A type 1 CCK receptor mutant that mimics the dysfunction observed for wild type receptor in a high cholesterol environment [Protein Structure and Folding]

May 13th, 2014 by Desai, A. J., Harikumar, K. G., Miller, L. J.

Cholecystokinin (CCK) stimulates the type 1 CCK receptor (CCK1R) to elicit satiety after a meal. Agonists with this activity, while potentially useful for treatment of obesity, also can have side effects and toxicities of concern, making development of an intrinsically inactive positive allosteric modulator (PAM) quite attractive. PAMs also have the potential to correct the defective receptor-G protein coupling observed in the high membrane cholesterol environment described in metabolic syndrome. Current model systems to study CCK1R in such an environment are unstable and expensive to maintain. We now report that the Y140A mutation within a cholesterol-binding motif and the conserved, class A GPCR-specific E/DRY signature sequence results in ligand binding and activity characteristics similar to wild type CCK1R in a high cholesterol environment. This is true for natural CCK, as well as ligands with distinct chemistries and activity profiles. Additionally, the Y140A construct also behaved like CCK1R in high cholesterol in regard to its internalization, sensitivity to a non-hydrolyzable GTP analogue, and anisotropy of a bound fluorescent CCK analogue. Chimeric CCK1R/CCK2R constructs that systematically changed the residues in the allosteric ligand-binding pocket were studied in the presence of Y140A. This established increased importance of unique residues within TM3 and reduced importance of TM2 for binding in the presence of this mutation, with the agonist trigger likely pulled away from its Leu356 target on TM7. The distinct conformation of this intramembranous pocket within Y140A CCK1R provides an opportunity to normalize this using a small molecule allosteric ligand, thereby providing safe and effective correction of the coupling defect in metabolic syndrome.
  • Posted in Journal of Biological Chemistry, Publications
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