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. 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). Posttranslational processing of CCK involves sulfation of the tyrosine at position seven from the C-terminal and ␣-amidation of the C-terminal phenylalanine residue (1). Studies using chemically synthesized fragments have shown that the C-terminal sulfated and amidated octapeptide Asp-Tyr(SO 3 H)-Met-GlyTrp-Met-Asp-Phe-NH 2 (Fig. 1) exhibits the full spectrum of biological activity. However, fragments as small as the C-terminal tetrapeptide Trp-Met-Asp-Phe-NH 2 , which CCK has in common with the related peptide gastrin, retain biological activity (2).The actions of CCK are mediated by membrane receptors that are divided into two subtypes, the CCK-A and the CCK-B/gastrin (3). 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␣...
Solid-Phase Synthesis of Chiral 3-Substituted Quinazoline-2,4-diones.-A series of N-urethane precursors such as (I) or (III) are prepared and subjected to cyclization in the presence of base (DBU, NaOH or NEt3). The desired quinazolines are obtained in good to excellent yield without racemization. The successful results are transformed to analogous syntheses on a polymer support. -(GOUILLEUX, L.; FEHRENTZ, J.-A.; WINTERNITZ, F.; MARTINEZ, J.; Tetrahedron Lett. 37 (1996) 39, 7031-7034; Lab. Aminoacid., Pept. Proteines, CNRS, Fac. Pharm., Univ. Montpellier I et II, F-34060 Montpellier, Fr.; EN)
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