Date of Award

5-2001

Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Bender, Jeffrey R.

Abstract

The role of membrane ICAM-1 in intercellular adhesion, through interaction with the leukocyte beta2 integrin receptors LFA-1 and Mac-1, is well established. However, until recently, ICAM-1 was widely regarded as being simply an "anchor" molecule, promoting, for example, firm adhesion of lymphocytes to vascular endothelial cells. The demonstration that ICAM-1 is connected to the actin-based cytoskeleton through interactions with subcortical actin-binding proteins, such as alpha-actinin, enhanced this concept. However, ICAM-1 has been increasingly shown to have signaling properties of its own. Engagement of ICAM-1 extracellularly, either through its natural counter-receptors, LFA-1/Mac-1, or through antibody mediated clustering, can produce numerous changes within the cell. Recent evidence now confirms that ICAM-1 crosslinking induces multiple activation responses including: (1) phosphorylation of several cytoplasmic proteins, including cortactin, cdc2 kinase and lyn kinase; (2) activation of the Erk 1/2 MAP kinases; and (3) induction of various functional cellular responses such as oxidative burst from mononuclear leukocytes, neutrophil activation, and rho activation in endothelial cells. Although we know of several downstream targets of ICAM-1 engagement, very little is known about upstream events, i.e., the most proximal components of the signaling cascade involving direct protein interactions with the ICAM-1 cytodomain, or through indirect interactions in a multimeric unit.The specific aim of this project is to study the proximal aspects of ICAM-1 signaling. The hypothesis is that ICAM-1-mediated signal transduction requires interactions between currently unidentified intracellular proteins and the cytoplasmic domain of ICAM-1. In these studies, TAXREB107, a recently discovered protein, was specifically associated with the ICAM-1 cytodomain in GST-pulldown experiments using human endothelial cell extracts. This association was biochemically confirmed by co-immunoprecipitations of recombinantly expressed TAXREB107 and the ICAM-1 cytodomain, as well as endogenous TAXREB107 and ICAM-1. Recombinant TAXREB107, expressed in a wide spectrum of mammalian cell types, greatly augmented the ERK 1/2 MAPK activation induced by activating antibody or natural ligand (fibrinogen) engagement of ICAM-1. This amplifying effect was abolished by co-expression of the ICAM-1, but not VCAM-1 cytodomain. Furthermore, immunofluorescence studies demonstrated that TAXREB107, which possesses DNA-binding capability, is predominantly located in the cytoplasm in non-activated CHO or human endothelial cells, and rapidly translocates to the nucleus upon ICAM-1 engagement. These data demonstrate an interaction between the ICAM-1 cytoplasmic tail and TAXREB107, and support that this interaction can lead to a productive amplification of proximal ICAM-1-mediated signaling. This may consequently stimulate gene expression of TAXREB-responsive genes or promote a spectrum of ERK-mediated cellular responses, also including gene activation.

Download
Open Access

This Article is Open Access

Share

COinS