Abstract:
:Understanding the structures of active and inactive agonist- and antagonist-bound receptor complexes is of great interest. In this work, we focus on position 30 of cholecystokinin (CCK) and its spatial approximation with the type A CCK receptor. For this, we developed two photoaffinity labeling probes, replacing the naturally occurring tryptophan with p-benzoyl-l-phenylalanine (Bpa) or p-nitro-phenylalanine (NO(2)-Phe). The Bpa probe was shown to represent an antagonist, whereas the NO(2)-Phe probe stimulated intracellular calcium as a fully efficacious agonist (EC(50) = 81 +/- 15 nM). Both ligands bound to the receptor specifically, although with lower affinity than CCK (K(i) values: Bpa probe, 270 +/- 72 nM; NO(2)-Phe probe, 180 +/- 40 nM). Both probes covalently labeled the receptor in an efficient manner. The Bpa antagonist labeled the receptor in two distinct regions as identified by cyanogen bromide cleavage, with labeled bands migrating at M(r) = 25,000 and 4500. The former represented the glycosylated amino-terminal fragment, with the site of attachment further localized by endoproteinase Lys-C cleavage to the region between Asn(10) and Lys(37). The latter was shown to represent the first extracellular loop using further cleavage and sequencing of the wild-type and a mutant receptor. Following the same approach, the NO(2)-Phe agonist probe was shown to also label the first extracellular loop region. Radiochemical sequencing identified that the Bpa antagonist probe labeled receptor residue Lys(105), whereas the NO(2)-Phe agonist probe labeled residue Leu(99). These data extend our understanding of the molecular basis of binding and the conformational states of this important receptor.
journal_name
Mol Pharmacoljournal_title
Molecular pharmacologyauthors
Dong M,Hadac EM,Pinon DI,Miller LJdoi
10.1124/mol.105.012179keywords:
subject
Has Abstractpub_date
2005-06-01 00:00:00pages
1892-900issue
6eissn
0026-895Xissn
1521-0111pii
mol.105.012179journal_volume
67pub_type
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