CONSTITUTIVE ACTIVITY OF GLUCAGON RECEPTOR MUTANTS

Increased constitutive activity has been observed in the PTH receptor in association with naturally occurring mutations of two residues that are conserved between members of the glucagon/vasoactive intestinal p eptide/calcitonin 7TM receptor family. Here, the corresponding residue s of the glucagon receptor, His(178) and Thr(352), were probed by muta genesis. An elevated level of basal cAMP production was observed after the exchange of His(178) into Arg, but not for the exchange into Lys, Ala, or Glu. However, for all of these His(178) substitutions, an inc reased binding affinity for glucagon was observed [dissociation consta nt (K-d) ranging from 1.1-6.4 nM, wild type: K-d = 12.0 nM]. A further increase in cAMP production was observed for the [H178R] construct up on stimulation with glucagon, albeit the EC50 surprisingly was increas ed approximately 10-fold relative to the wild-type receptor. Substitut ion of Thr(352), located at the intracellular end of transmembrane seg ment VI, with Ala led to a slightly elevated basal cAMP level, while t he introduction of Pro or Ser at this position affected rather the bin ding affinity of glucagon or the EC50 for stimulation of cAMP producti on. The large extracellular segment, which is essential for glucagon b inding, was not required for constitutive activation of the glucagon r eceptor as the introduction of the [H178R] mutation into an N-terminal ly truncated construct exhibited an elevated basal level of cAMP produ ction. The analog des-His(1)-[Glu(9)]glucagon amide, which in vivo is a glucagon antagonist, was an agonist on both the wild-type and the [H 178R] receptor and did not display any activity as an inverse agonist. It is concluded that the various phenotypes displayed by the constitu tively active glucagon receptor mutants reflect the existence of multi ple agonist-preferring receptor conformers, which include functionally active as well as inactive states. This view agrees with a recent mul ti-state extension of the ternary complex model for 7TM receptor activ ation.