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

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-His¹-[Glu⁹]glucagon amide (glucagon-NH₂). Alanine substitution in TM (transmembrane) helix 2 of Arg¹⁷³ or of His¹⁷⁷ detrimentally affected glucagon and glucagon-NH₂ response maxima. TM2 receptor mutant, Phe¹⁸¹-Ala, displayed reduced maximum cAMP accumulation in response to glucagon-NH₂. Thr³⁵³-Cys (TM6) and Glu⁴⁰⁶-Ala (TM7) receptors demonstrated constitutive activity and enhanced EC₅₀ values for glucagon-NH₂; Arg³⁴⁶-Ala (TM6) and Asn⁴⁰⁴-Ala (TM7) receptors were activated by sub-fmol glucagon concentrations, yet were not constitutively active and demonstrated wild-type receptor-like EC₅₀ values for glucagon-NH₂. Unlike Arg³⁴⁶-Ala receptors, Thr³⁵³-Cys, Asn⁴⁰⁴-Ala and Glu⁴⁰⁶-Ala receptors demonstrated improved EC₅₀ 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-NH₂ affinity, Arg³⁴⁶-Ala, Thr³⁵³-Cys, Asn⁴⁰⁴-Ala and Glu⁴⁰⁶-Ala receptors displayed glucagon-NH₂ 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.