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Agonists of the secretin receptor have potential applications for diseases of the cardiovascular, gastrointestinal, and metabolic systems, yet no clinically-active non-peptidyl agonists of this receptor have yet been developed. In the current work, we have identified a new small molecule lead compound with this pharmacological profile. We have prepared and characterized a systematic structure-activity series around this thiadiazole scaffold to better understand the molecular determinants of its activity. We were able to enhance the in vitro activity and to maintain the specificity of the parent compound. We found the most active candidate to be quite stable in plasma, although it was metabolized by hepatic microsomes. This chemical probe should be useful for in vitro studies and needs to be tested for in vivo pharmacological activity. This could be an important lead toward the development of a first-in-class orally active agonist of the secretin receptor, which could be useful for multiple disease states., Competing Interests: Declaration of competing interest We declare no potential conflicts of interest with respect to research, authorship, and/or publication of this article., (Copyright © 2024. Published by Elsevier Inc.)

Development of optimal therapeutics for disease states that can be associated with increased membrane cholesterol requires better molecular understanding of lipid modulation of the drug target. Type 1 cholecystokinin receptor (CCK1R) agonist actions are affected by increased membrane cholesterol, enhancing ligand binding and reducing calcium signaling, while agonist actions of the closely related CCK2R are not. In this work, we identified a set of chimeric human CCK1R/CCK2R mutations that exchange the cholesterol sensitivity of these 2 receptors, providing powerful tools when expressed in CHO and HEK-293 model cell lines to explore mechanisms. Static, low energy, high-resolution structures of the mutant CCK1R constructs, stabilized in complex with G protein, were not substantially different, suggesting that alterations to receptor dynamics were key to altered function. We reveal that cholesterol-dependent dynamic changes in the conformation of the helical bundle of CCK receptors affects both ligand binding at the extracellular surface and G protein coupling at the cytosolic surface, as well as their interrelationships involved in stimulus-response coupling. This provides an ideal setting for potential allosteric modulators to correct the negative impact of membrane cholesterol on CCK1R., Competing Interests: PMS and AC are co-founders and shareholders of Septerna Inc. and DACRA Tx. DW is a shareholder in Septerna Inc. and co-founder and shareholder of DACRA Tx., (Copyright: © 2024 Harikumar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The functional significance of the interactions between proteins in living cells to form short-lived quaternary structures cannot be overemphasized. Yet, quaternary structure information is not captured by current methods, neither can those methods determine structure within living cells. The dynamic versatility, abundance, and functional diversity of G protein-coupled receptors (GPCRs) pose myriad challenges to existing technologies but also present these proteins as the ideal testbed for new technologies to investigate the complex inter-regulation of receptor-ligand, receptor-receptor, and receptor-downstream effector interfaces in living cells. Here, we present development and use of a novel method capable of overcoming existing challenges by combining distributions (or spectrograms) of FRET efficiencies from populations of fluorescently tagged proteins associating into oligomeric complexes in live cells with diffusion-like trajectories of FRET donors and acceptors obtained from molecular dynamics (MD) simulations. Our approach provides an atom-level picture of the binding interfaces within oligomers of the human secretin receptor (hSecR) in live cells and allows for extraction of mechanistic insights into the function of GPCRs oligomerization. This FRET-MD spectrometry approach is a robust platform for investigating protein-protein binding mechanisms and opens a new avenue for investigating stable as well as fleeting quaternary structures of any membrane proteins in living cells.

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., (© 2024. The Author(s).)

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.

Obesity has become a prevailing health burden globally and particularly in the US. It is associated with many health problems, including cardiovascular disease, diabetes and poorer mental health. Hence, there is a high demand to find safe and effective therapeutics for sustainable weight loss. Cholecystokinin (CCK) has been implicated as one of the first gastrointestinal hormones to reduce overeating and suppress appetite by activating the type 1 cholecystokinin receptor (CCK1R). Several drug development campaigns have focused on finding CCK1R-specific agonists, which showed promising efficacy for reducing meal size and weight, but fell short on FDA approval, likely due to side effects associated with potent, long-lasting activation of CCK1Rs. Positive allosteric modulators (PAMs) without inherent agonist activity have been proposed to overcome the shortcomings of traditional, orthosteric agonists and restore CCK1R signaling in failing physiologic systems. However, drug discovery campaigns searching for such novel acting CCK1R agents remain limited. Here we report a high-throughput screening effort and the establishment of a testing funnel, which led to the identification of novel CCK1R modulators. We utilized IP-One accumulation to develop robust functional equilibrium assays tailored to either detect PAMs, agonists or non-specific activators. In addition, we established the CCK1R multiplex PAM assay as a novel method to evaluate functional selectivity capable of recording CCK1R-induced cAMP accumulation and β-arrestin recruitment in the same well. This selection and arrangement of methods enabled the discovery of three scaffolds, which we characterized and validated in an array of functional and binding assays. We found two hits incorporating a tetracyclic scaffold that significantly enhanced CCK signaling at CCK1Rs without intrinsically activating CCK1Rs in an overexpressing system. Our results demonstrate that a well-thought-out testing funnel can identify small molecules with a distinct pharmacological profile and provides an important milestone for the development of novel potential treatments of obesity., Competing Interests: Declaration of Conflicting Interest We declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (Copyright © 2022. Published by Elsevier Inc.)

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 His 1 and Ser 2 and seventh transmembrane segment (TM7) receptor residue E373. We interrogated these interactions using systematic structure-activity analysis of peptide and receptor. His 1 was critical for binding and cAMP responses, but its orientation was not critical, and substitution could independently modify affinity and efficacy. Ser 2 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., (Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics.)

Drugs useful in prevention/treatment of obesity could improve health. Cholecystokinin (CCK) is a key regulator of appetite, working through the type 1 CCK receptor (CCK1R); however, full agonists have not stimulated more weight loss than dieting. We proposed an alternate strategy to target this receptor, while reducing likelihood of side effects and/or toxicity. Positive allosteric modulators (PAMs) with minimal intrinsic agonist activity would enhance CCK action, while maintaining spatial and temporal characteristics of physiologic signaling. This could correct abnormal stimulus-activity coupling observed in a high-cholesterol environment observed in obesity. We utilized high-throughput screening to identify a molecule with this pharmacological profile and studied its basis of action. Compound 1 was a weak partial agonist, with PAM activity to enhance CCK action at CCK1R, but not CCK2R, maintained in both normal and high cholesterol. Compound 1 (10 µM) did not exhibit agonist activity or stimulate internalization of CCK1R. It enhanced CCK activity by slowing the off-rate of bound hormone, increasing its binding affinity. Computational docking of Compound 1 to CCK1R yielded plausible poses. A radioiodinatable photolabile analogue retained Compound 1 pharmacology and covalently labeled CCK1R Thr 211 , consistent with one proposed pose. Our study identifies a novel, selective, CCK1R PAM that binds to the receptor to enhance action of CCK-8 and CCK-58 in both normal and disease-mimicking high-cholesterol environments. This facilitates the development of compounds that target the physiologic spatial and temporal engagement of CCK1R by CCK that underpins its critical role in metabolic regulation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Harikumar, Coudrat, Desai, Dong, Dengler, Furness, Christopoulos, Wootten, Sergienko, Sexton and Miller.)

G protein-coupled receptors (GPCRs) are known to be modulated by membrane cholesterol levels, but whether or not the effects are caused by specific receptor-cholesterol interactions or cholesterol's general effects on the membrane is not well-understood. We performed coarse-grained molecular dynamics (CGMD) simulations coupled with structural bioinformatics approaches on the β 2 -adrenergic receptor (β 2 AR) and the cholecystokinin (CCK) receptor subfamily. The β 2 AR has been shown to be sensitive to membrane cholesterol and cholesterol molecules have been clearly resolved in numerous β 2 AR crystal structures. The two CCK receptors are highly homologous and preserve similar cholesterol recognition motifs but despite their homology, CCK 1 R shows functional sensitivity to membrane cholesterol while CCK 2 R does not. Our results offer new insights into how cholesterol modulates GPCR function by showing cholesterol interactions with β 2 AR that agree with previously published data; additionally, we observe differential and specific cholesterol binding in the CCK receptor subfamily while revealing a previously unreported Cholesterol Recognition Amino-acid Consensus (CRAC) sequence that is also conserved across 38% of class A GPCRs. A thermal denaturation assay (LCP-T m ) shows that mutation of a conserved CRAC sequence on TM7 of the β 2 AR affects cholesterol stabilization of the receptor in a lipid bilayer. The results of this study provide a better understanding of receptor-cholesterol interactions that can contribute to novel and improved therapeutics for a variety of diseases., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) regulates metabolism in response to the cellular energy states. Under energy stress, AMP stabilizes the active AMPK conformation, in which the kinase activation loop (AL) is protected from protein phosphatases, thus keeping the AL in its active, phosphorylated state. At low AMP:ATP (adenosine triphosphate) ratios, ATP inhibits AMPK by increasing AL dynamics and accessibility. We developed conformation-specific antibodies to trap ATP-bound AMPK in a fully inactive, dynamic state and determined its structure at 3.5-angstrom resolution using cryo-electron microscopy. A 180° rotation and 100-angstrom displacement of the kinase domain fully exposes the AL. On the basis of the structure and supporting biophysical data, we propose a multistep mechanism explaining how adenine nucleotides and pharmacological agonists modulate AMPK activity by altering AL phosphorylation and accessibility., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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 serotonin 1A receptor specific radiolabeled agonist for binding to the receptor. Interestingly, these fluorescent ligands display a high environmental sensitivity of their fluorescence. Importantly, the human serotonin 1A 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.

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., Competing Interests: The authors have declared that no competing interests exist.

Cholecystokinin is a gastrointestinal peptide hormone with important roles in metabolic physiology and the maintenance of normal nutritional status, as well as potential roles in the prevention and management of obesity, currently one of the dominant causes of direct or indirect morbidity and mortality. In this review, we discuss the roles of this hormone and its receptors in maintaining nutritional homeostasis, with a particular focus on appetite control. Targeting this action led to the development of full agonists of the type 1 cholecystokinin receptor that have so far failed in clinical trials for obesity. The possible reasons for clinical failure are discussed, along with alternative pharmacologic strategies to target this receptor for prevention and management of obesity, including development of biased agonists and allosteric modulators. Cellular cholesterol is a natural modulator of the type 1 cholecystokinin receptor, with elevated levels disrupting normal stimulus-activity coupling. The molecular basis for this is discussed, along with strategies to overcome this challenge with a corrective positive allosteric modulator. There remains substantial scope for development of drugs to target the type 1 cholecystokinin receptor with these new pharmacologic strategies and such drugs may provide new approaches for treatment of obesity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Miller, Harikumar, Wootten and Sexton.)

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.

The secretin receptor (SCTR) is a prototypic Class B1 G protein-coupled receptor (GPCR) that represents a key target for the development of therapeutics for the treatment of cardiovascular, gastrointestinal, and metabolic disorders. However, no non-peptidic molecules targeting this receptor have yet been disclosed. Using a high-throughput screening campaign directed at SCTR to identify small molecule modulators, we have identified three structurally related scaffolds positively modulating SCTRs. Here we outline a comprehensive study comprising a structure-activity series based on commercially available analogs of the three hit scaffold sets A (2-sulfonyl pyrimidines), B (2-mercapto pyrimidines) and C (2-amino pyrimidines), which revealed determinants of activity, cooperativity and specificity. Structural optimization of original hits resulted in analog B2, which substantially enhances signaling of truncated secretin peptides and prolongs residence time of labeled secretin up to 13-fold in a dose-dependent manner. Furthermore, we found that investigated compounds display structural similarity to positive allosteric modulators (PAMs) active at the glucagon-like peptide-1 receptor (GLP-1R), and we were able to confirm cross-recognition of that receptor by a subset of analogs. Studies using SCTR and GLP-1R mutants revealed that scaffold A, but not B and C, likely acts via two distinct mechanisms, one of which constitutes covalent modification of Cys-347 GLP-1R known from GLP-1R-selective modulators. The scaffolds identified in this study might not only serve as novel pharmacologic tools to decipher SCTR- or GLP-1R-specific signaling pathways, but also as structural leads to elucidate allosteric binding sites facilitating the future development of orally available therapeutic approaches targeting these receptors., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The secretin receptor (SCTR), a prototypical class B G protein-coupled receptor (GPCR), exerts its effects mainly by activating Gαs proteins upon binding of its endogenous peptide ligand secretin. SCTRs can be found in a variety of tissues and organs across species, including the pancreas, stomach, liver, heart, lung, colon, kidney, and brain. Beyond that, modulation of SCTR-mediated signaling has therapeutic potential for the treatment of multiple diseases, such as heart failure, obesity, and diabetes. However, no ligands other than secretin and its peptide analogs have been described to regulate SCTRs, probably due to inherent challenges in family B GPCR drug discovery. Here we report creation of a testing funnel that allowed targeted detection of SCTR small-molecule activators. Pursuing the strategy to identify positive allosteric modulators (PAMs), we established a unique primary screening assay employing a mixture of three orthosteric stimulators that was compared in a screening campaign testing 12,000 small-molecule compounds. Beyond that, we developed a comprehensive set of secondary assays, such as a radiolabel-free target engagement assay and a NanoBiT (NanoLuc Binary Technology)-based approach to detect β-arrestin-2 recruitment, all feasible in a high-throughput environment as well as capable of profiling ligands and hits regarding their effect on binding and receptor function. This combination of methods enabled the discovery of five promising scaffolds, four of which have been validated and further characterized with respect to their allosteric activities. We propose that our results may serve as starting points for developing the first in vivo active small molecules targeting SCTRs.

The class B secretin GPCR (SecR) has broad physiological effects, with target potential for treatment of metabolic and cardiovascular disease. Molecular understanding of SecR binding and activation is important for its therapeutic exploitation. We combined cryo-electron microscopy, molecular dynamics, and biochemical cross-linking to determine a 2.3 Å structure, and interrogate dynamics, of secretin bound to the SecR:Gs complex. SecR exhibited a unique organization of its extracellular domain (ECD) relative to its 7-transmembrane (TM) core, forming more extended interactions than other family members. Numerous polar interactions formed between secretin and the receptor extracellular loops (ECLs) and TM helices. Cysteine-cross-linking, cryo-electron microscopy multivariate analysis and molecular dynamics simulations revealed that interactions between peptide and receptor were dynamic, and suggested a model for initial peptide engagement where early interactions between the far N-terminus of the peptide and SecR ECL2 likely occur following initial binding of the peptide C-terminus to the ECD.

The secretin receptor is a prototypic class B GPCR with substantial and broad pharmacologic importance. The aim of this project was to develop a high affinity selective antagonist as a new and important pharmacologic tool and to aid stabilization of this receptor in an inactive conformation for ultimate structural characterization. Amino-terminal truncation of the natural 27-residue ligand reduced biological activity, but also markedly reduced binding affinity. This was rationally and experimentally overcome with lactam stabilization of helical structure and with replacement of residues with natural and unnatural amino acids. A key new step in this effort was the replacement of peptide residue Leu 22 with L-cyclohexylalanine (Cha) to enhance potential hydrophobic interactions with receptor residues Leu 31 , Val 34 , and Phe 92 that were predicted from molecular modeling. Alanine-replacement mutagenesis of these residues markedly affected ligand binding and biological activity. The optimal antagonist ligand, (Y 10 ,c[E 16 ,K 20 ],I 17 ,Cha 22 ,R 25 )sec(6-27), exhibited high binding affinity (4 nM), similar to natural secretin, and exhibited no demonstrable biological activity to stimulate cAMP accumulation, intracellular calcium mobilization, or β-arrestin-2 translocation. It acts as an orthosteric competitive antagonist, predicted to bind within the peptide-binding groove in the receptor extracellular domain. The analogous peptide that was one residue longer, retaining Thr 5 , exhibited partial agonist activity, while further truncation of even a single residue (Phe 6 ) reduced binding affinity. This sec(6-27)-based peptide will be an important new tool for pharmacological and structural studies., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Cholecystokinin cholescintigraphy is used clinically to quantify gallbladder ejection fraction as an indicator of functional gallbladder disorder. It can also provide the opportunity to quantify an individual's responsiveness to the physiologic stimulant of gallbladder contraction, cholecystokinin, which is a major regulator of appetite and postprandial satiety. Methods: In the current work, we use cholecystokinin cholescintigraphy to quantify the kinetics of gallbladder emptying, including average and peak rates, in response to a standard cholecystokinin infusion. Results: We demonstrated that patients with no gallstones or biliary obstruction who empty their gallbladders completely in response to cholecystokinin, having an ejection fraction greater than 80%, exhibit a broad range of sensitivity to this hormone. Three distinct kinetic profiles were observed, with those most sensitive to cholecystokinin achieving the earliest peak and the fastest rate of gallbladder emptying, whereas those least sensitive to cholecystokinin have the latest peak and the slowest rate of emptying. Conclusion: Patients can have abnormal cholecystokinin stimulus-activity coupling as an effect of endogenous negative allosteric modulation by membrane cholesterol. This was predicted in ex vivo studies but has not, to our knowledge, previously been demonstrated in vivo. This type of kinetic analysis provides a tool to quantify cholecystokinin responsiveness in patients and identify patients who might benefit from a drug that would positively modulate cholecystokinin action to improve their appetite regulation and to better control their weight., (© 2020 by the Society of Nuclear Medicine and Molecular Imaging.)

Frizzled receptors (FZDs) are class-F G-protein-coupled receptors (GPCRs) that function in Wnt signalling and are essential for developing and adult organisms 1,2 . 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.

γ-Secretase epsilon-cleavage assay is derived from the cell-based Tango assay (Kang et al ., 2015), and is a fast and sensitive method to determine the initial cleavage of C99 by γ-secretase. In this protocol, we use HTL cells, which are HEK293 cells with a stably integrated luciferase reporter under the control of the bacterial tetO operator element, in which C99 C terminally fused to a reversed tetracyclin-inducible activator (rTA) transcriptional activator is expressed. Endogenous or transfected γ-secretase cleaves a C terminally fused rTA transcriptional activator from C99, allowing rTA to move to the nucleus to activate a luciferase reporter gene as a measurement for γ-secretase cleavage activity., Competing Interests: The authors declare no conflict of interest.

The Tango assay is a protein-protein interaction assay, in which a transcription factor (rTA) is fused to a membrane-bound protein via a linker that contains a cleavage site for TEV protease, whereas a soluble interaction partner is fused to TEV protease (Barnea et al. , 2008). Association between the two interaction partners leads to an efficient cleavage of the transcription factor, allowing it to translocate to the nucleus and activate a luciferase reporter gene as measurement of the interactions. In this modified assay, we fused one copy of the membrane-spanning amyloid precursor protein (APP) C99 region to TEV site-rTA (C99-TEV site-rTA) and a second copy to TEV protease (C99-TEV) to analyze intramembrane C99-C99 interaction in live cells., Competing Interests: The authors declare no conflict of interest.

The bioluminescence resonance energy transfer (BRET) assay can be used as an indicator of molecular approximation and/or interaction. A significant resonance energy transfer signal is generated when the acceptor, having the appropriate spectral overlap with the donor emission, is approximated with the donor. In the example provided, proteins tagged with bioluminescent Renilla luciferase (Rlu) as donor and yellow fluorescent protein (YFP) as acceptor were co-expressed in cells. This pair of donor and acceptor have an approximate Förster distance of 4.4 nm, providing the optimal working distance (Dacres et al ., 2010). This technique can be used to explore the time-course of specific molecular interactions that occur in living cells., Competing Interests: Authors have no conflicts of interest or competing interests.

Pulldown assay is a conventional method to determine protein-protein interactions in vitro . Expressing a protein of interest with two different tags allows testing whether both versions can be captured via one of the two tags as homooligomeric complex. This protocol is based on streptavidin bead capture of a biotinylated protein and co-associated Flag-tagged protein using Streptavidin MagBeads., Competing Interests: The authors declare no conflict of interest.

One of the hallmarks of Alzheimer's disease is the formation of extracellular amyloid plaques that consist mainly of abnormally aggregated forms of amyloid β (Aβ) 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 Aβ production and changes in the aggregation-determining Aβ42/Aβ40 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 ( 43 TVIV 46 ) 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 Aβ42/40 ratio., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Class B guanine nucleotide-binding protein (G protein)-coupled receptors form symmetrical homodimeric complexes along the lipid face of transmembrane segment 4 (TM4) and can form heterodimeric complexes, although their structure is unknown. The current study demonstrates that the lipid face of TM4 is also the predominant determinant for formation of heteroreceptor complexes between two class B receptors, secretin receptor (SecR) and glucagonlike peptide-1 receptor (GLP-1R), which are expressed on pancreatic islet cells. Because these receptors use the same interface for formation of homo- and heteroreceptor complexes, competitive forces may affect expression of different complexes. Assessment of SecR and GLP-1R dimeric complexes via recombinant expression in Chinese hamster ovary cells revealed that homodimeric receptor complexes were more stable than the heterodimeric complexes, and the homodimeric SecR/SecR is more stable than the GLP-1R/GLP-1R complex. Given the greater tendency for homodimeric compared with heterodimeric complex formation, the heteroreceptor complexes lacked the expression that might have been predicted by geometry alone. Nevertheless, cells coexpressing these receptors formed heterodimeric complexes that correlated with reduced intracellular calcium responses to secretin, but no change in the cyclic adenosine monophosphate responses to each natural agonist. This functional effect was confirmed in pancreatic islets isolated from wild-type and GLP-1R knockout mice. In these cells, the increased calcium response mediated by secretin in the absence of GLP-1R was paralleled by an increased glucose-dependent insulin response, indicating that the heterodimeric receptor complexes modulate secretin responses. Furthermore, the heterodimeric receptor complexes also mediated agonist-induced cross-receptor internalization, a process that could have broad functional significance in sites of natural receptor coexpression., (Copyright © 2017 Endocrine Society.)

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 Gα s 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., (© 2017 The Author(s).)

Wnt/β-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 (FZD 4 ) 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 FZD 4 CRDs and oligomerization of full-length FZD 4 , 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., (© 2017 DeBruine et al.; Published by Cold Spring Harbor Laboratory Press.)

The gastrointestinal (GI) tract has a central role in nutritional homeostasis, as location for food ingestion, digestion and absorption, with the gut endocrine system responding to and regulating these events, as well as influencing appetite. One key GI hormone with the full spectrum of these activities is cholecystokinin (CCK), a peptide released from neuroendocrine I cells scattered through the proximal intestine in response to fat and protein, with effects to stimulate gall bladder contraction and pancreatic exocrine secretion, to regulate gastric emptying and intestinal transit, and to induce satiety. There has been interest in targeting the type 1 CCK receptor (CCK1R) for drug development to provide non-caloric satiation as an aid to dieting and weight loss; however, there have been concerns about CCK1R agonists related to side effects and potential trophic impact on the pancreas. A positive allosteric modulator (PAM) of CCK action at this receptor without intrinsic agonist activity could provide a safer and more effective approach to long-term administration. In addition, CCK1R stimulus-activity coupling has been shown to be negatively affected by excess membrane cholesterol, a condition described in the metabolic syndrome, thereby potentially interfering with an important servomechanism regulating appetite. A PAM targeting this receptor could also potentially correct the negative impact of cholesterol on CCK1R function. We will review the molecular basis for binding natural peptide agonist, binding and action of small molecules within the allosteric pocket, and the impact of cholesterol. Novel strategies for taking advantage of this receptor for the prevention and management of obesity will be reviewed., Competing Interests: The authors declare no conflict of interest.

The involvement of secretin (SCT) and secretin receptor (SCTR) in regulating body water homeostasis is well established. Identified as one of the vasopressin (Vp)-independent mechanisms in fluid balance, SCT regulates aquaporin 2 (AQP2) in the kidney distal collecting duct cells through activating intracellular cAMP production. This ability to bypass Vp-mediated water reabsorption in kidney implicates SCT's potential to treat nephrogenic diabetes insipidus (NDI). Research on NDI in the past has largely been focused on the searching for mutations in vasopressin receptor 2 (AVPR2), while the functional relationship between SCTR, AVPR2 and NDI remains unclear. Here, we demonstrate the interaction between SCTR and AVPR2 to modulate cellular signaling in vitro. Interestingly, we show in this report that upon heteromer formation with SCTR, R137H, a NDI-causing AVPR2 mutant that is defective in trafficking to cell surface, can functionally be rescued. Our data may provide an explanation for this clinically mild case of NDI, and insights into the pathological development of NDI in the future., Competing Interests: The authors have declared that no competing interests exist.

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., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

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., (Copyright © 2015 the American Physiological Society.)