Differential docking of high-affinity peptide ligands to type A and B cholecystokinin receptors demonstrated by photoaffinity labeling

Maoqing Dong, Guangming Liu, Delia I. Pinon, Laurence J. Miller

Research output: Contribution to journalArticle

19 Scopus citations

Abstract

Type A and B cholecystokinin (CCK) receptors are highly homologous members of the class-I family of G protein-coupled receptors that bind CCK with high affinity. However, they have divergent structural specificities, with the type A receptor requiring seven carboxyl-terminal residues including a sulfated tyrosine and the type B receptor requiring only the carboxyl-terminal tetrapeptide. The aim of this work was to utilize affinity labeling to determine spatial approximations with photolabile p-benzoyl-L-phenylalanine (Bpa) residues sited at each end of CCK as docked at the type B CCK receptor, contrasting this with analogous work using similar probes docked at the type A receptor. Both probes were fully efficacious, potent agonists that stimulated intracellular calcium in receptor-bearing CHO-CCKBR cells (EC50 values: Bpa24 probe, 41 ± 9 pM; Bpa33 probe, 15 ± 3.3 pM). They bound specifically, with high affinity (Ki values: Bpa24 probe, 0.60 ± 0.17 nM; Bpa33 probe, 0.58 ± 0.11 nM). Cyanogen bromide cleavage of the covalently labeled receptor suggested the first extracellular loop as the region of labeling by each probe, distinct from the type A CCK receptor regions labeled using the same probes (third loop and amino-terminal tail, respectively). This was confirmed by subsequent enzymatic and chemical cleavage of labeled wild-type and mutant receptors. Sequential cycles of Edman degradation of labeled receptor fragments identified the specific residues within loop one labeled by each probe (Bpa 24 probe labeled Phe122; Bpa33 probe labeled Thr119). This provides a direct demonstration of distinct modes of docking the same high-affinity ligand to highly homologous receptors.

Original languageEnglish (US)
Pages (from-to)6693-6700
Number of pages8
JournalBiochemistry
Volume44
Issue number17
DOIs
StatePublished - May 3 2005

ASJC Scopus subject areas

  • Biochemistry

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