Importance of the 10–13 Region of Glucagon for Its Receptor Interactions and Activation of Adenylate Cyclase

The role of the Tyr¹⁰-Ser¹¹-Lys¹²-Tyr¹³ region of glucagon in the binding interaction and activation of the glucagon receptor was investigated by means of the synthetic glucagon analogues [Phe¹³]glucagonamide (2), [Phe¹⁰]glucagonamide (3), [Phe¹⁰]glucagon (4), [Phe^10,13]glucagon (5), [Pro¹¹]glucagon (6), [Pro¹¹,Gly¹²]glucagonamide (7), [Ala¹¹ glucagon (8), and [Oac^11-13]glucagonamide (9). These analogues were synthesized by solid-phase peptide synthesis on p-methylbenzhydrylamine or Merrifield resins with protected N^α-tert-butyloxycarbonyl amino acids. Purification by dialysis, cation-exchange chromatography, gel filtration, and preparative reverse-phase high-performance liquid chromatography (HPLC) gave products that proved homogeneous by thin-layer chromatography and HPLC and on analysis by amino acid analysis, by sequencing, and by a-chymotryptic peptide mapping with HPLC. Biological activities were examined by measurement of the stimulation of liver plasma membrane adenylate cyclase and by specific displacement of [¹²⁵I]glucagon from glucagon receptors. The results of these studies indicate that while the biological “message” region of glucagon is located elsewhere, the 10–13 region has multiple roles in the glucagon-glucagon receptor interaction: (1) this region provides functional groups for direct binding interaction with the receptor, and (2) this region interacts with the receptor in such a way as to allow the “transduction message” portion of glucagon to interact and activate the receptor.