STRUCTURE/FUNCTION OF A HEPATIC CEA BINDING PROTEIN

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Carcinoembryonic antigen (CEA) is involved in the development of hepatic metastases from colorectal cancers. The major hepatic CEA binding protein is 80kD Kupffer cell surface molecule. The binding site for the 80kD protein has been located to the junction of the N-terminal and first loop domains of CEA and is a pentapeptide (PELPK). Treatment of Kupffer cells with CEA results in the production of IL-1, IL-6 and TNF- alpha and two proteins of 115 and 125kD are rapidly phosphorylated when Kupffer cells are exposed to CEA. In this application we will also study the structure and function of the Kupffer cell 80kD carcinoembryonic antigen (CEA) peptide binding protein by further protein sequencing, production of antibodies and by cloning and expressing its gene from both rat and human Kupffer cell cDNA libraries using the available peptide sequences as the basis for probes. To examine the function of the CEA binding protein in metastasis we will examine the mechanism of activation of hepatic Kupffer cells by CEA and the synthetic ligand for the 80kD binding protein PELPK-albumin by a) Studying the consequences of ligand binding to the 80kD protein on Kupffer cells, on the metastatic proclivity of cancer cells in the hepatic sinusoid and the effects on adhesion to the hepatic endothelium. Using inhibitory antibodies to endothelial cells binding proteins we will identify the adhesion molecules involved. b) We will examine the effects of inhibition of signal transduction (specifically phosphorylation of tyrosine) on the regulation of metastatic potential of human colorectal cancer cell lines by CEA. c) We will use site directed mutagenesis to alter the five amino acid (PELPK) binding site for the 80kD protein to examine its effect on Kupffer cell response to CEA and on hepatic metastasis formation. We will also use a construct that has the first 75 amino acids of the CEA N-domain deleted. This CEA does not form homotypic aggregates but retains the PELPK sequence. This will allow us to determine if CEA produced to enhance metastatic potential. These studies will increase our knowledge of the role of the 80kD binding protein in the liver and its relationship with CEA in the mechanism by which colorectal cancer cells attach and invade in the hepatic sinusoid.