Abstract
Sulfation of the tyrosine at the seventh position from the C terminus of cholecystokinin (CCK) is crucial for CCK binding to the CCK-A receptor. Using three-dimensional modeling, we identified methionine 195 of the CCK-A receptor as a putative amino acid in interaction with the aromatic ring of the sulfated tyrosine of CCK. We analyzed the role played by the two partners of this interaction. The exchange of Met-195 for a leucine caused a minor decrease (2. 8-fold) on the affinity of the high affinity sites for sulfated CCK-9, a strong drop (73%) of their number, and a 30-fold decrease on the affinity of the low and very low affinity sites for sulfated CCK-9, with no change in their number. The mutation also caused a 54-fold decrease of the potency of the receptor to induce inositol phosphates production. The high affinity sites of the wild-type CCK-A receptor were highly selective (800-fold) toward sulfated versus nonsulfated CCK, whereas low and very low affinity sites were poorly selective (10- and 18-fold). In addition, the M195L mutant bound, and responded to, sulfated CCK analogues with decreased affinities and potencies, whereas it bound and responded to nonsulfated CCK identically to the wild-type receptor. Thus, Met-195 interacts with the aromatic ring of the sulfated tyrosine to correctly position the sulfated group of CCK in the binding site of the receptor. This interaction is essential for CCK-dependent transition of the CCK-A receptor to a high affinity state. Our data should represent an important step toward the identification of the residue(s) of the receptor in interaction with the sulfate moiety of CCK and the understanding of the molecular mechanisms that govern CCK-A receptor activation.
Highlights
The peptide cholecystokinin (CCK)1 is found throughout the gastrointestinal system and the central nervous system where it acts both as a hormone and a neurotransmitter [1]
We present in this work an optimized threedimensional model of the CCK agonist1⁄7CCK-A receptor complex, which enabled us to propose that the residue methionine 195, located in the second extracellular loop of the CCK-A receptor, may interact with the tyrosyl residue of CCK
The key amino acid of the receptor involved in this additional interaction is the methionine 195 located in the second extracellular loop of the CCK-A receptor
Summary
The peptide cholecystokinin (CCK) is found throughout the gastrointestinal system and the central nervous system where it acts both as a hormone and a neurotransmitter [1]. The cloning of the cDNA coding for these receptors has shown that they belong to the superfamily of G protein-coupled receptors which are characterized by seven transmembrane domains connected by intracellular and extracellular loops with an extracellular N-terminal and intracellular C-terminal [4, 5] Both receptor subtypes can exist in several affinity states for sulfated CCK and have in common the functional coupling to phospholipase-C, presumably via binding to a G␣q/11 heterotrimeric GTP-binding protein (6 –9). We present in this work an optimized threedimensional model of the CCK agonist1⁄7CCK-A receptor complex, which enabled us to propose that the residue methionine 195, located in the second extracellular loop of the CCK-A receptor, may interact with the tyrosyl residue of CCK Such interactions between an aromatic ring and neighboring functional groups have been previously found in several proteins
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