IDENTIFICATION OF AN ESSENTIAL SERINE RESIDUE IN GLUCAGON - IMPLICATION FOR AN ACTIVE-SITE TRIAD

Several glucagon analogs containing substitutions for serine have been synthesized to assess the role of the four serine residues in the hor mone. The strategic importance of His(1) has been confirmed, and we ha ve previously identified an aspartic acid critical for activity at pos ition 9. While these findings have led to a series of pure glucagon an tagonists, the details of specific glucagon-receptor interactions that switch on the ensuing signaling events are still not readily apparent . The requirement for serine was tested by the chemical synthesis of a series of analogs containing substitutions for the hydrophilic hydrox yl group in each of the highly conserved serine residues at positions 2, 8, 11, and 16 of glucagon. The resulting analogs were analyzed in r at hepatocyte membranes for their receptor-binding affinities as well as their abilities to stimulate adenylate cyclase. Positions 2 and 8 w ere the most sensitive to modification, where both binding and activit y were adversely affected. This is consistent with the notion that alt hough the sequence responsible for transduction lies in the amino-term inal half of glucagon, some residues at that end also contribute to bi nding affinity. Modifications at position 11 generated high-binding-af finity derivatives that were full or moderate agonists. In contrast, p osition 16 replacement analogs maintained significant receptor binding affinities while the agonist properties were almost completely lost, thus separating binding and transduction functions. Therefore, Ser(16) is a third critical residue that determines glucagon activity. It is postulated, but not proven, that a serine residue, together with His(1 ) and Asp(9), may participate in the putative active center of glucago n, which, upon initial recognition and binding to receptor, leads to t ransduction of the biological signal. A dependence of the glucagon act ion on a three-residue cooperative mechanism might be analogous to the charge relay scheme of serine proteases. It is suggested that, after binding to its receptor, glucagon becomes activated and functions like a coenzyme in catalyzing the specific hydrolysis of a peptide bond in the receptor, generating new amino and carboxyl end groups, and that one of these exposed chains may contact the GTP-binding protein and ac tivate it for further interaction with adenylate cyclase. This idea wa s supported by inhibition experiments with 4-amidinophenylmethanesulfo nyl fluoride (APMSF), a specific and irreversible inhibitor of serine proteases, which at a concentration of 5 mM completely suppressed cAMP formation by glucagon in liver membranes. cAMP formation was not affe cted if either glucagon or membranes were separately pretreated with A PMSF and then assayed.