Mutational analysis of the putative devazepide binding site of the CCKA receptor

Abstract

Recently a molecular model was proposed lor the binding site of the antagonist 3M -)-/V-(2,3-dihydro-l-mediyl-2-oxo-5~phenyl-l H-1,4-benzodia/epine-3 s I) I // mdole 2-c^'hoxamide (deva/epide) on the cholecystokinin-A (CCK A) receptor (Van der Bent et al., 1994. Drug Design Discov. 12, 129 14X). Fifteen amino acids were identified, including hydrophilic ones such as Seru '\ Asivw and S e r'71\ that might interact with the caihoxamide moiety in deva/epide. To provide mutational evidence for this model, wild-type and mutant receptors (S139A, N349A and S379A) were transiently expressed and compared with respect to the ability of devazepide to inhibit binding of radiolabelled cholecystokinin <2(> 33)^peptkle amide (CCK^S) mid CCK-H-evoked (V 1 mobilization. The data presented suggest the involvement of the three residues in antagonist binding, although to a different extent. However, it does not seem likely that hydrogen bonds are the driving force in view of the relatively minor changes in receptor affinity and activity. £9 1997 Elsevier Science B.V.

Arginine 336 and Asparagine 333 of the Human Cholecystokinin-A Receptor Binding Site Interact with the Penultimate Aspartic Acid and the C-terminal Amide of Cholecystokinin

“…In contrast, mutation of an Asn residue of the rat CCK-AR that corresponds to Asn-333 in the human CCK-AR was reported to affect receptor sensitivity to both agonist (CCK) and nonpeptide antagonist (L-364,718). The authors suggested that the observed effects were due to receptor expression default (33). The results from our study confirmed that exchange of Asn-333 for an Ala diminishes receptor expression at the cell surface.…”

Refinement of the Conformation of a Critical Region of Charge-Charge Interaction between Cholecystokinin and Its Receptor

“…The right includes the densitometric analysis of data from three independent experiments (means Ϯ S.E.M.). DTVSAEKHLSGT --HQGSDFGP of helices within the lipid bilayer (Smeets et al, 1997). It is possible that Glu or Asp substitution at position 197 altered the amino terminus conformation and interactions with the extracellular loops, thus enhancing access to the nonpeptidyl binding site while simultaneously disrupting critical determinants for peptide binding within the loop domains.…”

“…Rather than ruling out such an interaction, these results probably reflect conformational changes that prevent effective spatial approximation of the residues. Experimental evidence for this included the clear allosteric effect of charge reversal in the position of Arg 197 on facilitation of binding of the benzodiazepine antagonist, L-364,718, that binds within the helical bundle in the lipid bilayer (Smeets et al, 1997). These mutants also displayed enhanced sensitivity to trypsin degradation relative to the wild-type receptor.…”

Fluorescence Polarization Screening for Allosteric Small Molecule Ligands of the Cholecystokinin Receptor

“…15,16 Its fluorescence characteristics are described in the current work. These data reveal an emission maximum of the receptorbound state of this ligand at 333 nm, when excited by light at 290 nm.…”

“…14 One such class of nonpeptidyl ligands is the 1,5-benzodiazepine derivatives, which have been shown to dock within the intramembranous helical bundle domain. 15,16 This domain has been demonstrated to represent an allosteric site of action, which has been shown to be fully distinct from the orthosteric site of action of the natural peptide ligand of this receptor. 17 By definition, allosteric ligands bind to receptor sites that are distinct from the site of binding of the natural orthosteric ligand.…”