Pinon, Delia I.

Partial agonists are effective tools for advancing development of highly selective drugs and providing insights into molecular regulation of cellular functions. Here, we explore the impact of a partial agonist on key aspects of cholecystokinin (CCK) receptor regulation, its lateral mobility and cellular trafficking, in native pancreatic acinar cells and Chinese hamster ovary cells expressing CCK receptor (CHO-CCKR). We developed and characterized a novel fluorescent partial agonist, rhodamine-Gly-[(Nle28,31)CCK-26–32]-phenethyl ester, that binds specifically and with high affinity to CCK receptors. Such analogs are fully efficacious pancreatic acinar cell secretagogues without supramaximal inhibition that mobilize intracellular calcium with little or no increase in phospholipase C (PLC) activity. Despite minimal phosphorylation of CCK receptors in response to this partial agonist, receptor trafficking was the same as that observed with full agonist (CCK). This included normal internalization via clathrin-dependent endocytosis in CHO-CCKR cells and insulation on the surface of pancreatic acinar cells. Also, as with CCK-occupied receptor, fluorescence recovery after photobleaching of partial agonist-occupied receptor on the acinar cell surface demonstrated a marked temperature-dependent slowing of its rate of diffusion. This was similarly associated with resistance to acid-induced dissociation of ligand. Thus some key molecular regulatory mechanisms for CCK receptor internalization and insulation may be initiated by cellular signaling cascades that are not dependent on PLC activation or receptor phosphorylation.

The glucagon-like peptide-1 receptor (GLP-1R) is a family B G protein-coupled receptor and an important drug target for the treatment of type II diabetes, with activation of pancreatic GLP-1Rs eliciting glucose-dependent insulin secretion. Currently, approved therapeutics acting at this receptor are peptide based, and there is substantial interest in small molecule modulators for the GLP-1R. Using a variety of resonance energy transfer techniques, we demonstrate that the GLP-1R forms homodimers and that transmembrane helix 4 (TM4) provides the primary dimerization interface. We show that disruption of dimerization using a TM4 peptide, a minigene construct encoding TM4, or by mutation of TM4, eliminates G protein-dependent high-affinity binding to GLP-1(7-36)NH 2 but has selective effects on receptor signaling. There was <10-fold decrease in potency in cAMP accumulation or ERK1/2 phosphorylation assays but marked loss of intracellular calcium mobilization by peptide agonists. In contrast, there was near-complete abrogation of the cAMP response to an allosteric agonist, compound 2, but preservation of ERK phosphorylation. Collectively, this indicates that GLP-1R dimerization is important for control of signal bias. Furthermore, we reveal that two small molecule ligands are unaltered in their ability to allosterically modulate signaling from peptide ligands, demonstrating that these modulators act in cis within a single receptor protomer, and this has important implications for small molecule drug design.

While it is evident that the carboxyl‐terminal region of natural peptide ligands bind to the amino‐terminal domain of class B GPCRs, how their biologically critical amino‐terminal regions dock to the receptor is unclear. We utilize cysteine trapping to systematically explore spatial approximations among residues in the first five positions of secretin and in every position within the receptor extracellular loops (ECLs). Only Cys 2 and Cys 5 secretin analogues exhibited full activity and retained moderate binding affinity (IC 50 : 92±4 and 83±1 nM, respectively). When these peptides probed 61 human secretin receptor cysteine‐replacement mutants, a broad network of receptor residues could form disulfide bonds consistent with a dynamic ligand‐receptor interface. Two distinct patterns of disulfide bond formation were observed: Cys 2 predominantly labeled residues in the amino terminus of ECL2 and ECL3 (relative labeling intensity: Ser 340 , 94±7%; Pro 341 , 84±9%; Phe 258 , 73±5%; Trp 274 62±8%), and Cys 5 labeled those in the carboxyl terminus of ECL2 and ECL3 (Gln 348 , 100%; Ile 347 , 73±12%; Glu 342 , 59±10%; Phe 351 , 58±11%). These constraints were utilized in molecular modeling, providing improved understanding of the structure of the transmembrane bundle and interconnecting loops, the orientation between receptor domains, and the molecular basis of ligand docking. Key spatial approximations between peptide and receptor predicted by this model (H 1 ‐W 274 , D 3 ‐N 268 , G 4 ‐F 258 ) were supported by mutagenesis and residue‐residue complementation studies.—Dong, M., Xu, X., Ball, A. M., Makhoul, J. A., Lam, P. C.‐H., Pinon, D. I., Orry, A., Sexton, P. M., Abagyan, R., Miller, L. J. Mapping spatial approximations between the amino terminus of secretin and each of the extracellular loops of its receptor using cysteine trapping. FASEB J. 26, 5092–5105 (2012). www.fasebj.org

Fluorescence spectroscopic studies are powerful tools for the evaluation of receptor structure and the dynamic changes associated with receptor activation. Here, we have developed two chemically distinct fluorescent probes of the cholecystokinin (CCK) receptor by attaching acrylodan or a nitrobenzoxadiazole moiety to the amino terminus of a partial agonist CCK analogue. These two probes were able to bind to the CCK receptor specifically and with high affinity, and were able to elicit only submaximal intracellular calcium responses typical of partial agonists. The fluorescence characteristics of these probes were compared with those previously reported for structurally-related full agonist and antagonist probes. Like the previous probes, the partial agonist probes exhibited longer fluorescence lifetimes and increased anisotropy when bound to the receptor than when free in solution. The receptor-bound probes were not easily quenched by potassium iodide, suggesting that the fluorophores were protected from the extracellular aqueous milieu. The fluorescence characteristics of the partial agonist probes were quite similar to those of the analogous full agonist probes and quite distinct from the analogous antagonist probes. These data suggest that the partially activated conformational state of this receptor is more closely related to its fully active state than to its inactive state.