The knowledge of the binding sites of G protein-coupled
cholecystokinin receptors represents important insights
that may serve to understand their activation processes
and to design or optimize ligands. Our aim was to identify
the amino acid of the cholecystokinin-A receptor (CCK-AR)
binding site in an interaction with the sulfate of CCK,
which is crucial for CCK binding and activity. A three-dimensional
model of the [CCK-AR-CCK] complex was built.
In this model, Arg197 was the best candidate residue for
a ionic interaction with the sulfate of CCK. Arg197 was
exchanged for a methionine by site-directed mutagenesis.
Wild-type and mutated CCK-AR were transiently expressed
in COS-7 cells for pharmacological and functional analysis.
The mutated receptor on Arg197 did not bind the agonist radioligand
125I-BH-[Thr, Nle]-CCK-9; however, it
bound the nonpeptide antagonist [3H]-SR27,897
as the wild-type receptor. The mutant was ≅1,470-
and 3,200-fold less potent than the wild-type CCK-AR to
activate G proteins and to induce inositol phosphate production,
respectively. This is consistent with the 500-fold lower
potency and 800-fold lower affinity of nonsulfated CCK
relative to sulfated CCK on the wild-type receptor. These
data, together with those showing that the mutated receptor
failed to discriminate nonsulfated and sulfated CCK while
it retained other pharmacological features of the CCK-AR,
strongly support an interaction between Arg197 of the CCK-AR
binding site and the sulfate of CCK. In addition, the mutated
CCK-AR resembled the low affinity state of the wild-type
CCK-AR, suggesting that Arg197–sulfate interaction
regulates conformational changes of the CCK-AR that are
required for its physiological activation.