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

Abstract

A heterogeneity of CCK 2 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 CCK 2 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 CCK 8 . Activation of wild-type as well as F347A, D100N or K333M/K334T/R335L CCK 2 receptors by this ligand led to a similar arachidonic acid release which was blocked by pertussis toxin and the phospholipase A 2 inhibitor, mepacrine. Nevertheless, in contrast to the wild-type CCK 2 receptor, addition of CCK 8 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 CCK 2 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 A 2 . These data bring additional support at the molecular level to the existence of different affinity states of CCK 2 receptors suggested from the results of binding assays and behavioural studies.

Partial Agonism, Neutral Antagonism, and Inverse Agonism at the Human Wild-Type and Constitutively Active Cholecystokinin-2 Receptors

“…Our findings are in line with previous studies, which showed the importance of aromatic residues Tyr189 (TM4), Tyr192 (TM4), Phe227 (TM5), Phe342 (TM6), Trp346 (TM6), and Tyr350 (TM6) in the activation process of the CCK2R (Blaker et al, 1998;Jagerschmidt et al, 1998;Pommier et al, 2003;Langer et al, 2005). It is noteworthy that in other receptors, a hydrophobic cluster between transmembrane helices V and VI constrains the receptor in an inactive conformation via interhelical interactions and is involved in activation by agonists (Fanelli and De Benedetti, 2005).…”

“…Previous studies, including ours, provided evidence that the proximity of the phenylalanine side chain of CCK with the aromatic network of CCK2R binding site composed of amino acids Tyr189 (TM4), Tyr192 (TM4), Phe227 (TM5), Phe342 (TM6), Trp346 (TM6), and Tyr350 (TM6) was a key feature for CCK2R activation (Blaker et al, 1998;Jagerschmidt et al, 1998;Pommier et al, 2003;Langer et al, 2005). Analysis of the different complexes after docking and dynamic simulation procedure (Fig.…”

Molecular Mechanism Underlying Partial and Full Agonism Mediated by the Human Cholecystokinin-1 Receptor

“…3). Indeed, the triplebasic sequence Lys 308 -Lys 309 -Arg 310 , that is conserved in the CCK2R, has been shown to be without any importance on G␣ i /PLA2 activation, but its mutation abolished coupling to G␣ q /␣ 11 /PLC (35). As illustrated on Fig.…”

Cholecystokinin and Gastrin Receptors

“…Transmembrane residues of the CCK2R such as Asp of TM II, triple basic motif (KKR) at the COOH terminus of the third intracellular loop, and a Phe residue of TM VI were shown to play a key role in the coupling of CCK2R to phospholipase C (222,383,554). Interestingly, mutation of two of these amino acids or motifs, namely, the Phe of TM VI and the KKR motif of the third intracellular loops, were also demonstrated to be without importance for CCK2R-induced stimulation of arachidonic acid release, supporting distinct mechanisms of CCK2R coupling to phospholipase C and phospholipase A 2 (383). The chemical nature of residue 121 within TM III of the CCK1R seems to be very important for activation of the receptor.…”