tagged and mutant forms of J-ra when transfected into human intestinal epithelial cells (Caco-2). Transient transfection experiments will be carried out to determine if transfected proteins are appropriately expressed. Subsequently, expansion of stabely transfected cell lines and detailed functional assessment of tight junction assembly-permeability/regulation will be carried out in Boston in the applicants Boston lab which is well suited for these functional studies. On the second trip (June 1, 1994 - October 30, 1994) results from the formalized functional screening of mutants will provide a basis for more detailed mutagenesis/chimaera construction aimed at identifying sequence information which imparts junction-specific targeting and/or controls junction assemble/permeability/regulation. Subsequently, a second cycle of functional screening of stabel transfected cell lines will begin in the applicants Boston laboratory. These studies will introduce the applicant to molecular structure/function analyses, may provide key insights into tight junction assemble and/or regulation, and will provide a springboard for long-term collaborative interactions between the host and applicant laboratories. It is anticipated that this project will be an important component of the ongoing NIH-funded research for the next 5-7 years in the Division of Gastrointestinal Pathology at the Brigham and Women' Hospital.
