Inositol Monophosphate Accumulation Research Articles

Loss-of-Function Thr347Ala Variant in the G Protein Subunit-α11 Causes Familial Hypocalciuric Hypercalcemia 2 (FHH2).

To date, only four loss-of-function variants in the GNA11 gene encoding the G protein subunit α11 (Gα11) leading to familial hypocalciuric hypercalcemia 2 (FHH2) have been characterized. Gα11 is involved in calcium-sensing receptor (CaSR) signaling, and loss-of-function variants in GNA11 lead to reduced agonist potency at CaSR and an FHH phenotype. We have identified a family with a heterozygous GNA11 Thr347Ala variant and characterized its impact on calcium homeostasis in FHH2 patients and the signaling properties of CaSR through the Gα11-Thr347Ala variant in vitro. The index patient and her family had clinical, biochemical, and genetic analyses performed. The expression levels of Gα11 and the cell-surface expression levels of CaSR in human embryonic kidney 293A Gq/11 knock-out cells (ΔGq/11-HEK293A) co-transfected with CaSR and Gα11 (wild type (WT) or Thr347Ala) were determined, and the functional properties exhibited by calcium at CaSR were characterized in an inositol monophosphate (IP1) accumulation assay. Heterozygous carriers of the GNA11 Thr347Ala variant had mild asymptomatic hypercalcemia, hypocalciuria, and inappropriately high normal parathyroid hormone (PTH) levels considering their elevated serum calcium levels. Whereas the variant did not impact Gα11 expression or CaSR cell surface expression levels, calcium displayed a moderately but significantly lower agonist potency at CaSR/Gα11-Thr347Ala-transfected cells compared with CaSR/Gα11-WT-transfected cells in the IP1 accumulation assay (EC50 values of 5.67 mM and 4.38 mM, respectively). This identification of a novel GNA11 variant causing FHH2 substantiates the important role of Gα11 for CaSR signaling and Ca2+ homeostasis.

Abstract 671: G-protein coupled receptor (GPCR) screening library identifies sertindole, an antipsychotic, as potential therapeutic option to target GNAQ mutation Q209L in uveal melanoma

Abstract Background: Uveal melanoma is a rare but aggressive intraocular malignancy with limited therapeutic options. A hallmark genetic alteration associated with uveal melanoma is the mutation of the GNAQ gene, particularly the Q209L substitution in the Gαq protein, which contributes to oncogenic signaling and tumor progression. The GNAQ gene encodes for a Gαq protein that plays a critical role in signal transduction pathways involving G protein-coupled receptors (GPCRs). The Q209L substitution results in a gain-of-function mutation, leading to the chronic activation of downstream signaling pathways. This persistent signaling activity contributes to the uncontrolled growth of uveal melanoma cells. As G protein-coupled receptors (GPCRs) play a crucial role in this process, screening libraries of GPCR inhibitors has been explored as a potential therapeutic avenue. These libraries comprise a vast array of compounds with the potential to block or modulate GPCR activity. Identifying specific inhibitors that can effectively interfere with the oncogenic signaling cascades triggered by the mutated Gαq protein is a critical step in developing targeted therapies for uveal melanoma. Methods: GPCR library high throughput screening employed isogenic cell lines expressing wild type or mutant (Q209L) GNAQ. This process utilized IP-One assays to identify compounds affecting Gq-coupled receptors by detecting inositol monophosphate accumulation. Subsequently, promising hits were evaluated for their impact on inhibiting the growth and proliferation of uveal melanoma cells. Further analysis involved Western blotting to assess their potential in promoting autophagy and apoptosis, inhibiting downstream signaling pathways, and a migration assay. Finally, ROS generation was assessed using flow cytometry. Results: Using isogenic cell lines expressing GNAQ gene wild type and the Q209L mutant, a high-throughput screening of GPCR libraries identified sertindole among 5 hits. Sertindole showed a dose-dependent inhibition of Q209L-mutated uveal melanoma cells, significantly reducing cell viability, proliferation, and migration. It also suppressed key signaling pathways, including the critical MAPK pathway, and activated autophagy and apoptosis, likely via ROS generation. These findings suggest sertindole's multifaceted approach in suppressing cancer in uveal melanoma. Conclusion: Our study highlights the potential therapeutic value of sertindole in GNAQ Q209L-mutated uveal melanoma. Further research is needed to evaluate the efficacy of sertindole in preclinical mouse models of uveal melanoma. Citation Format: Shelby Rheinschmidt, Taylor Bargenquast, Sydney Adamson, Trason Thode, Alexis Weston, Tithi Ghosh Halder, Serina Ng, Harini Tulasi, Brian Durbin, Justin Moser, Mohan Kaadige, Raffaella Soldi, Sunil Sharma. G-protein coupled receptor (GPCR) screening library identifies sertindole, an antipsychotic, as potential therapeutic option to target GNAQ mutation Q209L in uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 671.

Identification of three elevenin receptors and roles of elevenin disulfide bond and residues in receptor activation in Aplysia californica

Neuropeptides are ubiquitous intercellular signaling molecules in the CNS and play diverse roles in modulating physiological functions by acting on specific G-protein coupled receptors (GPCRs). Among them, the elevenin signaling system is now believed to be present primarily in protostomes. Although elevenin was first identified from the L11 neuron of the abdominal ganglion in mollusc Aplysia californica, no receptors have been described in Aplysia, nor in any other molluscs. Here, using two elevenin receptors in annelid Platynereis dumerilii, we found three putative elevenin GPCRs in Aplysia. We cloned the three receptors and tentatively named them apElevR1, apElevR2, and apElevR3. Using an inositol monophosphate (IP1) accumulation assay, we demonstrated that Aplysia elevenin with the disulfide bond activated the three putative receptors with low EC50 values (ranging from 1.2 to 25 nM), supporting that they are true receptors for elevenin. In contrast, elevenin without the disulfide bond could not activate the receptors, indicating that the disulfide bond is required for receptor activity. Using alanine substitution of individual conserved residues other than the two cysteines, we showed that these residues appear to be critical to receptor activity, and the three different receptors had different sensitivities to the single residue substitution. Finally, we examined the roles of those residues outside the disulfide bond ring by removing these residues and found that they also appeared to be important to receptor activity. Thus, our study provides an important basis for further study of the functions of elevenin and its receptors in Aplysia and other molluscs.

Calcium-Sensing Receptor Negative Allosteric Modulators Oppose Contraction of Mouse Airways.

Asthma is a heterogeneous chronic airways disease with an unmet need for improved therapeutics in uncontrolled severe disease. The calcium-sensing receptor (CaSR) is a G protein-coupled receptor upregulated in asthma. The CaSR agonist, spermine, is also increased in asthmatic airways and contributes to bronchoconstriction. CaSR negative allosteric modulators (NAMs) oppose chronic airway inflammation, remodelling and hyperresponsiveness in murine and guinea pig asthma models, but whether CaSR NAMs are effective acute bronchodilators compared to standard-of-care has not yet been established. Further, the ability of different classes of NAMs to inhibit spermine-induced CaSR signaling or MCh-induced airway contraction has not been quantified. Here, we show CaSR NAMs differentially inhibit spermine-induced intracellular calcium mobilisation and inositol monophosphate accumulation in HEK293 cells stably expressing the CaSR. NAMs reverse methacholine (MCh)-mediated airway contraction in mouse precision cut lung slices with similar maximal relaxation compared to the standard treatment, salbutamol. Of note, the bronchodilator effects of CaSR NAMs are maintained under conditions of β2 adrenergic receptor desensitization when salbutamol efficacy is abolished. Furthermore, overnight treatment with some, but not all, CaSR NAMs prevents MCh-mediated bronchoconstriction. These findings further support the CaSR as a putative drug target, and NAMs as alternative or adjunct bronchodilators, in asthma.

Characterization of an Aplysia vasotocin signaling system and actions of posttranslational modifications and individual residues of the ligand on receptor activity.

The vasopressin/oxytocin signaling system is present in both protostomes and deuterostomes and plays various physiological roles. Although there were reports for both vasopressin-like peptides and receptors in mollusc Lymnaea and Octopus, no precursor or receptors have been described in mollusc Aplysia. Here, through bioinformatics, molecular and cellular biology, we identified both the precursor and two receptors for Aplysia vasopressin-like peptide, which we named Aplysia vasotocin (apVT). The precursor provides evidence for the exact sequence of apVT, which is identical to conopressin G from cone snail venom, and contains 9 amino acids, with two cysteines at position 1 and 6, similar to nearly all vasopressin-like peptides. Through inositol monophosphate (IP1) accumulation assay, we demonstrated that two of the three putative receptors we cloned from Aplysia cDNA are true receptors for apVT. We named the two receptors as apVTR1 and apVTR2. We then determined the roles of post-translational modifications (PTMs) of apVT, i.e., the disulfide bond between two cysteines and the C-terminal amidation on receptor activity. Both the disulfide bond and amidation were critical for the activation of the two receptors. Cross-activity with conopressin S, annetocin from an annelid, and vertebrate oxytocin showed that although all three ligands can activate both receptors, the potency of these peptides differed depending on their residue variations from apVT. We, therefore, tested the roles of each residue through alanine substitution and found that each substitution could reduce the potency of the peptide analog, and substitution of the residues within the disulfide bond tended to have a larger impact on receptor activity than the substitution of those outside the bond. Moreover, the two receptors had different sensitivities to the PTMs and single residue substitutions. Thus, we have characterized the Aplysia vasotocin signaling system and showed how the PTMs and individual residues in the ligand contributed to receptor activity.

Rigorous Characterization of Allosteric Modulation of the Human Metabotropic Glutamate Receptor 1 Reveals Probe- and Assay-Dependent Pharmacology.

Allosteric modulation of metabotropic glutamate receptor subtype 1 (mGlu1) represents a viable therapeutic target for treating numerous central nervous system disorders. Although multiple chemically distinct mGlu1 positive (PAMs) and negative (NAMs) allosteric modulators have been identified, drug discovery paradigms have not included rigorous pharmacological analysis. In the present study, we hypothesized that existing mGlu1 allosteric modulators possess unappreciated probe-dependent or biased pharmacology. Using human embryonic kidney 293 (HEK293A) cells stably expressing human mGlu1, we screened mGlu1 PAMs and NAMs from divergent chemical scaffolds for modulation of different mGlu1 orthosteric agonists in intracellular calcium (iCa2+) mobilization and inositol monophosphate (IP1) accumulation assays. Operational models of agonism and allosterism were used to derive estimates for important pharmacological parameters such as affinity, efficacy, and cooperativity. Modulation of glutamate and quisqualate-mediated iCa2+ mobilization revealed probe dependence at the level of affinity and cooperativity for both mGlu1 PAMs and NAMs. We also identified the previously described mGlu5 selective NAM PF-06462894 as an mGlu1 NAM with a different pharmacological profile from other NAMs. Differential profiles were also observed when comparing ligand pharmacology between iCa2+ mobilization and IP1 accumulation. The PAMs Ro67-4853 and CPPHA displayed apparent negative cooperativity for modulation of quisqualate affinity, and the NAMs CPCCOEt and PF-06462894 had a marked reduction in cooperativity with quisqualate in IP1 accumulation and upon extended incubation in iCa2+ mobilization assays. These data highlight the importance of rigorous assessment of mGlu1 modulator pharmacology to inform future drug discovery programs for mGlu1 allosteric modulators. SIGNIFICANCE STATEMENT: Metabotropic glutamate receptor subtype 1 (mGlu1) positive and negative allosteric modulators have therapeutic potential in multiple central nervous system disorders. We show that chemically distinct modulators display differential pharmacology with different orthosteric ligands and across divergent signaling pathways at human mGlu1. Such complexities in allosteric ligand pharmacology should be considered in future mGlu1 allosteric drug discovery programs.

Pharmacological characterization of novel small molecule agonists and antagonists for the orphan receptor GPR139

The orphan G protein-coupled receptor GPR139 is predominantly expressed in the central nervous system and has attracted considerable interest as a therapeutic target. However, the biological role of this receptor remains somewhat elusive, in part due to the lack of quality pharmacological tools to investigate GPR139 function. In an effort to understand GPR139 signaling and to identify improved compounds, in this study we performed virtual screening and analog searches, in combination with multiple pharmacological assays. We characterized GPR139-dependent signaling using previously published reference agonists in Ca2+ mobilization and inositol monophosphate accumulation assays, as well as a novel real-time GPR139 internalization assay. For the four reference agonists tested, the rank order of potency was conserved across signaling and internalization assays: JNJ-63533054 > Compound 1a » Takeda > AC4 > DL43, consistent with previously reported values. We noted an increased efficacy of JNJ-63533054-mediated inositol monophosphate signaling and internalization, relative to Compound 1a. We then performed virtual screening for GPR139 agonist and antagonist compounds that were screened and validated in GPR139 functional assays. We identified four GPR139 agonists that were active in all assays, with similar or reduced potency relative to known compounds. Likewise, compound analogs selected based on GPR139 agonist and antagonist substructure searches behaved similarly to their parent compounds. Thus, we have characterized GPR139 signaling for multiple new ligands using G protein-dependent assays and a new real-time internalization assay. These data add to the GPR139 tool compound repertoire, which could be optimized in future medical chemistry campaigns.

Differences across sexes on head-twitch behavior and 5-HT2A receptor signaling in C57BL/6J mice

Psychedelics, also known as classical hallucinogens, affect processes related to perception, cognition and sensory processing mostly via the serotonin 5-HT2A receptor (5-HT2AR). This class of psychoactive substances, which includes lysergic acid diethylamide (LSD), psilocybin, mescaline and the substituted amphetamine 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), is receiving renewed attention for their potential therapeutic properties as it relates to psychiatric conditions such as depression and substance use disorders. Current studies focused on the potentially clinical effects of psychedelics on human subjects tend to exclude sex as a biological variable. Much of the understanding of psychedelic pharmacology is derived from rodent models, but most of this preclinical research has only focused on male mice. Here we tested the effects of DOI on head-twitch behavior (HTR) – a mouse behavioral proxy of human psychedelic potential – in male and female mice. DOI elicited more HTR in female as compared to male C57BL/6J mice, a sex-specific exacerbated behavior that was not observed in 129S6/SvEv animals. Volinanserin (or M100907) – a 5-HT2AR antagonist – fully prevented DOI-induced HTR in male and female C57BL/6J mice. Accumulation of inositol monophosphate (IP1) in the frontal cortex upon DOI administration showed no sex-related effect in C57BL/6J mice. However, the pharmacokinetic properties of DOI differed among sexes – brain and plasma concentrations of DOI were lower 30 and 60 min after drug administration in female as compared to male C57BL/6J mice. Together, these results suggest strain-dependent and sex-related differences in the behavioral and pharmacokinetic profiles of the 5-HT2AR agonist DOI in C57BL/6J mice, and support the importance of studying sex as a biological variable in preclinical psychedelic research.

Abstract 851: Functional dissection of GNAQ and GNA11 oncogenic mutations identifies potential targeted therapy

Abstract Background: Oncogenic variants of GNAQ and GNA11 arising in embryonic or fetal development lead to a spectrum of congenital mosaic disorders including Sturge-Weber syndrome (SWS) and Phakomatosis Pigmentovascularis (PPV). Both SWS and PPV are characterised by vascular malformations in brain, skin and eye, with worsening neurological sequelae after birth suggesting a potential treatment window. Although the genetic causes are understood, and previous data in non-disease-specific cell lines have suggested MAPK activation, the functional effects of the mutations in vascular endothelium have not been studied. The characteristic finding of intracerebral intravascular calcification led us to hypothesise that intra-cellular calcium-signalling disturbances leading to localised imbalances in calcium homeostasis may be involved in disease pathogenesis. Methods: Stable cell lines were established to study cell signalling downstream of GNAQ c.548G>A, p.(R183Q) or GNA11 c.547C>T, p.(R183C) variants in endothelial cells. We re-expressed mutant GNAQ or GNA11 alleles in HEK293 cells in which both endogenous genes had been knocked out for validation purposes. Constitutive calcium signaling was evaluated by measuring inositol-monophosphate accumulation and by a NFAT-luciferase reporter assay, while ligand-stimulated calcium signaling was monitored over time following incubation with intra-cellular calcium probe Fluo-8. In a parallel complementary study, serum calcium and related indices were assayed in 35 patients with SWS or PPV, consented for research under appropriate approvals. Results: GNAQ and GNA11 variants led to marked constitutive calcium signalling in vascular endothelial cells, but not to MAPK activation. GNAQ-mutant endothelial cells also showed aberrant calcium signalling responses to GPCR ligand, which led in turn to sustained replenishment of intracellular calcium stores from the extracellular space. These calcium signalling defects could be rescued by a specific calcium channel inhibitor. Strikingly, and previously undescribed in these diseases, 43% of patients were found to have hypocalcaemia, and 20% hyperparathyroidism, currently presumed secondary but under further investigation. Conclusions: GNAQ mosaicism leads to constitutive and ligand-induced over-activation of intracellular calcium signalling, which increases influx of calcium from the extracellular to the intracellular space and could be the explanation for systemic hypocalcaemia in a substantial proportion of patients. These data have immediate implications for clinical management of these mosaic diseases, and shed light on the in vivo pathogenic actions of of GNAQ/GNA11 oncogenic variants. Citation Format: Davide Zecchin, Nicole Knoepfel, Anna Gluck, Mark Stevenson, Kate E. Lines, Satyamaanasa Polubothu, Noreen Muwanga-Nanyonjo, Sara Barberan-Martin, Fanourios Michailidis, Dale Bryant, Ulrike Loebel, Asuka Inoue, Robert Semple, Sarah Aylett, Rajesh V. Thakker, Veronica A. Kinsler. Functional dissection of GNAQ and GNA11 oncogenic mutations identifies potential targeted therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 851.

Tasipimidine—the pharmacological profile of a novel orally active selective α2A-adrenoceptor agonist

The pharmacological profile of tasipimidine, a novel orally active α2-adrenoceptor agonist developed for situational anxiety and fear in dogs, was studied in various in vitro and in vivo models. In the cell assays, tasipimidine demonstrated binding affinity and full agonism on the human α2A-adrenoceptors with a pEC50 of 7.57, while agonism on the α2B-and α2C-adrenoceptors and the rodent α2D-adrenoceptor was weaker, resulting in pEC50 values of 6.00, 6.29 and 6.56, respectively. Tasipimidine had a low binding affinity on the human α1-adrenoceptors. It had no functional effects in the LNCaP cells expressing endogenously the human α1A-adrenoceptors but was a weak agonist in the Chem-1 cells coexpressing Gα15 protein and α1A-adrenoceptors. In the recombinant CHO cells, although tasipimidine was a weak partial agonist in the inositol monophosphate accumulation assay, it was a full agonist in the intracellular [Ca2+] assay. No functional effects were observed on the human α1B-adrenoceptor, whereas in the rat α1A and α1B-adrenoceptors, tasipimidine was a weak partial agonist. In the rat vas deferens preparations, tasipimidine was a full agonist on the α2D-adrenoceptor but weak partial agonist on the α1-adrenoceptor. The receptor profile of tasipimidine indicated few secondary targets, and no functional effects were observed. Sedative effects of tasipimidine were demonstrated in vivo by the reduced acoustic startle reflex in rats with subcutaneous doses and decreased spontaneous locomotor activity in mice with subcutaneous and higher oral doses. It may be concluded that tasipimidine is an orally active and selective α2A-adrenoceptor agonist.

RAGE ligands stimulate angiotensin II type I receptor (AT1) via RAGE/AT1 complex on the cell membrane

The receptor for advanced glycation end-products (RAGE) and the G protein-coupled angiotensin II (AngII) type I receptor (AT1) play a central role in cardiovascular diseases. It was recently reported that RAGE modifies AngII-mediated AT1 activation via the membrane oligomeric complex of the two receptors. In this study, we investigated the presence of the different directional crosstalk in this phenomenon, that is, the RAGE/AT1 complex plays a role in the signal transduction pathway of RAGE ligands. We generated Chinese hamster ovary (CHO) cells stably expressing RAGE and AT1, mutated AT1, or AT2 receptor. The activation of two types of G protein α-subunit, Gq and Gi, was estimated through the accumulation of inositol monophosphate and the inhibition of forskolin-induced cAMP production, respectively. Rat kidney epithelial cells were used to assess RAGE ligand-induced cellular responses. We determined that RAGE ligands activated Gi, but not Gq, only in cells expressing RAGE and wildtype AT1. The activation was inhibited by an AT1 blocker (ARB) as well as a RAGE inhibitor. ARBs inhibited RAGE ligand-induced ERK phosphorylation, NF-κB activation, and epithelial–mesenchymal transition of rat renal epithelial cells. Our findings suggest that the activation of AT1 plays a central role in RAGE-mediated cellular responses and elucidate the role of a novel molecular mechanism in the development of cardiovascular diseases.

Detailed In Vitro Pharmacological Characterization of Clinically Tested Negative Allosteric Modulators of the Metabotropic Glutamate Receptor 5

Negative allosteric modulation of the metabotropic glutamate 5 (mGlu5) receptor has emerged as a potential strategy for the treatment of neurologic disorders. Despite the success in preclinical studies, many mGlu5 negative allosteric modulators (NAMs) that have reached clinical trials failed due to lack of efficacy. In this study, we provide a detailed in vitro pharmacological characterization of nine clinically and preclinically tested NAMs. We evaluated inhibition of l-glutamate-induced signaling with Ca2+ mobilization, inositol monophosphate (IP1) accumulation, extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, and real-time receptor internalization assays on rat mGlu5 expressed in HEK293A cells. Moreover, we determined association rates (kon) and dissociation rates (koff), as well as NAM affinities with [3H]methoxy-PEPy binding experiments. kon and koff values varied greatly between the nine NAMs (34- and 139-fold, respectively) resulting in long receptor residence times (>400 min) for basimglurant and mavoglurant, medium residence times (10-30 min) for AZD2066, remeglurant, and (RS)-remeglurant, and low residence times (<10 mins) for dipraglurant, F169521, F1699611, and STX107. We found that all NAMs inhibited l-glutamate-induced mGlu5 receptor internalization, generally with a similar potency to IP1 accumulation and ERK1/2 phosphorylation, whereas Ca2+ mobilization was less potently inhibited. Operational model of allosterism analyses revealed that dipraglurant and (RS)-remeglurant were biased toward (affinity) receptor internalization and away (cooperativity) from the ERK1/2 phosphorylation pathway, respectively. Our study is the first to measure mGlu5 NAM binding kinetics and negative allosteric modulation of mGlu5 receptor internalization and adds significant new knowledge about the molecular pharmacology of a diverse range of clinically relevant NAMs. SIGNIFICANCE STATEMENT: The metabotropic glutamate 5 (mGlu5) receptor is important in many brain functions and implicated in several neurological pathologies. Negative allosteric modulators (NAMs) have shown promising results in preclinical models but have so far failed in human clinical trials. Here we provide the most comprehensive and comparative molecular pharmacological study to date of nine preclinically/clinically tested NAMs at the mGlu5 receptor, which is also the first study to measure ligand binding kinetics and negative allosteric modulation of mGlu5 receptor internalization.

SAT-036 Extracellular Domain Calcium-Sensing Receptor (CaSR) Mutations Leading to Hypercalcemic and Hypocalcemic Disorders Cluster at the Homodimeric Interface

The calcium-sensing receptor (CaSR) is a homodimeric G-protein coupled receptor that plays a pivotal role in extracellular Ca2+ (Ca2+e) homeostasis, and loss- and gain-of-function mutations of the CaSR cause familial hypocalciuric hypercalcemia type 1 (FHH1) and autosomal dominant hypocalcemia type 1 (ADH1), respectively. Crystal structure analysis has shown that the CaSR has a large extracellular domain (ECD) comprising lobes 1 and 2, and a cysteine-rich domain (CRD), which connects the ECD to the transmembrane domain. The CaSR ECD binds Ca2+e at multiple sites, which induces conformational changes at the extracellular dimer interface that lead to intracellular signaling via the inositol trisphosphate (IP3) and mitogen-activated protein kinase (MAPK) pathways. To further elucidate the structure-function relationships of the ECD, we examined the location of all CaSR ECD mutations reported to-date in FHH1 and ADH1 probands using available CaSR crystal structures. These studies identified that 127 FHH1 and 66 ADH1 mutations affected ECD residues, with >50% of these mutations being located at the dimer interface. Mutations predicted to disrupt key CaSR dimer-dimer interactions included: a lobe 1 Tyr161Cys mutation, which impaired an interprotomer interaction with the lobe 1 Pro55 residue; a Ser171Asn mutation predicted to disrupt a lobe 2 interprotomer salt bridge, which forms upon agonist binding; and a Gly553Arg mutation, which altered interprotomer hydrophobic interactions within the CRD. We investigated the effect of the Tyr161Cys, Ser171Asn and Gly553Arg mutations on receptor dimerization using western blotting and showed that these mutant proteins all form dimers similar to the wild-type (WT) CaSR. Furthermore, we evaluated the effect of these mutations on CaSR function in HEK293 cells following stimulation with Ca2+e, by measuring accumulation of inositol monophosphate (IP1), which is an IP3 metabolite, and also by measuring serum response element (SRE)-containing luciferase reporter fold-change responses, which is an indicator of MAPK activation. The Tyr161Cys, Ser171Asn and Gly553Arg dimer interface mutations were shown to significantly impair CaSR-mediated signaling. Indeed, cells expressing the Tyr161Cys, Ser171Asn or Gly553Arg mutant CaSRs all showed a >30% reduction in IP1 accumulation and >50% reduction in SRE-reporter responses, respectively, compared to WT-expressing cells (p<0.05, results are from 3 independent experiments). Thus, these studies demonstrate that the majority of FHH1- and ADH1-causing CaSR ECD mutations are located at the dimer interface, but likely do not disrupt dimer formation. Instead, these mutations may potentially influence conformational changes that occur at the dimer interface upon CaSR activation.

Design, Synthesis, and Evaluation of a Diazirine Photoaffinity Probe for Ligand-Based Receptor Capture Targeting G Protein–Coupled Receptors

Chemoproteomic approaches to identify ligand-receptor interactions have gained popularity. However, identifying transmembrane receptors remains challenging. A new trifunctional probe to aid the nonbiased identification of such receptors was developed and synthesized using a convenient seven-step synthesis. This probe contained three functional groups: 1) an N-hydroxysuccinimide ester for ligand-coupling through free amines, 2) a diazirine moiety to capture the receptor of interest upon irradiation with UV light, and 3) a biotin group which allowed affinity purification of the final adduct using streptavidin. The interaction between the G protein–coupled tachykinin neurokinin 1 (NK1) receptor, expressed in an inducible manner, and the peptidic ligand substance P was used as a test system. Liquid chromatography–mass spectrometry analysis confirmed successful coupling of the probe to substance P, while inositol monophosphate accumulation assays demonstrated that coupling of the probe did not interfere substantially with the substance P–NK1 receptor interaction. Confocal microscopy and western blotting provided evidence of the formation of a covalent bond between the probe and the NK1 receptor upon UV activation. As proof of concept, the probe was used in full ligand-based receptor-capture experiments to identify the substance P–binding receptor via liquid chromatography–tandem mass spectrometry, resulting in the successful identification of only the NK1 receptor. This provides proof of concept toward general utilization of this probe to define interactions between ligands and previously unidentified plasma-membrane receptors.

Assessment of the Molecular Mechanisms of Action of Novel 4-Phenylpyridine-2-One and 6-Phenylpyrimidin-4-One Allosteric Modulators at the M1 Muscarinic Acetylcholine Receptors

Positive allosteric modulators (PAMs) that target the M1 muscarinic acetylcholine (ACh) receptor (M1 mAChR) are potential treatments for cognitive deficits in conditions such as Alzheimer disease and schizophrenia. We recently reported novel 4-phenylpyridine-2-one and 6-phenylpyrimidin-4-one M1 mAChR PAMs with the potential to display different modes of positive allosteric modulation and/or agonism but whose molecular mechanisms of action remain undetermined. The current study compared the pharmacology of three such novel PAMs with the prototypical first-generation PAM, benzyl quinolone carboxylic acid (BQCA), in a recombinant Chinese hamster ovary (CHO) cell line stably expressing the human M1 mAChR. Interactions between the orthosteric agonists and the novel PAMs or BQCA suggested their allosteric effects were solely governed by modulation of agonist affinity. The greatest degree of positive co-operativity was observed with higher efficacy agonists, whereas minimal potentiation was observed when the modulators were tested against the lower efficacy agonist, xanomeline. Each PAM was investigated for its effects on the endogenous agonist ACh on three different signaling pathways [extracellular signal-regulated kinases 1/2 phosphorylation, inositol monophosphate (IP1) accumulation, and β-arrestin-2 recruitment], revealing that the allosteric potentiation generally tracked with the efficiency of stimulus-response coupling, and that there was little pathway bias in the allosteric effects. Thus, despite the identification of novel allosteric scaffolds targeting the M1 mAChR, the molecular mechanism of action of these compounds is largely consistent with a model of allostery previously described for BQCA, suggesting that this may be a more generalized mechanism for M1 mAChR PAM effects than previously appreciated.

“Selective” Class C G Protein-Coupled Receptor Modulators Are Neutral or Biased mGlu5 Allosteric Ligands

Numerous positive and negative allosteric modulators (PAMs and NAMs) of class C G protein-coupled receptors (GPCRs) have been developed as valuable preclinical pharmacologic tools and therapeutic agents. Although many class C GPCR allosteric modulators have undergone subtype selectivity screening, most assay paradigms have failed to perform rigorous pharmacologic assessment. Using mGlu5 as a representative class C GPCR, we tested the hypothesis that allosteric modulator selectivity was based on cooperativity rather than affinity. Specifically, we aimed to identify ligands that bound to mGlu5 but exhibited neutral cooperativity with mGlu5 agonists. We additionally evaluated the potential for these ligands to exhibit biased pharmacology. Radioligand binding, intracellular calcium (iCa2+) mobilization, and inositol monophosphate (IP1) accumulation assays were undertaken in human embryonic kidney cells expressing low levels of rat mGlu5 (HEK293A-mGlu5-low) for diverse allosteric chemotypes. Numerous "non-mGlu5" class C GPCR allosteric modulators incompletely displaced allosteric mGlu5 radioligand [3H]methoxy-PEPy binding, consistent with a negative allosteric interaction. Affinity estimates for CPCCOEt (mGlu1 ligand), PHCCC (mGlu4 ligand), GS39783 (GABAB ligand), AZ12216052 (mGlu8 ligand), and CGP7930 (GABAB ligand) at mGlu5 were within 10-fold of their target receptor. Most class C GPCR allosteric modulators had neutral cooperativity with both orthosteric and allosteric mGlu5 agonists in functional assays; however, NPS2143 (calcium-sensing receptor (CaSR) NAM), cinacalcet (CaSR PAM), CGP7930, and AZ12216052 were partial mGlu5 agonists for IP1 accumulation, but not iCa2+ mobilization. By using mGlu5 as a model class C GPCR, we find that for many class C GPCR allosteric modulators, subtype selectivity is driven by cooperativity and misinterpreted owing to unappreciated bias.

Functional Authentication of a Novel Gastropod Gonadotropin-Releasing Hormone Receptor Reveals Unusual Features and Evolutionary Insight.

A gonadotropin-releasing hormone (GnRH)-like molecule was previously identified in a gastropod, Aplysia californica, and named ap-GnRH. In this study, we cloned the full-length cDNA of a putative ap-GnRH receptor (ap-GnRHR) and functionally authenticated this receptor as a bona fide ap-GnRHR. This receptor contains two potential translation start sites, each accompanied by a Kozak sequence, suggesting the translation of a long and a short form of the receptor is possible. The putative ap-GnRHR maintains the conserved structural motifs of GnRHR-like receptors and shares 45% sequence identity with the octopus GnRHR. The expression of the putative ap-GnRHR short form is ubiquitous in all tissues examined, whereas the long form is only expressed in parts of the central nervous system, osphradium, small hermaphroditic duct, and ovotestis. The cDNA encoding the long or the short receptor was transfected into the Drosophila S2 cell line and subject to a radioreceptor assay using 125I-labeled ap-GnRH as the radioligand. Further, the transfected cells were treated with various concentrations of ap-GnRH and measured for the accumulation of cAMP and inositol monophosphate (IP1). Radioreceptor assay revealed that only the long receptor bound specifically to the radioligand. Further, only the long receptor responded to ap-GnRH with an increased accumulation of IP1, but not cAMP. Our studies show that despite the more prevalent expression of the short receptor, only the long receptor is the functional ap-GnRHR. Importantly, this is only the second report on the authentication of a protostome GnRHR, and based on the function and the phylogenetic grouping of ap-GnRHR, we suggest that this receptor is more similar to protostome corazonin receptors than chordate GnRHRs.

Effects of amitriptyline, a tricyclic antidepressant, on smooth muscle reactivity in isolated rat trachea

This study was designed to investigate the action of amitriptyline, a tricyclic antidepressant, on airway smooth muscle reactivity and its underlying mechanisms. In isolated rat trachea, isometric force was recorded to examine the effects of amitriptyline on the contractile response to acetylcholine (ACh), electrical field stimulation (EFS), calyculin A (a myosin light chain phosphatase inhibitor), and sphingosylphosphorylcholine (SPC; a Rhokinase activator). In addition, inositol monophosphate (IP1) accumulation was measured to examine its effects on inositol 1, 4, 5-trisphosphate (IP(3)) production during stimulation with ACh. Amitriptyline inhibited the contractile responses to ACh, EFS, calyculin A, and SPC, with the concentrations of amitriptyline (mean +/- SD) required to exert 50% inhibition (IC(50)) being 4.3 +/- 1.3 microM, 3.2 +/- 1.6 microM, 256.4 +/- 106.4 microM, and 98.2 +/- 21.8 microM, respectively. In addition, amitriptyline (10 microM) eliminated the ACh (10 microM)-induced IP(1) accumulation. The results suggest that amitriptyline does not influence tracheal smooth muscle reactivity at clinical concentrations (<1 microM), but attenuates the reactivity at supraclinical concentrations (> or =1 microM). The attenuated response to ACh brought about by amitriptyline is presumably due, at least in part, to the inhibition of phosphatidylinositol (PI) metabolism. The ability of amitriptyline to inhibit the calyculin Ainduced contraction suggests that amitriptyline also inhibits the Ca(2+)-calmodulin-myosin light chain pathway independently of the inhibition of PI metabolism. Finally, the difference between the IC(50) values for SPC-induced contraction and those for calyculin A-induced contraction suggests that amitriptyline may also inhibit the Rho-kinase pathway.

Specific Leukotriene Receptors Couple to Distinct G Proteins to Effect Stimulation of Alveolar Macrophage Host Defense Functions

Leukotrienes (LTs) are lipid mediators implicated in asthma and other inflammatory diseases. LTB(4) and LTD(4) also participate in antimicrobial defense by stimulating phagocyte functions via ligation of B leukotriene type 1 (BLT1) receptor and cysteinyl LT type 1 (cysLT1) receptor, respectively. Although both Galpha(i) and Galpha(q) proteins have been shown to be coupled to both BLT1 and cysLT1 receptors in transfected cell systems, there is little known about specific G protein subunit coupling to LT receptors, or to other G protein-coupled receptors, in primary cells. In this study we sought to define the role of specific G proteins in pulmonary alveolar macrophage (AM) innate immune responses to LTB(4) and LTD(4). LTB(4) but not LTD(4) reduced cAMP levels in rat AM by a pertussis toxin (PTX)-sensitive mechanism. Enhancement of FcgammaR-mediated phagocytosis and bacterial killing by LTB(4) was also PTX-sensitive, whereas that induced by LTD(4) was not. LTD(4) and LTB(4) induced Ca(2+) and intracellular inositol monophosphate accumulation, respectively, highlighting the role of Galpha(q) protein in mediating PTX-insensitive LTD(4) enhancement of phagocytosis and microbicidal activity. Studies with liposome-delivered G protein blocking Abs indicated a dependency on specific Galpha(q/11) and Galpha(i3) subunits, but not Galpha(i2) or G(beta)gamma, in LTB(4)-enhanced phagocytosis. The selective importance of Galpha(q/11) protein was also demonstrated in LTD(4)-enhanced phagocytosis. The present investigation identifies differences in specific G protein subunit coupling to LT receptors in antimicrobial responses and highlights the importance of defining the specific G proteins coupled to heptahelical receptors in primary cells, rather than simply using heterologous expression systems.

Multiple signaling pathways involved in the effect of endothelin type b receptor in rat median eminence

We assessed the possible link between endothelin receptor mediated phosphoinositide breakdown and NO/cGMP signaling pathways in rat arcuate nucleus-median eminence fragments (AN-ME), brain structures known to contain a rich plexus of nitric oxide synthase (NOS)-containing neurons and fibers, together with densely arranged endothelin ETB-receptors-like immunoreactive fibres. Our data show that ET-1, ET-3 and the ETB-receptors agonist, IRL 1620, increased inositol monophosphate (InsP1) accumulation, NOS activity and cGMP formation, in a similar degree. The stimulatory effect of ETs on InsP1 accumulation and cGMP formation was inhibited by the phospholipase C (PLC) inhibitor, neomycin, and the absence of extracellular calcium, suggesting that calcium is involved in endothelin receptor-induced PLC activation. The L-arginine analog, L-NAME, inhibited ET-1 or IRL1620-stimulated cGMP formation. The ETA receptor antagonists BQ 123, did not alter, while the ETB receptor antagonists BQ788 inhibited ETs-induced increase in the PI metabolism, NOS activity and cGMP generation. Our data indicate that in AN-ME, ETB receptor signals through receptor-mediated calcium dependent-stimulation of phosphoinositide breakdown and activation of NOS/cGMP signaling pathway.