Use of photoaffinity probes containing poly(ethylene glycol) spacers for topographical mapping of the cholecystokinin receptor complex

Abstract

To further define the structure of the pancreatic cholecystokinin (CCK) receptor and the topographical distance relationships between its subunits, we developed a series of monofunctional photoaffinity probes in which a fixed receptor-binding domain was separated from a photolabile nitrophenylacetamido group by defined lengths of a flexible spacer. The well-characterized CCK receptor radioligand 125I-D-Tyr-Gly-[(Nle28,31)CCK-26-33] provided the receptor-binding component of the probes, while the polymer poly(ethylene glycol) (2, 4, 7, and 10 monomer units long) was used as the spacer. The patterns of affinity labeling of rat pancreatic plasma membranes were examined as a function of spacer length. This ranged from 7.3 to 16.2 A, as calculated by root-mean-square end-to-end distances and validated experimentally by time-resolved fluorescence resonance energy transfer measurements. All probes in the series specifically labeled the Mr = 85,000-95,000 glycoprotein with Mr = 42,000 core, which has been proposed to contain the hormone recognition site. In addition, when the spacer length reached 16.2 A, membrane proteins of Mr = 80,000 and Mr = 40,000 were specifically labeled. The product of endo-beta-N-acetylglucosaminidase F digestion of the Mr = 80,000 protein was Mr = 65,000, similar to a protein previously identified in affinity labeling experiments using a CCK-33-based probe. These observations are consistent with the Mr = 85,000-95,000 pancreatic protein representing the hormone-binding subunit of the CCK receptor, while proteins of Mr = 80,000 and Mr = 40,000 may represent noncovalently associated subunits sited within 16.2 A of the binding domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulation of a G protein-coupled receptor on the plasmalemmal surface of the pancreatic acinar cell.

“…The CCK analogue, GIy-([Nle28,al]CCK [26][27][28][29][30][31][32][33], and its rhodamine and colloidal gold-conjugates and radiolabeled forms were synthesized in our laboratory as we previously described (33,34,37). The fluorescent CCK analogue (Rho-CCK) has been demonstrated to be a fully efficacious agonist with slightly reduced potency compared with native CCK-8, but with high receptor binding affinity (37).…”

“…In this report, we have explored the nature of cellular handling of the cholecystokinin (CCK) receptor expressed as a native protein on the rat pancreatic acinar cell. This receptor has been the focus of a large number of studies of structure (25,(30)(31)(32)(33)38), function (26), and regulation (18,27), and desensitization of CCK responsiveness in these cells has been clearly demonstrated (1). The CCK receptor is a G protein--coupled receptor in the [~-adrenergic receptor family which is physiologically important in the hormonal regulation of zymogen secretion from the pancreatic acinar cell after ingestion of a meal.…”

Entropy-Driven Supramolecular Ring-Opening Polymerization of a Cyclic Hemoglobin Monomer for Constructing a Hemoglobin–PEG Alternating Polymer with Structural Regularity

“…On the other hand, the root-mean-squared end-to-end distances (rrms) of free random chains of 2, 5, 10, and 20 kDa PEGs can be estimated theoretically as 35, 51, 79, and 111 Å, respectively (Supporting Information). 49 Each d value was found to be fairly close to the corresponding rrms value, but ca. 1.5 times larger.…”

Preparation and characterization of conjugates of monoclonal antibodies and staphylococcal enterotoxin A using a new hydrophilic crosslinker

“…Polymers with ethylene oxide units, polyethylene glycols, have several advantages compared to other spacers. They do not interfere with the conformation of peptides or proteins to which they are attached and they have amphiphilic characteristics enhancing solubility in both water and polar organic solvents (23).…”