Eugene E. Marcantonio - US grants

The main objective of the proposed research plan is to investigate the mechanisms by which cell control their interactions with extracellular matrix and basement membranes during migration and differentiation in the normal and disease state. The integrin receptor heterodimer alpha1-beta1, a laminin/collagen receptor, has been chosen as a model system for this study because of several unique features. This molecular has a limited distribution in vivo, being present on activated lymphocytes, neuronal cells and in the mesangium of the kidney in adult tissues studied to date. This distribution implies a selective and specific function which is not fulfilled by other integrin collagen/laminin receptors (i.e., alpha2,3,and 6). This integrin can be induced on the surface of lymphocytes after activation in vitro, on PC12 cells in vitro, and in vivo on the synovial lymphocytes of patients with rheumatoid arthritis. Thus, this molecule probably is tightly regulated in vivo, and enables us to study the control of integrin function. cDNA clones for the human integrin alpha1 subunit will be used to produce fusion proteins and synthetic peptides for the generation of antibodies. The role of alpha1 in providing positional information in cell migrations crucial to normal development will be investigated by determining the temporal and spatial pattern of expression in mammalian embryos suing these probes. In particular, the presence of this integrin on migrating cells will allow us to determine if there are changes in expression levels from early in development, when the cells are migrating, to late, when they have reached their destination. Lastly, this integrin has shown different ligand specificities on varying cell types, binding laminin and collagen on some, while binding only collagen on others, a property which permits investigation of the factors both intrinsic and extrinsic to alpha1 which affect ligand specificity in cell migrations in vivo. These factors will be studied using intact, truncated and mutant forms of the molecule, the latter two generated by site-directed mutagenesis and heterologous eukaryotic expression systems. This research will enhance our knowledge of the mechanism of cell migration in both normal states such as development, and abnormal states such as tumor cell metastasis, and chronic inflammatory diseases; with the long term goal of determining the role of alpha1 and other integrins in the targeting of cells in disease states.

The broad goals of this proposal are to determine the roles for integrin receptors in the development and function of T-lymphocytes. A combination of in vivo and in vitro approaches have been chosen. In order to test the role of integrin receptor function in T cell development, transgenic mice have been produced with a dominant negative form of integrin. Specifically, this laboratory is using a construct with the active integrin beta 1 cytoplasmic domain connected to the extracellular domain of the tac antigen, which breaks the transmembrane connection between extracellular matrix and the cytoskeleton provided by integrin receptors. The proximal lck promoter has been chosen to direct thymic specific expression of this construct in transgenic mice, leading to a reduction in the number of mature, single positive thymocytes in these mice as compared to normal liftermates. Our hypothesis is that this deficit represent a lack of positive selection in the transgenic mice, and we will test that hypothesis using transgenic mice with monoallelic expression of a T cell receptor (TCR) with a defined antigen recognition. Depending on the genetic background, conditions can be established in which mature thymocytes are produced only by positive selection of the monoallelic clones. Since positive selection has been shown to require p2lras, transgenic mice expressing a dominant negative form of the adaptor protein Shc will be produced, which should block integrin signaling to p21 ras. In vitro, molecular approaches to dissect integrin signaling pathways in co-stimulatory function of T cells will be done. Using recently developed reporter constructs, the role of versus FAK activation in of the TCR integrins have in co-stimulation on selected ECM substratum, and the role this signaling could play in chronic inflammatory diseases.

DESCRIPTION (Adpated from applicant's abstract): The primary goal of this renewal proposal is to determine the mechanism and consequences of intracellular signals from the integrin a1b1. Upon ligand binding, this receptor produces multiple signals which organize the actin cytoskeleton and stimulant cell growth. Previous studies have shown that swapping the cytoplasmic domains of a1 and beta1 leads to an integrin with defective signaling despite its capacity to bind ligand. An insertion within the cytoplasmic domain of the beta subunit can restore the signaling properties of this receptor. This model will be used to study the mechanism of the transmembrane conformation change occurring upon signaling. The second aim will compare wild type and defective receptors to dissect the proximal aspects of signaling via FAK. In the defective receptor FAK is activated yet the chimeric receptor does not phosphorylate CAS. Studies will focus upon cSrc and its ability to mediate the signaling between FAK and CAS. Mutant forms of cSrc will be expressed to determine if they will rescue the defective receptors. The third aim will focus on a1b1 growth signals through the adaptor Shc. Studies in Jurkat cells found that the receptor tyrosine phosphatase CD45 and the Src kinase Lck are required and the Src kinase Fyn is not sufficient in the these cells, in contrast to fibroblasts. Studies will address the mechanism of Src activation and Shc signaling. Finally, studies will address the consequences of the above mentioned growth signals using the two collagen receptors a1b1 and a2b1, which differ in their ability to phosphorylate Shc. This system will be use to examine the nature of integrin signals involved in the synergy with peptide growth factor receptor signals.