Evidence to support a spectrum of active states for the glucagon receptor

Abstract

The ternary complex model suggests that G-protein-coupled receptors resonate between inactive (R) and active (R*) forms. Physiologically, R sites ordinarily predominate with a few R* sites giving rise to basal activity. Agonists recognize, stabilize and increase the R* population, thus altering intracellular activity. There is evidence to suggest the possibility of a spectrum of conformations between R and R*. Our aim is to study the consequences of putative GR (glucagon receptor)-activating mutations using glucagon and partial agonist des-His(1)-[Glu(9)]glucagon amide (glucagon-NH(2)). Alanine substitution in TM (transmembrane) helix 2 of Arg(173) or of His(177) detrimentally affected glucagon and glucagon-NH(2) response maxima. TM2 receptor mutant, Phe(181)-Ala, displayed reduced maximum cAMP accumulation in response to glucagon-NH(2). Thr(353)-Cys (TM6) and Glu(406)-Ala (TM7) receptors demonstrated constitutive activity and enhanced EC(50) values for glucagon-NH(2); Arg(346)-Ala (TM6) and Asn(404)-Ala (TM7) receptors were activated by sub-fmol glucagon concentrations, yet were not constitutively active and demonstrated wild-type receptor-like EC(50) values for glucagon-NH(2). Unlike Arg(346)-Ala receptors, Thr(353)-Cys, Asn(404)-Ala and Glu(406)-Ala receptors demonstrated improved EC(50) values for glucagon, whereas their maximal responses to and their affinity for glucagon were comparable with the wild-type receptor. In contrast, despite slightly reduced glucagon-NH(2) affinity, Arg(346)-Ala, Thr(353)-Cys, Asn(404)-Ala and Glu(406)-Ala receptors displayed glucagon-NH(2) response maxima that exceeded those seen for wild-type receptors. Interestingly, we observed biphasic glucagon-mediated signalling responses. Our results are consistent with the concept of different agonists promoting the formation of distinct active states from partially active R*(low) to fully active R*(high) forms.