Receptor Binding and Adenylate Cyclase Activities of Glucagon Analogues Modified in the N-Terminal Region

In this study, we determined the ability of four N-terminally modified derivatives of glucagon, [3-Me-His¹,Arg¹²]^-, [Phe¹,Arg¹²]^-, [D-Ala⁴,Arg¹²]-, and [D-Phe⁴]glucagon, to compete with ¹²⁵I-glucagon for binding sites specific for glucagon in hepatic plasma membranes and to activate the hepatic adenylate cyclase system, the second step involved in producing many of the physiological effects of glucagon. Relative to the native hormone, 3-Me-His¹,Arg¹²]glucagon binds approximately twofold greater to hepatic plasma membranes but is fivefold less potent in the adenylate cyclase assay. [Phe¹,Arg¹²] glucagon binds threefold weaker and is also approximately fivefold less potent in adenylate cyclase activity. In addition, both analogues are partial agonists with respect to adenylate cyclase. These results support the critical role of the N-terminal histidine residue in eliciting maximal transduction of the hormonal message. [D-Ala⁴,Arg¹²]glucagon and [D-Phe⁴] glucagon, analogues designed to examine the possible importance of a β-bend conformation in the N-terminal region of glucagon for binding and biological activities, have binding potencies relative to glucagon of 31% and 69%, respectively. [D-Ala⁴,Arg¹²]glucagon is a partial agonist in the adenylate cyclase assay system having a fourfold reduction in potency, while the [D-Phe⁴] derivative is a full agonist essentially equipotent with the native hormone. These results do not necessarily support the role of an N-terminal β-bend in glucagon receptor recognition. With respect to in vivo glycogenolysis activities, all of the analogues have previously been reported to be full agonists. The partial agonism of [3-Me-His¹,Arg¹²]-, [Phe¹,Arg¹²]-, and [D-Ala⁴,Arg¹²]glucagon for adenylate cyclase activity in isolated liver plasma membranes observed in this study is not modulated by changes in the guanosine triphosphate (GTP) concentration. In addition, the receptor binding dose-response curve for [Phe¹,Arg¹²]glucagon is shifted to the right in the presence of GTP to the same extent as that seen with the native hormone. Thus, the partial agonism demonstrated by these three analogues in this study is not due to a lack of modulation by GTP of the receptor binding and adenylate cyclase activities measured on liver plasma membranes. The in vivo degradation rates for glucagon and [D-Phe⁴]glucagon, half-lives of 5.3 and 7.5 min, respectively, were determined in this study. This slightly slower rate of degradation for [D-Phe⁴] glucagon is not sufficient to account for its highly potent glycogenolytic activity seen in vivo. The lack of correlation between the in vitro adenylate cyclase and the in vivo glucose release activities for these compounds is discussed.