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.
Evidence to support a spectrum of active states for the glucagon receptor
N. Strudwick, N. Bhogal, N.A. Evans, F.E. Blaney, J.B.C. Findlay; Evidence to support a spectrum of active states for the glucagon receptor. Biochem Soc Trans 1 November 2004; 32 (6): 1037–1039. doi: https://doi.org/10.1042/BST0321037
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