Cholecystokinin (CCK) is an important gastrointestinal and neural polypeptide with a diverse spectrum of biological activity, however, many basic questions remain about the structure and function of CCK receptors. The objective of the proposed research is to isolate a cDNA clone for the CCK receptor and to use this cDNA to analyze the structure, function and tissue specific expression of the CCK receptor. We have recently found that Xenopus laevis oocytes injected with mRNA from AR42J cells, a pancreatic acinar cell line, express functional CCK receptors that can be detected by their ability to increase 45Ca2+ efflux. In the proposed research AR42J cell mRNA will be used to construct cDNA libraries in vectors which allow synthesis of RNA in vitro and the CCK receptor cDNA will be isolated by functional expression in oocytes. Nucleotide sequence data will be obtained from the isolated CCK receptor cDNA and used to determine the primary structure and to predict the secondary structure, hydropathicity, and potential sites for glycosylation, disulfide cross-linking and phosphorylation of the CCK receptor. Expression of the CCK receptor, by injection of transcribed RNA into Xenopus oocytes and by subcloning the cDNA into eukaryotic expression vectors and transfecting a variety of cell lines, will provide valuable information about CCK receptor function. Exogenously expressed CCK receptors will be tested for their abilities to bind CCK, internalize CCK, increase phosphoinositide turnover, increase intracellular Ca2+ release, and interact with guanine nucleotide binding proteins. Functional domains of the CCK receptor will then be elucidated both by perturbation (e.g. site-directed mutagenesis) leading to loss of function, and by construction of chimeric receptors leading to acquisition of functions. The cloned CCK receptor cDNA will also be used to determine the tissue specificity of CCK receptor expression. mRNA prepared from a variety of tissues will be analyzed by Northern blot hybridization. Differences between the cloned CCK receptor cDNA and homologous mRNAs found in other tissue will be further elucidated using RNase protection and primer extension methods.
