The role of segment 32-47 of cholecystokinin receptor type A in CCK8 binding: Synthesis, nuclear magnetic resonance, circular dichroism and fluorescence studies

The segment 32-47 of the N-terminal extracellular domain of the type A cholecystokinin receptor, CCK_A-R(32-47), was synthesized and structurally characterized in a membrane mimicking environment by CD, NMR and molecular dynamics calculations. The region of CCK_A-R(32-47) encompassing residues 39-46 adopted a well-defined secondary structure in the presence of DPC micelles, whereas the conformation of the N-terminal region (segment 32-37) could not be uniquely defined by the NOE derived distance constraints because of local flexibility. The conformation of the binding domain of CCK_A-R(32-47) was different from that found for the intact N-terminal receptor tail, CCK_A -R(1-47). To assess whether CCK_A-R(32-47) was still able to bind the nonsulfated cholecystokinin C-terminal octapeptide, CCK8, a series of titrations was carried out in SDS and DPC micelles, and the binding interaction was followed by fluorescence spectroscopy. These titrations gave no evidence for complex formation, whereas a high binding affinity was found between CCK_A-R(1-47) and CCK8. The different affinities for the ligand shown by CCK_A-R(32-47) and CCK_A-R(1-47) were paralleled by different interaction modes between the receptor segments and the micelles. The interaction 6of CCK_A-R(32-47) with DPC micelles was much weaker than that of CCK_A-R(1-47), because the former receptor segment lacks proper stabilizing contacts with the micelle surface. In the case of SDS micelles CCK_A-R(32-47) was found to form non-micellar adducts with the detergent that prevented the onset of a functionally significant interaction between the receptor segment and the micelle. It is concluded that tertiary structure interactions brought about by the 1-31 segment play a key role in the stabilization of the membrane bound, biologically active conformation of the N-terminal extracellular tail of the CCK_A receptor.