CD8 has been shown to be critically involved in class I MHC-restricted T cell development and antigen recognition function. CD8 co-receptors exist as either CD8alpha/alpha homodimers or CD8alphabeta molecules through the use of leucine zipper (LC) sequences that permit the secretion and formation of soluble Ig-like domains of the CD8alpha/alpha homodimer which led to crystallization of the mCD8alpha/alpha/Kb complex and diffracted to a 2.8 A resolution. The precise molecular interaction of CD8alphabeta is still ill-defined. In the present proposal a detailed analysis of CD8alphabeta peptide/MHC interaction will be performed utilizing a well-characterized complex of a vascular stromatitis virus octapeptide (VSV8) bound tot he Kb molecule. First, CD8alphabeta will be engineered for secretion in eukaryotic cells (Lec3.2.8.1 CHO) which synthesize homogeneous glycans and whose glycoproteins can be readily deglycosylated using Endo-H. Material will be provided for X-ray crystallography by itself as well as complexed with homogeneously VSV8-loaded Kb molecules. CD8alphabeta-LZ will also be complexed with various Fag fragments of mAbs directed against the LZ, CD8alpha or CD8beta. Biophysical analysis of the CD8-VSV8/Kb interaction will be performing using surface plasmon resonance. The affinity of the CD8alphabeta ectodomains and Ig segments for VSV8/Kb, TCRs and TCR-VSV8/kappaB and beta2M will created by PCR method and used in functional studies to probe the basis of CD8-peptide/MHC interaction. The function of the stalk region of CD8alphabeta will be dissected by structural swap between co-receptor molecules and the precise interaction between CD8alpha-beta and Kb will be defined by mutagenesis. Third, the function of the cytoplasmic domain of CD8alpha or CD8beta in the co-receptor function will be dissected by mutagenesis experiments. The signal transduction molecules involved in co-receptor function of CD8alpha or CD8beta will be examined by biochemical analysis. In parallel, the potential molecules involved in signal transduction of CD8beta will be cloned in the yeast two hybrid system.
