Direct demonstration of unique mode of natural peptide binding to the type 2 cholecystokinin receptor using photoaffinity labeling

Abstract

Direct analysis of mode of peptide docking using intrinsic photoaffinity labeling has provided detailed insights for the molecular basis of cholecystokinin (CCK) interaction with the type 1 CCK receptor. In the current work, this technique has been applied to the closely related type 2 CCK receptor that also binds the natural full agonist peptide, CCK, with high affinity. A series of photolabile CCK analog probes with sites of covalent attachment extending from position 26 through 32 were characterized, with the highest affinity analogs that possessed full biological activity utilized in photoaffinity labeling. The position 29 probe, incorporating a photolabile benzoyl-phenylalanine in that position, was shown to bind with high affinity and to be a full agonist, with potency not different from that of natural CCK, and to covalently label the type 2 CCK receptor in a saturable, specific and efficient manner. Using proteolytic peptide mapping, mutagenesis, and radiochemical Edman degradation sequencing, this probe was shown to establish a covalent bond with type 2 CCK receptor residue Phe¹²⁰ in the first extracellular loop. This was in contrast to its covalent attachment to Glu³⁴⁵ in the third extracellular loop of the type 1 CCK receptor, directly documenting differences in mode of docking this peptide to these receptors.

Keywords: Cholecystokinin; G protein-coupled receptors; Ligand binding; Photoaffinity labeling; Type 2 cholecystokinin receptor.

Fig.1

Primary structure of the Bpa²⁹ probe. Shown are primary amino acid sequences of natural CCK-8 (CCK-26-33) and the Bpa²⁹ probe. Natural residues are illustrated in clear circles, whereas modified residues are filled with grey, or with black in the case of the photolabile Bpa site of covalent labeling.

Fig. 2

Functional characterization of CCK probes incorporating photolabile residues in a variety of positions. Shown in the left panel are competition-binding curves using CCK and all listed photolabile CCK probes to compete for the binding of the CCK-like radioligand to CHO-CCK2R membranes. Values illustrated represent binding as percentages of saturable binding in the absence of the competing peptide, and are expressed as the means ± S.E.M. of data from three independent experiments performed in duplicate. The order of the peptides in the list corresponds to their order of highest to lowest affinity. Shown in the right panel are intracellular calcium responses to increasing concentrations of CCK and all the listed photolabile CCK probes in these cells. Data points represent the means ± S.E.M. of data from three independent experiments performed in duplicate, normalized relative to the maximal responses to CCK. The order of the peptides in the list corresponds to their order of highest to lowest potency.

Fig. 3

Photoaffinity labeling of the type 2 CCK receptor. Shown are representative autoradiographs of 10% SDS-PAGE gels used to separate the products of photoaffinity labeling of membranes from CHOCCK2R cells with the Bpa²⁹ probe in the presence of increasing concentrations of competing CCK, as well as the deglycosylation product by PNGase F (left panel). As a control, labeling of the non-receptor-bearing CHO cell membranes in the absence of competitor is also shown. Also shown is the densitometric analysis of data from three similar experiments, with data points expressed as means ± S.E.M (right panel).

Fig. 4

CNBr cleavage of wild type and mutant type 2 CCK receptor. Shown is a diagram of the predicted sites of CNBr cleavage of the human type 2 CCK receptor and the calculated masses of resultant fragments. Shown is a representative autoradiograph of a 10% NuPAGE gel used to separate the products of CNBr cleavage of the type 2 CCK receptor labeled with the Bpa²⁹ probe (middle panel). This cleavage resulted in a labeled band migrating at approximate M_r =4,000 that did not shift after deglycosylation by PNGase F. CNBr cleavage of the labeled wild type and M134L mutant receptor membranes are compared in the panel on the right. The band from the M134L mutant receptor was larger than that from the wild type receptor, migrating at approximate M_r = 8,500.

Fig. 5

Identification of receptor residue labeled with the Bpa²⁹ probe. Shown is a representative profile of radioactivity eluted during Edman degradation of the labeled band. The peak in radioactivity was found in cycle 3, corresponding to the labeling of receptor residue Phe¹²⁰ within the first extracellular loop of the type 2 CCK receptor.

Fig. 6

Graphic representation of sites of photoaffinity labeling of the type 1 and type 2 CCK receptors through photolabile moieties spread along the peptide pharmacophore. Shown is a two-dimensional representation of the topology of the CCK receptors with sites of covalent labeling highlighted. Colors are utilized to code for labeled residues within the amino terminus (green), ECL1 (yellow), ECL2 (blue), and ECL3 (red).