The objective of this proposal are to prepare and characterize derivatives of glucagon and to investigate their structure and interactions with the glucagon receptor in order to understand the mechanisms by which the hormone carries out its various functions in the normal and the diabetic state. The derivatives will be prepared by chemical modification, synthesis and/or semisynthesis, characterized chemically and physically, evaluated for kind and quantity of contaminants, and assayed for binding and activation. Nalpha-biotinyl, Nepsilon-acetimidoglucagon and Nalpha-maltoglucagon previously prepared are partial agonists. Modifications of the imidazole ring of His-1 should produce an effective antagonist. Extracellular cleavage sites whose modification could lead to a long acting antagonist will be sought. Nalpha-biotinyl, Nepsilon-acetimidoglucagon binding to glucagon specific receptors will be monitored by avidin labelled with enzymes, fluorescent tags or gold and evaluated both chemically and histochemically to identify secondary target cells. The derivative will be used to monitor internalization and receptor recycling. We will examine the hypothesis that internalization is followed by cleavage at Arg17-Arg18 to yield separate fragments which bind to and activate intracellular receptors. Binding of glucagon to synaptosomal receptors will be characterized. We expect to determine whether this synaptosomal binding results in adenylate cyclase activation. Glucagon binding protein of hepatocytes and synaptosomes will be solubilized, electrophoresed, isolated, and chemically characterized by tryptic cleavage and HPLC identification of the cleavage products, by end group analysis and partial sequence determination using exopeptidase cleavage and precolumn derivatization amino acid analysis. The results should lead to a greater understanding of the abnormalities of the diabetic state and a potential way of modifying these abnormalities.
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