Wnt5a promotes Frizzled-4 signalosome assembly by stabilizing cysteine-rich domain dimerization.

Wnt/ß-catenin signaling is activated when extracellular Wnt ligands bind Frizzled (FZD) receptors at the cell membrane. Wnts bind FZD cysteine-rich domains (CRDs) with high affinity through a palmitoylated N-terminal "thumb" and a disulfide-stabilized C-terminal "index finger," yet how these binding events trigger receptor activation and intracellular signaling remains unclear. Here we report the crystal structure of the Frizzled-4 (FZD4) CRD in complex with palmitoleic acid, which reveals a CRD tetramer consisting of two cross-braced CRD dimers. Each dimer is stabilized by interactions of one hydrophobic palmitoleic acid tail with two CRD palmitoleoyl-binding grooves oriented end to end, suggesting that the Wnt palmitoleoyl group stimulates CRD-CRD interaction. Using bioluminescence resonance energy transfer (BRET) in live cells, we show that WNT5A stimulates dimerization of membrane-anchored FZD4 CRDs and oligomerization of full-length FZD4, which requires the integrity of CRD palmitoleoyl-binding residues. These results suggest that FZD receptors may form signalosomes in response to Wnt binding through the CRDs and that the Wnt palmitoleoyl group is important in promoting these interactions. These results complement our understanding of lipoprotein receptor-related proteins 5 and 6 (LRP5/6), Dishevelled, and Axin signalosome assembly and provide a more complete model for Wnt signalosome assembly both intracellularly and at the membrane.

Single-molecule FRET imaging of GPCR dimers in living cells.

Class C G protein-coupled receptors (GPCRs) are known to form stable homodimers or heterodimers critical for function, but the oligomeric status of class A and B receptors, which constitute >90% of all GPCRs, remains hotly debated. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful approach with the potential to reveal valuable insights into GPCR organization but has rarely been used in living cells to study protein systems. Here, we report generally applicable methods for using smFRET to detect and track transmembrane proteins diffusing within the plasma membrane of mammalian cells. We leverage this in-cell smFRET approach to show agonist-induced structural dynamics within individual metabotropic glutamate receptor dimers. We apply these methods to representative class A, B and C receptors, finding evidence for receptor monomers, density-dependent dimers and constitutive dimers, respectively.

Structures of the human cholecystokinin 1 (CCK1) receptor bound to Gs and Gq mimetic proteins provide insight into mechanisms of G protein selectivity.

G protein-coupled receptors (GPCRs) are critical regulators of cellular function acting via heterotrimeric G proteins as their primary transducers with individual GPCRs capable of pleiotropic coupling to multiple G proteins. Structural features governing G protein selectivity and promiscuity are currently unclear. Here, we used cryo-electron microscopy (cryo-EM) to determine structures of the cholecystokinin (CCK) type 1 receptor (CCK1R) bound to the CCK peptide agonist, CCK-8 and 2 distinct transducer proteins, its primary transducer Gq, and the more weakly coupled Gs. As seen with other Gq/11-GPCR complexes, the Gq-α5 helix (αH5) bound to a relatively narrow pocket in the CCK1R core. Surprisingly, the backbone of the CCK1R and volume of the G protein binding pocket were essentially equivalent when Gs was bound, with the Gs αH5 displaying a conformation that arises from "unwinding" of the far carboxyl-terminal residues, compared to canonically Gs coupled receptors. Thus, integrated changes in the conformations of both the receptor and G protein are likely to play critical roles in the promiscuous coupling of individual GPCRs.

Crystal structure of the Frizzled 4 receptor in a ligand-free state.

Frizzled receptors (FZDs) are class-F G-protein-coupled receptors (GPCRs) that function in Wnt signalling and are essential for developing and adult organisms1,2. As central mediators in this complex signalling pathway, FZDs serve as gatekeeping proteins both for drug intervention and for the development of probes in basic and in therapeutic research. Here we present an atomic-resolution structure of the human Frizzled 4 receptor (FZD4) transmembrane domain in the absence of a bound ligand. The structure reveals an unusual transmembrane architecture in which helix VI is short and tightly packed, and is distinct from all other GPCR structures reported so far. Within this unique transmembrane fold is an extremely narrow and highly hydrophilic pocket that is not amenable to the binding of traditional GPCR ligands. We show that such a pocket is conserved across all FZDs, which may explain the long-standing difficulties in the development of ligands for these receptors. Molecular dynamics simulations on the microsecond timescale and mutational analysis uncovered two coupled, dynamic kinks located at helix VII that are involved in FZD4 activation. The stability of the structure in its ligand-free form, an unfavourable pocket for ligand binding and the two unusual kinks on helix VII suggest that FZDs may have evolved a novel ligand-recognition and activation mechanism that is distinct from that of other GPCRs.

Secretin Amino-Terminal Structure-Activity Relationships and Complementary Mutagenesis at the Site of Docking to the Secretin Receptor.

Class B1 G protein-coupled receptors are activated by peptides, with amino-terminal regions critical for biologic activity. Although high resolution structures exist, understanding of key features of the peptide activation domain that drive signaling is limited. In the secretin receptor (SecR) structure, interactions are observed between peptide residues His1 and Ser2 and seventh transmembrane segment (TM7) receptor residue E373. We interrogated these interactions using systematic structure-activity analysis of peptide and receptor. His1 was critical for binding and cAMP responses, but its orientation was not critical, and substitution could independently modify affinity and efficacy. Ser2 was also critical, with all substitutions reducing peptide affinity and functional responses proportionally. Mutation of E373 to conserved acidic Asp (E373D), uncharged polar Gln (E373Q), or charge-reversed basic Arg (E373R) did not alter receptor expression, with all exhibiting secretin-dependent cAMP accumulation. All position 373 mutants displayed reduced binding affinities and cAMP potencies for many peptide analogs, although relative effects of position 1 peptides were similar whereas position 2 peptides exhibited substantial differences. The peptide including basic Lys in position 2 was active at SecR having acidic Glu in position 373 and at E373D while exhibiting minimal activity at those receptors in which an acidic residue is absent in this position (E373Q and E373R). In contrast, the peptide including acidic Glu in position 2 was equipotent with secretin at E373R while being much less potent than secretin at wild-type SecR and E373D. These data support functional importance of a charge-charge interaction between the amino-terminal region of secretin and the top of TM7. SIGNIFICANCE STATEMENT: This work refines our molecular understanding of the activation mechanisms of class B1 G protein-coupled receptors. The amino-terminal region of secretin interacts with the seventh transmembrane segment of its receptor with structural specificity and with a charge-charge interaction helping to drive functional activation.

Structure and function of Norrin in assembly and activation of a Frizzled 4-Lrp5/6 complex.

Norrin is a cysteine-rich growth factor that is required for angiogenesis in the eye, ear, brain, and female reproductive organs. It functions as an atypical Wnt ligand by specifically binding to the Frizzled 4 (Fz4) receptor. Here we report the crystal structure of Norrin, which reveals a unique dimeric structure with each monomer adopting a conserved cystine knot fold. Functional studies demonstrate that the novel Norrin dimer interface is required for Fz4 activation. Furthermore, we demonstrate that Norrin contains separate binding sites for Fz4 and for the Wnt ligand coreceptor Lrp5 (low-density lipoprotein-related protein 5) or Lrp6. Instead of inducing Fz4 dimerization, Norrin induces the formation of a ternary complex with Fz4 and Lrp5/6 by binding to their respective extracellular domains. These results provide crucial insights into the assembly and activation of the Norrin-Fz4-Lrp5/6 signaling complex.

Environment-Sensitive Fluorescence of 7-Nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-Labeled Ligands for Serotonin Receptors.

Serotonin is a neurotransmitter that plays a crucial role in the regulation of several behavioral and cognitive functions by binding to a number of different serotonin receptors present on the cell surface. We report here the synthesis and characterization of several novel fluorescent analogs of serotonin in which the fluorescent NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl) group is covalently attached to serotonin. The fluorescent ligands compete with the serotonin1A receptor specific radiolabeled agonist for binding to the receptor. Interestingly, these fluorescent ligands display a high environmental sensitivity of their fluorescence. Importantly, the human serotonin1A receptor stably expressed in CHO-K1 cells could be specifically labeled with one of the fluorescent ligands with minimal nonspecific labeling. Interestingly, we show by spectral imaging that the NBD-labeled ligand exhibits a red edge excitation shift (REES) of 29 nm when bound to the receptor, implying that it is localized in a restricted microenvironment. Taken together, our results show that NBD-labeled serotonin analogs offer an attractive fluorescent approach for elucidating the molecular environment of the serotonin binding site in serotonin receptors. In view of the multiple roles played by the serotonergic systems in the central and peripheral nervous systems, these fluorescent ligands would be useful in future studies involving serotonin receptors.

Dimerization of the transmembrane domain of amyloid precursor protein is determined by residues around the γ-secretase cleavage sites.

One of the hallmarks of Alzheimer's disease is the formation of extracellular amyloid plaques that consist mainly of abnormally aggregated forms of amyloid ß (Aß) peptides. These peptides are generated by γ-secretase-catalyzed cleavage of a dimeric membrane-bound C-terminal fragment (C99) of the amyloid precursor protein. Although C99 homodimerization has been linked to Aß production and changes in the aggregation-determining Aß42/Aß40 ratio, the motif through which C99 dimerizes has remained controversial. Here, we have used two independent assays to gain insight into C99 homodimerization in the context of the membrane of live cells: bioluminescence resonance energy transfer and Tango membrane protein-protein interaction assays, which were further confirmed by traditional pull-down assays. Our results indicate a four-amino acid region within the C99 transmembrane helix (43TVIV46) as well as its local secondary structure as critical determinants for homodimerization. These four amino acids are also a hot spot of familial Alzheimer's disease-linked mutations that both decrease C99 homodimerization and γ-secretase cleavage and alter the initial cleavage site to increase the Aß42/40 ratio.

Spatial intensity distribution analysis quantifies the extent and regulation of homodimerization of the secretin receptor.

Previous studies have indicated that the G-protein-coupled secretin receptor is present as a homodimer, organized through symmetrical contacts in transmembrane domain IV, and that receptor dimerization is critical for high-potency signalling by secretin. However, whether all of the receptor exists in the dimeric form or if this is regulated is unclear. We used measures of quantal brightness of the secretin receptor tagged with monomeric enhanced green fluorescent protein (mEGFP) and spatial intensity distribution analysis to assess this. Calibration using cells expressing plasma membrane-anchored forms of mEGFP initially allowed us to demonstrate that the epidermal growth factor receptor is predominantly monomeric in the absence of ligand and while wild-type receptor was rapidly converted into a dimeric form by ligand, a mutated form of this receptor remained monomeric. Equivalent studies showed that, at moderate expression levels, the secretin receptor exists as a mixture of monomeric and dimeric forms, with little evidence of higher-order complexity. However, sodium butyrate-induced up-regulation of the receptor resulted in a shift from monomeric towards oligomeric organization. In contrast, a form of the secretin receptor containing a pair of mutations on the lipid-facing side of transmembrane domain IV was almost entirely monomeric. Down-regulation of the secretin receptor-interacting G-protein Gαs did not alter receptor organization, indicating that dimerization is defined specifically by direct protein-protein interactions between copies of the receptor polypeptide, while short-term treatment with secretin had no effect on organization of the wild-type receptor but increased the dimeric proportion of the mutated receptor variant.

Structure and Function of Cross-class Complexes of G Protein-coupled Secretin and Angiotensin 1a Receptors.

Complexes of secretin (SecR) and angiotensin 1a (Atr1a) receptors have been proposed to be functionally important in osmoregulation, providing an explanation for overlapping and interdependent functions of hormones that bind and activate different classes of GPCRs. However, the nature of these cross-class complexes has not been well characterized and their signaling properties have not been systematically explored. We now use competitive inhibition of receptor bioluminescence resonance energy transfer and bimolecular fluorescence complementation to establish the dominant functionally important state as a symmetrical homodimeric form of SecR decorated by monomeric Atr1a, interacting through lipid-exposed faces of Atr1a TM1 and TM4. Conditions increasing prevalence of this complex exhibited negative allosteric modulatory impact on secretin-stimulated cAMP responses at SecR. In contrast, activating Atr1a with full agonist in such a complex exhibited a positive allosteric modulatory impact on the same signaling event. This modulation was functionally biased, with secretin-stimulated calcium responses unaffected, whereas angiotensin-stimulated calcium responses through the complex were reduced or absent. Further supporting this interpretation, Atr1a with mutations of lipid-exposed faces of TM1 and TM4 that did not affect its ability to bind or signal, could be expressed in the same cell as SecR, yet not exhibit either the negative or positive allosteric impact on cAMP observed with the inactive or activated states of wild type Atr1a on function, and not interfere with angiotensin-stimulated calcium responses like complexes with Atr1a. This may provide a more selective means of exploring the physiologic functional impact of this cross-class receptor complex without interfering with the function of either component receptor.

A type 1 cholecystokinin receptor mutant that mimics the dysfunction observed for wild type receptor in a high cholesterol environment.

Cholecystokinin (CCK) stimulates the type 1 CCK receptor (CCK1R) to elicit satiety after a meal. Agonists with this activity, although potentially useful for treatment of obesity, can also have side effects and toxicities of concern, making the development of an intrinsically inactive positive allosteric modulator quite attractive. Positive allosteric modulators also have the potential to correct the defective receptor-G protein coupling observed in the high membrane cholesterol environment described in metabolic syndrome. Current model systems to study CCK1R in such an environment are unstable and expensive to maintain. We now report that the Y140A mutation within a cholesterol-binding motif and the conserved, class A G protein-coupled receptor-specific (E/D)RY signature sequence results in ligand binding and activity characteristics similar to wild type CCK1R in a high cholesterol environment. This is true for natural CCK, as well as ligands with distinct chemistries and activity profiles. Additionally, the Y140A construct also behaved like CCK1R in high cholesterol in regard to its internalization, sensitivity to a nonhydrolyzable GTP analog, and anisotropy of a bound fluorescent CCK analog. Chimeric CCK1R/CCK2R constructs that systematically changed the residues in the allosteric ligand-binding pocket were studied in the presence of Y140A. This established increased importance of unique residues within TM3 and reduced the importance of TM2 for binding in the presence of this mutation, with the agonist trigger likely pulled away from its Leu(356) target on TM7. The distinct conformation of this intramembranous pocket within Y140A CCK1R provides an opportunity to normalize this by using a small molecule allosteric ligand, thereby providing safe and effective correction of the coupling defect in metabolic syndrome.

Impact of ursodeoxycholic acid on a CCK1R cholesterol-binding site may contribute to its positive effects in digestive function.

Dysfunction of the type 1 cholecystokinin (CCK) receptor (CCK1R) as a result of increased gallbladder muscularis membrane cholesterol has been implicated in the pathogenesis of cholesterol gallstones. Administration of ursodeoxycholic acid, which is structurally related to cholesterol, has been shown to have beneficial effects on gallstone formation. Our aims were to explore the possible direct effects and mechanism of action of bile acids on CCK receptor function. We studied the effects of structurally related hydrophobic chenodeoxycholic acid and hydrophilic ursodeoxycholic acid in vitro on CCK receptor function in the setting of normal and elevated membrane cholesterol. We also examined their effects on a cholesterol-insensitive CCK1R mutant (Y140A) disrupting a key site of cholesterol action. The results show that, similar to the impact of cholesterol on CCK receptors, bile acid effects were limited to CCK1R, with no effects on CCK2R. Chenodeoxycholic acid had a negative impact on CCK1R function, while ursodeoxycholic acid had no effect on CCK1R function in normal membranes but was protective against the negative impact of elevated cholesterol on this receptor. The cholesterol-insensitive CCK1R mutant Y140A was resistant to effects of both bile acids. These data suggest that bile acids compete with the action of cholesterol on CCK1R, probably by interacting at the same site, although the conformational impact of each bile acid appears to be different, with ursodeoxycholic acid capable of correcting the abnormal conformation of CCK1R in a high-cholesterol environment. This mechanism may contribute to the beneficial effect of ursodeoxycholic acid in reducing cholesterol gallstone formation.

Transmembrane peptides as unique tools to demonstrate the in vivo action of a cross-class GPCR heterocomplex.

Angiotensin (ANGII) and secretin (SCT) share overlapping, interdependent osmoregulatory functions in brain, where SCT peptide/receptor function is required for ANGII action, yet the molecular basis is unknown. Since receptors for these peptides (AT1aR, SCTR) are coexpressed in osmoregulatory centers, a possible mechanism is formation of a cross-class receptor heterocomplex. Here, we demonstrate such a complex and its functional importance to modulate signaling. Association of AT1aR with SCTR reduced ability of SCT to stimulate cyclic adenosine monophosphate (cAMP), with signaling augmented in presence of ANGII or constitutively active AT1aR. Several transmembrane (TM) peptides of these receptors were able to affect their conformation within complexes, reducing receptor BRET signals. AT1aR TM1 affected only formation and activity of the heterocomplex, without effect on homomers of either receptor, and reduced SCT-stimulated cAMP responses in cells expressing both receptors. This peptide was active in vivo by injection into mouse lateral ventricle, thereby suppressing water-drinking behavior after hyperosmotic shock, similar to SCTR knockouts. This supports the interpretation that active conformation of AT1aR is a key modulator of cAMP responses induced by SCT stimulation of SCTR. The SCTR/AT1aR complex is physiologically important, providing differential signaling to SCT in settings of hyperosmolality or food intake, modulated by differences in levels of ANGII.

Glucagon-like peptide-1 receptor dimerization differentially regulates agonist signaling but does not affect small molecule allostery.

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.

Molecular basis for benzodiazepine agonist action at the type 1 cholecystokinin receptor.

Understanding the molecular basis of drug action can facilitate development of more potent and selective drugs. Here, we explore the molecular basis for action of a unique small molecule ligand that is a type 1 cholecystokinin (CCK) receptor agonist and type 2 CCK receptor antagonist, GI181771X. We characterize its binding utilizing structurally related radioiodinated ligands selective for CCK receptor subtypes that utilize the same allosteric ligand-binding pocket, using wild-type receptors and chimeric constructs exchanging the distinct residues lining this pocket. Intracellular calcium assays were performed to determine biological activity. Molecular models for docking small molecule agonists to the type 1 CCK receptor were developed using a ligand-guided refinement approach. The optimal model was distinct from the previous antagonist model for the same receptor and was mechanistically consistent with the current mutagenesis data. This study revealed a key role for Leu(7.39) that was predicted to interact with the isopropyl group in the N1 position of the benzodiazepine that acts as a "trigger" for biological activity. The molecular model was predictive of binding of other small molecule agonists, effectively distinguishing these from 1065 approved drug decoys with an area under curve value of 99%. The model also selectively enriched for agonist compounds, with 130 agonists identified by ROC analysis when seeded in 2175 non-agonist ligands of the type 1 CCK receptor (area under curve 78%). Benzodiazepine agonists in this series docked in consistent pose within this pocket, with a key role played by Leu(7.39), whereas the role of this residue was less clear for chemically distinct agonists.

Impact of secretin receptor homo-dimerization on natural ligand binding.

Class B G protein-coupled receptors can form dimeric complexes important for high potency biological effects. Here, we apply pharmacological, biochemical, and biophysical techniques to cells and membranes expressing the prototypic secretin receptor (SecR) to gain insights into secretin binding to homo-dimeric and monomeric SecR. Spatial proximity between peptide and receptor residues, probed by disulfide bond formation, demonstrates that the secretin N-terminus moves from adjacent to extracellular loop 3 (ECL3) at wild type SecR toward ECL2 in non-dimerizing mutants. Analysis of fluorescent secretin analogs demonstrates stable engagement of the secretin C-terminal region within the receptor extracellular domain (ECD) for both dimeric and monomeric receptors, while the mid-region exhibits lower mobility while docked at the monomer. Moreover, decoupling of G protein interaction reduces mobility of the peptide mid-region at wild type receptor to levels similar to the mutant, whereas it has no further impact on the monomer. These data support a model of peptide engagement whereby the ability of SecR to dimerize promotes higher conformational dynamics of the peptide-bound receptor ECD and ECLs that likely facilitates more efficient G protein recruitment and activation, consistent with the higher observed functional potency of secretin at wild type SecR relative to the monomeric mutant receptor.

Molecular basis for binding and subtype selectivity of 1,4-benzodiazepine antagonist ligands of the cholecystokinin receptor.

Allosteric binding pockets in peptide-binding G protein-coupled receptors create opportunities for the development of small molecule drugs with substantial benefits over orthosteric ligands. To gain insights into molecular determinants for this pocket within type 1 and 2 cholecystokinin receptors (CCK1R and CCK2R), we prepared a series of receptor constructs in which six distinct residues in TM2, -3, -6, and -7 were reversed. Two novel iodinated CCK1R- and CCK2R-selective 1,4-benzodiazepine antagonists, differing only in stereochemistry at C3, were used. When all six residues within CCK1R were mutated to corresponding CCK2R residues, benzodiazepine selectivity was reversed, yet peptide binding selectivity was unaffected. Detailed analysis, including observations of gain of function, demonstrated that residues 6.51, 6.52, and 7.39 were most important for binding the CCK1R-selective ligand, whereas residues 2.61 and 7.39 were most important for binding CCK2R-selective ligand, although the effect of substitution of residue 2.61 was likely indirect. Ligand-guided homology modeling was applied to wild type receptors and those reversing benzodiazepine binding selectivity. The models had high predictive power in enriching known receptor-selective ligands from related decoys, indicating a high degree of precision in pocket definition. The benzodiazepines docked in similar poses in both receptors, with C3 urea substituents pointing upward, whereas different stereochemistry at C3 directed the C5 phenyl rings and N1 methyl groups into opposite orientations. The geometry of the binding pockets and specific interactions predicted for ligand docking in these models provide a molecular framework for understanding ligand selectivity at these receptor subtypes. Furthermore, the strong predictive power of these models suggests their usefulness in the discovery of lead compounds and in drug development programs.

Mechanism of Action and Structure-Activity Relationships of Tetracyclic Small Molecules Acting as Universal Positive Allosteric Modulators of the Cholecystokinin Receptor.

As part of an ongoing effort to develop a drug targeting the type 1 cholecystokinin receptor (CCK1R) to help prevent and/or treat obesity, we recently performed a high throughput screening effort of small molecules seeking candidates that enhanced the action of the natural agonist, CCK, thus acting as positive allosteric modulators without exhibiting intrinsic agonist action. Such probes would be expected to act in a temporally finite way to enhance CCK action to induce satiety during and after a meal and potentially even modulate activity at the CCK1R in a high cholesterol environment present in some obese patients. The current work focuses on the best scaffold, representing tetracyclic molecules identified through high throughput screening we previously reported. Extensive characterization of the two top "hits" from the previous effort demonstrated them to fulfill the desired pharmacologic profile. We undertook analog-by-catalog expansion of this scaffold using 65 commercially available analogs. In this effort, we were able to eliminate an off-target effect observed for this scaffold while retaining its activity as a positive allosteric modulator of CCK1R in both normal and high cholesterol membrane environments. These insights should be useful in the rational medicinal chemical enhancement of this scaffold and in the future development of candidates to advance to pre-clinical proof-of-concept and to clinical trials.

Ligand-induced internalization of the type 1 cholecystokinin receptor independent of recognized signaling activity.

Receptor ligands, identified as antagonists, based on the absence of stimulation of signaling, can rarely stimulate receptor internalization. d-Tyr-Gly-[(Nle(28,31),d-Trp(30))CCK-26-32]-2-phenylethyl ester (d-Trp-OPE) is such a ligand that binds to the cholecystokinin (CCK) receptor and stimulates internalization. Here, the molecular basis of this trafficking event is explored, with the assumption that ligand binding initiates conformational change, exposing an epitope to direct endocytosis. Ligand-stimulated internalization was studied morphologically using fluorescent CCK and d-Trp-OPE. d-Trp-OPE occupation of Chinese hamster ovary cell receptors stimulated internalization into the same region as CCK. Arrestin-biased action was ruled out using morphological translocation of fluorescent arrestin 2 and arrestin 3, moving to the membrane in response to CCK, but not d-Trp-OPE. Possible roles of the carboxyl terminus were studied using truncated receptor constructs, eliminating the proline-rich distal tail, the serine/threonine-rich midregion, and the remainder to the vicinal cysteines. None of these constructs disrupted d-Trp-OPE-stimulated internalization. Possible contributions of transmembrane segments were studied using competitive inhibition with peptides that also had no effect. Intracellular regions were studied with a similar strategy using coexpressing cell lines. Peptides corresponding to ends of each loop region were studied, with only the peptide at the carboxyl end of the third loop inhibiting d-Trp-OPE-stimulated internalization but having no effect on CCK-stimulated internalization. The region contributing to this effect was refined to peptide 309-323, located below the recognized G protein-association motif. While a receptor in which this segment was deleted did internalize in response to d-Trp-OPE, it exhibited abnormal ligand binding and did not signal in response to CCK, suggesting an abnormal conformation and possible mechanism of internalization distinct from that being studied. This interpretation was further supported by the inability of peptide 309-323 to inhibit its d-Trp-OPE-stimulated internalization. Thus the 309-323 region of the type 1 CCK receptor affects antagonist-stimulated internalization of this receptor, although its mechanism and interacting partner are not yet clear.

Differential sensitivity of types 1 and 2 cholecystokinin receptors to membrane cholesterol.

Recent studies indicate that membrane cholesterol can associate with G protein-coupled receptors (GPCRs) and affect their function. Previously, we reported that manipulation of membrane cholesterol affects ligand binding and signal transduction of the type 1 cholecystokinin receptor (CCK1R), a Class A GPCR. We now demonstrate that the closely related type 2 cholecystokinin receptor (CCK2R) does not share this cholesterol sensitivity. The sequences of both receptors reveal almost identical cholesterol interaction motifs in analogous locations in transmembrane segments two, three, four, and five. The disparity in cholesterol sensitivity between these receptors, despite their close structural relationship, provides a unique opportunity to define the possible structural basis of cholesterol sensitivity of CCK1R. To evaluate the relative contributions of different regions of CCK1R to cholesterol sensitivity, we performed ligand binding studies and biological activity assays of wild-type and CCK2R/CCK1R chimeric receptor-bearing Chinese hamster ovary cells after manipulation of membrane cholesterol. We also extended these studies to site-directed mutations within the cholesterol interaction motifs. The results contribute to a better understanding of the structural requirements for cholesterol sensitivity in CCK1R and provides insight into the function of other cholesterol-sensitive Class A GPCRs.