Oligomerization of the Class II G protein-coupled secretin receptor has been reported, but the molecular basis for this and its functional significance have not been determined. In the current work, we have examined the possible contribution of each of the transmembrane (TM) segments of this receptor to its homo-oligomerization, using the method of competitive disruption screening for inhibition of receptor bioluminescence resonance energy transfer signal. TM IV was the only segment that was found to disrupt receptor bioluminescence resonance energy transfer. Evaluation of predicted interhelical and lipidexposed faces of this TM segment demonstrated that its lipidexposed face represented the determinant for oligomerization. This was further confirmed by mutagenesis of the intact secretin receptor. Morphological FRET was utilized to demonstrate that secretin receptor oligomerization occurred at the cell surface and that this oligomerization was disrupted by mutating Gly 243 and Ile 247 , key residues within the lipid-exposed face of TM IV. Although disruption of the receptor oligomerization interface had no effect on secretin binding parameters, it reduced the ability of secretin to stimulate intracellular cAMP. This supports a clear functional effect of oligomerization of this receptor. Such an effect might be particularly relevant to clinical situations in which this receptor is overexpressed, such as in certain neoplasms.Dimerization of plasma membrane receptors represents a timely theme of substantial interest and potential importance (1). For single transmembrane tyrosine kinase receptors, dimerization has been shown to provide a molecular mechanism for cross-molecular phosphorylation and receptor regulation (2). For some G protein-coupled receptors, dimerization or oligomerization has also been demonstrated, although the rules for establishment of such complexes, the effects of ligand binding to these complexes, and its functional implications have been quite varied (3-6). The role of such complexes in normal physiology is also unclear, with most demonstrations of G protein-coupled receptor oligomerization occurring in the setting of receptor overexpression systems (7,8) We previously demonstrated the oligomerization of the Class II G protein-coupled secretin receptor, based on a positive bioluminescence resonance energy transfer (BRET) 2 signal from tagged receptors expressed in COS cells that was structurally specific, not shared with similar levels of overexpression of a Class I G protein-coupled receptor with the secretin receptor (9). Subsequently, using the same technique and complementing this with morphologic fluorescence resonance energy transfer (FRET) analysis, the secretin receptor oligomerization was shown to occur constitutively at the cell surface, without being disrupted by secretin agonist ligand binding (10). Further, the secretin receptor was shown to be capable of forming similar hetero-oligomers with structurally related VPAC1 and VPAC2 receptors (10). Of interest, expression of a secre...

]secretin-27 probe was a fully efficacious agonist, with a potency to stimulate cAMP accumulation by Chinese hamster ovary SecR cells similar to that of natural secretin (EC 50 ‫؍‬ 68 ؎ 22 pM analogue and 95 ؎ 25 pM secretin). It bound specifically and with high affinity (K i ‫؍‬ 5.0 ؎ 1.1 nM) and covalently labeled the M r ‫؍‬ 57,000-62,000 secretin receptor. Cyanogen bromide cleavage of the receptor yielded a major labeled fragment of apparent M r ‫؍‬ 19,000 that shifted to M r ‫؍‬ 9,000 after deglycosylation. This was most consistent with either of two glycosylated domains within the amino-terminal tail of the receptor. Immunoprecipitation with antibody directed to epitope tags incorporated into each of the candidate domains established that the fragment at the amino terminus of the receptor was the site of labeling. This was further localized to the amino-terminal 30 residues of the receptor by additional proteolysis of this fragment with endoproteinase Lys-C. This provides the first direct demonstration of a contact between a secretin-like agonist and its receptor and will contribute a useful constraint to the modeling of this interaction.The secretin receptor is prototypic of a recently recognized family (Class II) of guanine nucleotide-binding protein (G protein) 1 -coupled receptors (1). Members of this family are believed to have the seven-transmembrane segment topology typical of the superfamily, but they share Ͻ12% homology with the extensively studied Class I receptors in the rhodopsin/␤-adrenergic receptor family, and they lack the signature sequences of this family (2, 3). Secretin family receptors have long aminoterminal domains incorporating six highly conserved Cys residues, believed to contribute to disulfide bonds that help define the family (3, 4). Indeed, this complex domain has been suggested to play a key role in agonist binding, as suggested by receptor mutagenesis studies (5-9). Other extracellular loop domains have also been implicated in complementary roles for agonist binding and receptor activation (4,5,7,10). Natural ligands for this family of receptors are all peptides longer than 27 residues, with structure-activity series suggesting the presence of diffuse pharmacophoric domains (3). Although this large diffuse pharmacophore nicely complements the multiple domains predicted to be outside the membrane bilayer, there is no working model to predict how the two molecules might interact.In this work, we attempt to establish an initial constraint that will contribute to the development of a model for the interaction of secretin with its receptor. We do this through photoaffinity labeling. This has the theoretical advantage of directly probing the domain adjacent to the photolabile residue within the probe after it binds to the receptor. Using this approach, we have successfully identified two binding contacts between photolabile analogues of cholecystokinin and its receptor (11,12).In this work, we have developed an analogue of secretin that incorporates a site for radioiodination an...

Abstract. Receptor molecules play a major role in the desensitization of agonist-stimulated cellular responses. For G protein-coupled receptors, rapid desensitization occurs via receptor phosphorylation, sequestration, and internalization, yet the cellular compartments in which these events occur and their interrelationships are unclear. In this work, we focus on the cholecystokinin (CCK) receptor, which has been well characterized with respect to phosphorylation. We have used novel fluorescent and electron-dense CCK receptor ligands and an antibody to probe receptor localization in a CCK receptor-bearing CHO cell line. In the unstimulated state, receptors were diffusely distributed over the plasmalemma. Agonist occupation stimulated endocytosis via both clathrin-dependent and independent pathways. The former was predominant, leading to endosomal and lysosomal compartments, as well as recycling to the plasmalemma. The clathrin-independent processes led to a smooth vesicular compartment adjacent to the plasmalemma resembling caveolae, which did not transport ligand deeper within the cell. Potassium depletion largely eliminated clathrin-dependent endocytosis, while not interfering with agoniststimulated receptor movement into subplasmalemmal smooth vesicle compartments. These cellular endocytic events can be related to the established cycle of CCK receptor phosphorylation and dephosphorylation, which we have previously described (Klueppelberg,

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Distinct spatial approximations between residues within the secretin pharmacophore and its receptor can provide important constraints for modeling this agonist-receptor complex. We previously used a series of probes incorporating photolabile residues into positions 6, 12, 13, 14, 18, 22, and 26 of the 27-residue peptide and demonstrated that each covalently labeled a site within the receptor amino terminus. Although supporting a critical role of this domain for ligand binding, it does not explain the molecular mechanism of receptor activation. Here, we developed probes having photolabile residues at the amino terminus of secretin to explore possible approximations with a different receptor domain. The first probe incorporated a photolabile pbenzoyl-L-phenylalanine into the position of His 1 of rat secretin ([Bpa 1 ,Tyr 10 ]secretin-27). Because His 1 is critical for function, we also positioned a photolabile Bpa as an amino-terminal extension, in positions ؊1 (rat [Bpa ؊1 ,Tyr 10 ]secretin-27) and ؊2 (rat [Bpa ؊2 ,Gly ؊1 ,-Tyr 10 ]secretin-27). Each analog was shown to be a full agonist, stimulating cAMP accumulation in receptorbearing Chinese hamster ovary-SecR cells in a concentration-dependent manner, with the position ؊2 probe being most potent. They bound specifically and saturably, although the position 1 analog had lowest affinity, and all were able to label the receptor efficiently. Sequential specific cleavage, purification, and sequencing demonstrated that the sites of covalent attachment for each probe were high within the sixth transmembrane segment. This suggests that secretin binding may exert tension between the receptor amino terminus and the transmembrane domain to elicit a conformational change effecting receptor activation.The secretin receptor is prototypic of the Class B family of guanine nucleotide-binding protein (G protein)-coupled receptors which includes many important drug targets. Understanding of the molecular basis of ligand binding of receptors is important for the rational design of receptor-active drugs. However, the molecular basis of ligand binding of Class B receptors is less well understood than that of members of the Class A family, such as rhodopsin and the adrenergic receptor. This likely reflects the facts that natural ligands for Class B G protein-coupled receptors are relatively large peptides with diffuse phamacophoric domains and that these receptors have long and complex amino-terminal domains that are important for binding. Both of these interacting domains are flexible, with active conformations that have not been clearly defined.One of the distinct characteristics of the Class B receptor family is the long amino terminus that exceeds 120 residues in length. It contains 6 conserved Cys residues that have been demonstrated to form intradomain disulfide bonds (1-5) and to be critical for ligand binding. These disulfide bonds could provide key constraints for building a model of the secretin receptor, but definitive mapping of these bonds in an active receptor has not...

Insight into the molecular basis of cholecystokinin (CCK) binding to its receptor has come from receptor mutagenesis and photoaffinity labeling studies, with both contributing to the current hypothesis that the acidic Tyr-sulfate-27 residue within the peptide is situated adjacent to basic Arg 197 in the second loop of the receptor. Here, we refine our understanding of this region of interaction by examining a structure-activity series of these positions within both ligand and receptor and by performing three-dimensional molecular modeling of key pairs of modified ligand and receptor constructs. The important roles of Arg 197 and Tyr-sulfate-27 were supported by the marked negative impact on binding and biological response with their natural partner molecule when the receptor residue was replaced by acidic Asp or Glu and when the peptide residue was replaced by basic Arg, Lys, p-amino-Phe, p-guanidino-Phe, or p-methylamino-Phe. Complementary ligand-receptor chargeexchange experiments were unable to regain the lost function.This was supported by the molecular modeling, which demonstrated that the charge-reversed double mutants could not form a good interaction without extensive rearrangement of receptor conformation. The models further predicted that R197D and R197E mutations would lead to conformational changes in the extracellular domain, and this was experimentally supported by data showing that these mutations decreased peptide agonist and antagonist binding and increased nonpeptidyl antagonist binding. These receptor constructs also had increased susceptibility to trypsin degradation relative to the wild-type receptor. In contrast, the relatively conservative R197K mutation had modest negative impact on peptide agonist binding, again consistent with the modeling demonstration of loss of a series of stabilizing inter-and intramolecular bonds. The strong correlation between predicted and experimental results support the reported refinement in the three-dimensional structure of the CCK-occupied receptor.

Fluorescence is a powerful biophysical tool for the analysis of the structure and dynamics of proteins. Here, we have developed two series of new fluorescent probes of the cholecystokinin (CCK) receptor, representing structurally related peptide agonists and antagonists. Each ligand had one of three distinct fluorophores (Alexa 488 , nitrobenzoxadiazolyl, or acrylodan) incorporated in analogous positions at the amino terminus just outside the hormone's pharmacophore. All of the probes bound to the CCK receptor specifically and with high affinity, and intracellular calcium signaling studies showed the chemically modified peptides to be fully biologically active. Quenching by iodide and measurement of fluorescence spectra, anisotropy, and lifetimes were used to characterize the response of the fluorescence of the probe in the peptide-receptor complex for agonists and antagonists. All three fluorescence indicators provided the same insights into differences in the environment of the same indicator in the analogous position for agonist and antagonist peptides bound to the CCK receptor. Each agonist had its fluorescence quenched more easily and showed lower anisotropy (higher mobility of the probe) and shorter lifetime than the analogous antagonist. Treatment of agonist-occupied receptors with a non-hydrolyzable GTP analogue shifted the receptor into its inactive low affinity state and increased probe fluorescence lifetimes toward values observed with antagonist probes. These data are consistent with a molecular conformational change associated with receptor activation that causes the amino terminus of the ligand (situated above transmembrane segment six) to move away from its somewhat protected environment and toward the aqueous milieu.Cell surface receptors present on essentially every excitable cell of the body are important targets for pharmacotherapy. A detailed understanding of the structure of these molecules and the molecular basis of their activation should contribute to the rational design and refinement of ligands for these receptors. Receptor-bound, environmentally sensitive fluorescent reporters can provide information regarding ligand-binding domains (1). In this work, we utilize this approach to gain insight into agonist-and antagonist-binding domains of the type A cholecystokinin (CCK) 1 receptor, a physiologically important member of the rhodopsin/␤-adrenergic receptor family of guanine nucleotide-binding protein (G protein)-coupled receptors.The superfamily of G protein-coupled receptors represents the largest group of membrane receptors. They are remarkable for the diversity in structure of the natural agonist ligands that can activate them and initiate intracellular signaling cascades. These range in size from small photons and odorants to peptides, proteins, and even large viral particles. Tertiary structure determination by x-ray crystallography has provided the most incisive insight into the structure of superfamily members, which bind small ligands in the intramembranous helical bundle domain ...