Further evidence that the CCK₂ receptor is coupled to two transduction pathways using site-directed mutagenesis

A heterogeneity of CCK₂ receptors has been reported which could correspond to different states of coupling to G proteins and/or association with different second messenger systems. To investigate these hypotheses, the wild-type CCK₂ receptor and three mutants F347A, D100N and K333M/K334T/R335L, expected to modify the coupling of the G protein with the third intracellular loop of the receptor, were transfected into Cos-7 cells and their binding and signalling properties were evaluated using the natural ligand CCK₈. Activation of wild-type as well as F347A, D100N or K333M/K334T/R335L CCK₂ receptors by this ligand led to a similar arachidonic acid release which was blocked by pertussis toxin and the phospholipase A₂ inhibitor, mepacrine. Nevertheless, in contrast to the wild-type CCK₂ receptor, addition of CCK₈ to cells transfected with the F347A or K333M/K334T/R335L mutants did not result in the production of inositol phosphates while the maximum increase in this second messenger formation was reduced by 30% with the D100N mutant. Taken together, these results suggest that the CCK₂ receptor is coupled to two G proteins and that Phe347 and the cluster of basic residues K333/K334/R335 probably play a key role in G_q protein stimulation leading to inositol phosphate production but not in activation of the G protein coupled to phospholipase A₂. These data bring additional support at the molecular level to the existence of different affinity states of CCK₂ receptors suggested from the results of binding assays and behavioural studies.