A Type 1 Cholecystokinin Receptor Mutant That Mimics the Dysfunction Observed for Wild Type Receptor in a High Cholesterol Environment

• Published in: The Journal of biological chemistry Vol. 289; no. 26; pp. 18314 - 18326
• Main Authors: Desai, Aditya J., Harikumar, Kaleeckal G., Miller, Laurence J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 27.06.2014; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Animals; CHO Cells; Cholecystokinin; Cholesterol - chemistry; Cholesterol - metabolism; Cricetulus; G Protein-coupled Receptor (GPCR); Humans; Kinetics; Membrane Lipid; Models, Molecular; Mutation, Missense; Peptide Hormone; Protein Binding; Radioreceptor Assays; Receptor Binding; Receptor, Cholecystokinin A - chemistry; Receptor, Cholecystokinin A - genetics; Receptor, Cholecystokinin A - metabolism; Signal Transduction; Signal Transduction; Receptor Binding; Membrane Lipid; Radioreceptor Assays; Cholecystokinin; G Protein-coupled Receptor (GPCR); Peptide Hormone
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M114.570200

Cholecystokinin (CCK) stimulates the type 1 CCK receptor (CCK1R) to elicit satiety after a meal. Agonists with this activity, although potentially useful for treatment of obesity, can also have side effects and toxicities of concern, making the development of an intrinsically inactive positive allosteric modulator quite attractive. Positive allosteric modulators 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 G protein-coupled receptor-specific (E/D)RY 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 nonhydrolyzable GTP analog, and anisotropy of a bound fluorescent CCK analog. 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 the 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 by using a small molecule allosteric ligand, thereby providing safe and effective correction of the coupling defect in metabolic syndrome. Background: Type 1 CCK receptor function is affected by a high cholesterol environment. Results: The CCK1R Y140A mutant has ligand binding and activation characteristics similar to wild type CCK1R in high cholesterol. Conclusion: This mutant mimics CCK1R structure in high cholesterol. Significance: This mutant represents a powerful and unique tool for identification of ligands to correct the abnormal conformation of CCK1R in high cholesterol.