Despite significant progress in recent years in elucidating the intracellular biochemical consequences of T cell activation via the T cell receptor (TCR), the precise mechanism driving the initial communication of a signal across the membrane upon receptor ligation remains unknown. TCR clustering, coreceptor recruitment and conformational changes in the receptor have all been proposed to play important roles in signal initiation, but no widely accepted model has emerged. The structure and stoichiometry of the complete TCR/CD3 complex expressed at the T cell surface also remains an open question, and models involving a TCR valency of greater than one have been proposed. The goal of this project is to define the structural features that drive the assembly of the TCR/CD3 complex, and to examine the triggering mechanisms based on an understanding of the subunit interactions and the stoichiometry of the complex at the cell surface. An in vitro translation and assembly system will be one of the major tools for these studies, and the investigators have developed new methods for the preparation of ER microsomes that permit complex assembly experiments as well as approaches for isolation of key assembly intermediates. The preliminary data demonstrate unusual interactions between basic and acidic transmembrane residues of the TCR and associated CD3 chains. Assembly of each of the three CD3 dimers with the TCR requires two acidic CD3 and one basic TCR residue in the membrane, and in each case these three transmembrane residues appear to interact. The major hypothesis of this project is that these distinctive interactions among potentially charged transmembrane residues are critical not only in the assembly of the alpha-beta, gamma-delta and pre-TCR, but also in guiding the conformational changes that are induced upon receptor triggering. Specifically, these interaction points between the TCR and CD3 subunits in the membrane may be at the center of conformational movements of both extracellular and cytoplasmic domains that bring the signaling modules into close proximity upon receptor engagement. Such a conformational change may be induced by recruitment of TCR/CD3 complexes to lipid rafts, which have an increased thickness relative to the surrounding plasma membrane due to the presence of sphingolipids and cholesterol. ? ? ? ?
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