Differential docking of high-affinity peptide ligands to type A and B cholecystokinin receptors demonstrated by photoaffinity labeling.
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 (EC(50) values: Bpa(24) probe, 41 +/- 9 pM; Bpa(33) probe, 15 +/- 3.3 pM). They bound specifically, with high affinity (K(i) values: Bpa(24) probe, 0.60 +/- 0.17 nM; Bpa(33) 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 Phe(122); Bpa(33) probe labeled Thr(119)). This provides a direct demonstration of distinct modes of docking the same high-affinity ligand to highly homologous receptors.
Dong
Maoqing
M
Mayo Clinic Scottsdale, Cancer Center and Department of Molecular Pharmacology and Experimental Therapeutics, Scottsdale, Arizona 85259, USA.
Liu
Guangming
G
Pinon
Delia I
DI
Miller
Laurence J
LJ
D000595:Amino Acid Sequence; D000818:Animals; D001577:Benzophenones / Q000737:chemistry / Q000378:metabolism*; D016466:CHO Cells; D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006224:Cricetinae; D005110:Extracellular Space / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010449:Peptide Mapping; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D019920:Photoaffinity Labels / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D043886:Receptor, Cholecystokinin A / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D043887:Receptor, Cholecystokinin B / Q000819:agonists / Q000235:genetics / Q000378:metabolism*
C488060:4-benzoylphenylalanine; D001577:Benzophenones; D008024:Ligands; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D043886:Receptor, Cholecystokinin A; D043887:Receptor, Cholecystokinin B; D010649:Phenylalanine