Two molecules believed to regulate the adhesion of lymphocytes to the specialized endothelium of lymph nodes and related lymphoid structures will be studied to define the functional domains of the molecules required for successful endothelial interaction. A saturation mutagenesis/antibody selection scheme will be used to derive monoclonal antibody epitope loss variants which will be screened for loss of function in two lymphocyte binding assays. One of these, the lymphocyte transmigration assay, relies on cultured node endothelial cells; for this and related purposes, primary cultures of rat, monkey, and human peripheral and mesenteric node endothelial cells will be established. The cultured cells will be used as targets for transfectants bearing mutated homing receptors and for soluble forms of the homing receptors created by gene fusion of the extracellular domains, or subdomains, to tag sequences derived from human or mouse immunoglobulin constant regions. An attempt will be made to biochemically define the target molecules with which the homing receptors interact; because these molecules need not be proteins, biochemical fractionation schemes are proposed to help establish whether carbohydrate recognition (suggested by the lectin or link-protein homologies of the homing receptors) or protein recognition fulfills the dominant role in determining homing specificity. However if a protein component is involved, either as a scaffolding structure or for direct interaction with the homing receptors, polyclonal or monoclonal antibodies will be generated against the protein component, and the appropriate cDNA will be isolated from a library generated from the node endothelial cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK043031-02
Application #
3244291
Study Section
Allergy and Immunology Study Section (ALY)
Project Start
1990-07-01
Project End
1995-06-30
Budget Start
1991-07-01
Budget End
1992-06-30
Support Year
2
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Massachusetts General Hospital
Department
Type
DUNS #
City
Boston
State
MA
Country
United States
Zip Code
02199
Sun, Yuhua; Gershengorn, Marvin C (2002) Correlation between basal signaling and internalization of thyrotropin-releasing hormone receptors: evidence for involvement of similar receptor conformations. Endocrinology 143:2886-92
Fukumura, D; Xavier, R; Sugiura, T et al. (1998) Tumor induction of VEGF promoter activity in stromal cells. Cell 94:715-25
Xavier, R; Brennan, T; Li, Q et al. (1998) Membrane compartmentation is required for efficient T cell activation. Immunity 8:723-32
Kolanus, W; Nagel, W; Schiller, B et al. (1996) Alpha L beta 2 integrin/LFA-1 binding to ICAM-1 induced by cytohesin-1, a cytoplasmic regulatory molecule. Cell 86:233-42
Ting, A T; Pimentel-Muinos, F X; Seed, B (1996) RIP mediates tumor necrosis factor receptor 1 activation of NF-kappaB but not Fas/APO-1-initiated apoptosis. EMBO J 15:6189-96
Pouyani, T; Seed, B (1995) PSGL-1 recognition of P-selectin is controlled by a tyrosine sulfation consensus at the PSGL-1 amino terminus. Cell 83:333-43
Stanger, B Z; Leder, P; Lee, T H et al. (1995) RIP: a novel protein containing a death domain that interacts with Fas/APO-1 (CD95) in yeast and causes cell death. Cell 81:513-23
Aruffo, A; Kolanus, W; Walz, G et al. (1991) CD62/P-selectin recognition of myeloid and tumor cell sulfatides. Cell 67:35-44
Aruffo, A; Melnick, M B; Linsley, P S et al. (1991) The lymphocyte glycoprotein CD6 contains a repeated domain structure characteristic of a new family of cell surface and secreted proteins. J Exp Med 174:949-52
Walz, G; Aruffo, A; Kolanus, W et al. (1990) Recognition by ELAM-1 of the sialyl-Lex determinant on myeloid and tumor cells. Science 250:1132-5