Val J Watts’s research while affiliated with Purdue University West Lafayette and other places

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Publications (193)


Figure 2. mu-opioid receptor (MOR) agonist DAMGO-mediated inhibition of cAMP activity in HEK-AC1 cells. A-B) Brightfield (A) and GFP (B) images of HEK-AC1 cells expressing MOR and cADDis cAMP biosensor. C) Time-lapse images before (pre) and after stimulation of AC1 by 1 µM A23187, subsequent inhibition of AC activity by 1 µM DAMGO and maximal (Max) AC stimulation (10 µM FSK in the presence of 100 µM IBMX and 10 µM naloxone). Scale bar: 20 µm (A-C). D-I) cAMP kinetics following stimulation with 1 µM forskolin (FSK) or 5 µM A23187 (A23) followed by the addition of DAMGO as indicated. Bar
Figure 3. cAMP overshoot after chronic treatment with DAMGO in HEK-AC1 cells. A-C) Cells expressing cAMP-sensor and the MOR were treated with DAMGO or vehicle for 18 hr and AC activity was stimulated with 0.5 μM forskolin (FSK). Bar graphs showing cAMP overshoot peak and total responses. D-F) Cells were pretreated with vehicle or DAMGO for 18 hrs in the absence or presence of naloxone (NAL) or pertussis toxin (PTX) or MLN4924 as indicated followed by AC1 activation using A23187. Bar graphs showing peak and total cAMP overshoot responses. All error bars denote the SEM. For FSK stimulation (panel A-C), N=3; n=4 (Veh), n=4 (DAMGO); For A23187 stimulation (panel D-F) N=3; n=4 (all treatment groups). ShapiroWilk test of normality, followed by two-tailed unpaired t-test (panel B-C), or one-way ANOVA
Figure 4. Regulation of endogenous adenylyl cyclase activity in SH-SY5Y neuroblastoma cells. A-B) Expression of cADDis cAMP biosensor in SH-SY5Y cells. C-F) Time lapse images
Figure 5. cAMP overshoot in SH-SY5Y cells stimulated with the calcium ionophore A23187. A-C) Wild-type SH-SY5Y cells or D-F) SH-SY5Y cells expressing recombinant AC1 and MOR stimulated were pretreated with vehicle or DAMGO alone for 18 hr in the absence or presence of naloxone (NAL), pertussis toxin (PTX), MLN4924 or βark-CT transduction as indicated. AC activity was stimulated using A23187 in the presence of naloxone (NAL). (A, D) Average ΔF/F traces following A23187 stimulation for wild-type (A) or recombinant AC1-expressing (D) SH-SY5Y cells. B-C and E-F) Peak and total cAMP (AUCs) for all ROIs for the A23187 response (30 minutes). All bars denote average, and all error bars denote the SEM. For panel A-C N=4,
Figure 6. Decreased diazoxide-induced potassium flux in SH-SY5Y cells after chronic opioid stimulation. Photomicrographs of cells (A) Before thallium (Tl+) (B) After thallium (Tl+) in non-drug exposed cells, (C) Before and (D) after thallium addition in the prescence of the KATP channel opener, diazoxide (100uM). (E) Potassium flux measurements comparing the peak response compared to baseline, using thallium-based assay and confocal microscopy on SH-SY5Y cells. Exposure to diazoxide, a SUR1-selective KATP channel subtype agonist, increases potassium flux compared to baseline. Prior exposure of a KATP channel antagonist, glyburide, decreases potassium flux back untreated levels. Chronic administration of morphine at 0.5 uM and 10 uM over 72 hours, also decreases potassium flux. (F) Data in A presented as area under the curve. (G) Pre-exposure to a EPAC inhibitor, ESI-09, significantly increases potassium flux after morphine treatment (10uM, 72 hrs). (H) Inhibition of AC1 by pre-exposure

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Inhibition of adenylyl cyclase 1 (AC1) and exchange protein directly activated by cAMP (EPAC) restores ATP-sensitive potassium (KATP) channel activity after chronic opioid exposure
  • Preprint
  • File available

February 2025

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8 Reads

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Sabbir Alam

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Kayla Johnson

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[...]

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Val J Watts

Prolonged exposure to Gαi/o receptor agonists such as opioids can lead to a sensitization of adenylyl cyclases (ACs), resulting in heterologous sensitization or cyclic AMP (cAMP) overshoot. The molecular consequences of cAMP overshoot are not well understood, but this adaptive response is suggested to play a critical role in the development of opioid tolerance and withdrawal. We found that genetic reduction of AC1 and simultaneous upregulation of ATP-sensitive potassium (KATP) channel subunits, SUR1 or Kir6.2, significantly attenuated morphine tolerance and reduced naloxone-precipitated withdrawal. In vitro models utilized an EPAC2-GFP-cAMP biosensor to investigate sensitization of adenylyl cyclase in SH-SY5Y neuroblastoma cells and HEKΔAC3/6 knockout cells. Acute application of DAMGO significantly decreased the cAMP signal from the EPAC2-GFP-cAMP biosensor, while chronic DAMGO administration resulted in enhanced cAMP production following AC stimulation. Inhibition of cAMP overshoot was observed with naloxone (NAL), pertussis toxin (PTX), and the neddylation inhibitor, MLN4924 (Pevonedistat), as well as co-expression of β-adrenergic receptor kinase C-terminus (βARK-CT). After establishment of the AC1-EPAC sensitization in the in vitro models, we found that inhibition of AC1 or EPAC enhanced potassium channel activity after chronic morphine treatment, using a thallium-based assay in SH-SY5Y cells. Similar data were obtained in mouse dorsal root ganglia (DRG) after chronic morphine treatment. This study presents evidence for investigating further AC1 signaling as a target for opioid tolerance and withdrawal, by increasing EPAC activity and affecting potassium channels downstream of opioid receptors.

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Figure 3. Human GPR17 long isoform negatively regulates GLP-1 secretion from GLUTag cells. (A) GLP-1 secretion was measured from control-or hGPR17L-expressing GLUTag cells that were treated with either vehicle or 10 µM forskolin and 10 µM IBMX (F/I). Data represent mean ± SEM of six independent experiments and were analyzed by paired t test comparing control and hGPR17L expressing cells. *, p < 0.05. (B) The effect of 100 nM MDL29,951 or 100 µM MDL29,951 treatment on F/I-stimulated GLP-1 secretion. Data represent mean ± SEM of six independent experiments and were analyzed by one sample t test compared to 100. ***, p < 0.001. (C) Effect of 100 µM MDL29,951 on GLP-1 secretion stimulated by varying concentrations of forskolin together with 10 µM IBMX. Data represent mean ± SEM of two independent experiments. (D) F/I-stimulated GLP-1 secretion was measured upon treatment with indicated concentrations of MDL29,951 in the presence or absence of 10 µM HAMI3379 in GLUTag cells expressing hGPR17L. Data represent four independent experiments, and statistical analysis was conducted using unpaired t tests between vehicle and HAMI3379-treated cells for each concentration of MDL29,951. *, p < 0.05.
List of compounds and relevant molecular functions.
G Protein-Coupled Receptor 17 Inhibits Glucagon-like Peptide-1 Secretion via a Gi/o-Dependent Mechanism in Enteroendocrine Cells

December 2024

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18 Reads

Biomolecules

Gut peptides, including glucagon-like peptide-1 (GLP-1), regulate metabolic homeostasis and have emerged as the basis for multiple state-of-the-art diabetes and obesity therapies. We previously showed that G protein-coupled receptor 17 (GPR17) is expressed in intestinal enteroendocrine cells (EECs) and modulates nutrient-induced GLP-1 secretion. However, the GPR17-mediated molecular signaling pathways in EECs have yet to be fully deciphered. Here, we expressed the human GPR17 long isoform (hGPR17L) in GLUTag cells, a murine EEC line, and we used the GPR17 synthetic agonist MDL29,951 together with pharmacological probes and genetic approaches to quantitatively assess the contribution of GPR17 signaling to GLP-1 secretion. Constitutive hGPR17L activity inhibited GLP-1 secretion, and MDL29,951 treatment further inhibited this secretion, which was attenuated by treatment with the GPR17 antagonist HAMI3379. MDL29,951 promoted both Gi/o and Gq protein coupling to mediate cyclic AMP (cAMP) and calcium signaling. hGPR17L regulation of GLP-1 secretion appeared to be Gq-independent and dependent upon Gi/o signaling, but was not correlated with MDL29,951-induced whole-cell cAMP signaling. Our studies revealed key signaling mechanisms underlying the role of GPR17 in regulating GLP-1 secretion and suggest future opportunities for pharmacologically targeting GPR17 with inverse agonists to maximize GLP-1 secretion.


Discovery of Novel and Selective GPR17 Antagonists as Pharmacological Tools for Developing New Therapeutic Strategies in Diabetes and Obesity

December 2024

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18 Reads

G protein coupled receptors (GPCRs) are promising targets for diabetes and obesity therapy due to their roles in metabolism and excellent potential for pharmacological manipulation. We previously reported that Gpr17 ablation in the brain-gut axis leads to improved metabolic homeostasis, suggesting GPR17 antagonism could be developed for diabetes and obesity treatment. Here, we performed high throughput screening (HTS) and identified two new GPR17 antagonists (compound 978 and 527). Both compounds antagonized downstream Gαi/o, Gαq and β-arrestin signaling with high selectivity for GPR17, but not the closely related purinergic and cysteinyl leukotriene receptors. The molecular mechanisms of antagonism were revealed through Schild analysis, structure-activity relationship (SAR) studies and homology modelling. Compound 978 and its analog (793) attenuated GPR17 signaling and promoted glucagon-like peptide-1 (GLP-1) secretion in enteroendocrine cells. In summary, we identified selective GPR17 antagonists through HTS, which represent promising pharmacological tools for developing new therapeutic strategies in diabetes and obesity. Significance Our work highlights the therapeutic potential of GPR17 antagonism in the treatment of diabetes and obesity by leveraging its role in metabolic regulation. In previous studies, we have shown that Gpr17 ablation improves metabolic homeostasis, and here we expanded our research by identifying two novel small molecule antagonists of GPR17 through high-throughput screening. The compounds inhibited multiple downstream signaling pathways of GPR17 with high selectivity over other closely related receptors. Of particular significance, compound 978 and its analogs not only attenuated GPR17 signaling but also increased glucagon-like peptide-1 (GLP-1) secretion, a critical hormone for glucose homeostasis and appetite regulation. These findings shed new light into the molecular mechanisms of GPR17 antagonism and introduce valuable pharmacological tools for further exploration of therapeutic strategies in diabetes and obesity.


G protein-coupled receptor 17 inhibits glucagon-like peptide-1 secretion via a Gi/o-dependent mechanism in enteroendocrine cells

November 2024

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18 Reads

Gut peptides, including glucagon-like peptide-1 (GLP-1), regulate metabolic homeosta-sis and have emerged as the basis for multiple state-of-the-art diabetes and obesity therapies. We previously showed that G protein-coupled receptor 17 (GPR17) is expressed in intestinal entero-endocrine cells (EECs) and modulates nutrient-induced GLP-1 secretion. However, the GPR17-mediated molecular signaling pathways in EECs have yet to be fully deciphered. Here, we expressed the human GPR17 long isoform (hGPR17L) in GLUTag cells, a murine EEC line, and we used the GPR17 synthetic agonist MDL29,951 together with pharmacological probes and ge-netic approaches to quantitatively assess the contribution of GPR17 signaling to GLP-1 secretion. Constitutive hGPR17L activity inhibited GLP-1 secretion, and MDL29,951 treatment further in-hibited this secretion, which was attenuated by treatment with the GPR17 antagonist HAMI3379. MDL29,951 promoted both Gi/o and Gq protein coupling to mediate cyclic AMP (cAMP) and calcium signaling. hGPR17L regulation of GLP-1 secretion was Gq-independent and dependent upon Gi/o signaling, but was not correlated with MDL29,951-induced whole-cell cAMP signal-ing. Our studies revealed key signaling mechanisms underlying the role of GPR17 in regulating GLP-1 secretion and suggest future opportunities for pharmacologically targeting GPR17 with inverse agonists to maximize GLP-1 secretion.



Germline mutations in a G protein identify signaling cross-talk in T cells

September 2024

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191 Reads

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1 Citation

Science

Humans with monogenic inborn errors responsible for extreme disease phenotypes can reveal essential physiological pathways. We investigated germline mutations in GNAI2 , which encodes G αi2 , a key component in heterotrimeric G protein signal transduction usually thought to regulate adenylyl cyclase–mediated cyclic adenosine monophosphate (cAMP) production. Patients with activating G αi2 mutations had clinical presentations that included impaired immunity. Mutant G αi2 impaired cell migration and augmented responses to T cell receptor (TCR) stimulation. We found that mutant G αi2 influenced TCR signaling by sequestering the guanosine triphosphatase (GTPase)–activating protein RASA2, thereby promoting RAS activation and increasing downstream extracellular signal–regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)–AKT S6 signaling to drive cellular growth and proliferation.


A molecular mechanism to diversify Ca signaling downstream of Gs protein-coupled receptors

September 2024

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237 Reads

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6 Citations

A long-held tenet in inositol-lipid signaling is that cleavage of membrane phosphoinositides by phospholipase Cβ (PLCβ) isozymes to increase cytosolic Ca²⁺ in living cells is exclusive to Gq- and Gi-sensitive G protein-coupled receptors (GPCRs). Here we extend this central tenet and show that Gs-GPCRs also partake in inositol-lipid signaling and thereby increase cytosolic Ca²⁺. By combining CRISPR/Cas9 genome editing to delete Gαs, the adenylyl cyclase isoforms 3 and 6, or the PLCβ1-4 isozymes, with pharmacological and genetic inhibition of Gq and G11, we pin down Gs-derived Gβγ as driver of a PLCβ2/3-mediated cytosolic Ca²⁺ release module. This module does not require but crosstalks with Gαs-dependent cAMP, demands Gαq to release PLCβ3 autoinhibition, but becomes Gq-independent with mutational disruption of the PLCβ3 autoinhibited state. Our findings uncover the key steps of a previously unappreciated mechanism utilized by mammalian cells to finetune their calcium signaling regulation through Gs-GPCRs.




Structure of adenylyl cyclase 5 in complex with Gβγ offers insights into ADCY5-related dyskinesia

April 2024

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78 Reads

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2 Citations

Nature Structural & Molecular Biology

The nine different membrane-anchored adenylyl cyclase isoforms (AC1–9) in mammals are stimulated by the heterotrimeric G protein, Gαs, but their response to Gβγ regulation is isoform specific. In the present study, we report cryo-electron microscope structures of ligand-free AC5 in complex with Gβγ and a dimeric form of AC5 that could be involved in its regulation. Gβγ binds to a coiled-coil domain that links the AC transmembrane region to its catalytic core as well as to a region (C1b) that is known to be a hub for isoform-specific regulation. We confirmed the Gβγ interaction with both purified proteins and cell-based assays. Gain-of-function mutations in AC5 associated with human familial dyskinesia are located at the interface of AC5 with Gβγ and show reduced conditional activation by Gβγ, emphasizing the importance of the observed interaction for motor function in humans. We propose a molecular mechanism wherein Gβγ either prevents dimerization of AC5 or allosterically modulates the coiled-coil domain, and hence the catalytic core. As our mechanistic understanding of how individual AC isoforms are uniquely regulated is limited, studies such as this may provide new avenues for isoform-specific drug development.


Citations (55)


... Although MAPKs' activation via growth factors is well understood [4], less is known of its activation by GPCRs. A recent study demonstrated that G αi2 may influence TCR signaling by sequestering RASA2, thereby promoting RAS activation and stimulating ERK and PI3K to induce proliferation [36]. Another mechanism by which GqPCR induces JNK activation and apoptosis has been researched by our group in the past few years [37][38][39] and is discussed in a review in this Special Issue [3]. ...

Reference:

Special Issue: MAPK Signaling Cascades in Human Health and Diseases
Germline mutations in a G protein identify signaling cross-talk in T cells
  • Citing Article
  • September 2024

Science

... In Darrestin and DGs, the responses were right shifted so much that the dose-response curves were not completed, and the pharmacological parameters could not be determined, suggesting that arrestin and Gs are involved in calcium signaling. GPCR-Gs participate in calcium signaling either via Gq or Gbc directly activating inositol signaling, leading to increased cytosolic calcium [92]. GPCR also facilitates extracellular signal-regulated kinase (ERK) phosphorylation, via several different pathways, which could be dependent on G protein, arrestin, Ras, Rap, PKC, and tyrosine kinases [93]. ...

A molecular mechanism to diversify Ca signaling downstream of Gs protein-coupled receptors

... The cyclases are comprised of two major regions -catalytic (green) and transmembrane (TM, blue) regions that are linked by~40-amino acid extended helices (grey). Both catalytic and TM regions are assembled from two halves that are separated in the primary sequence [6,7]: subdomains C1A and C2A form a pseudo-dimer that constitutes the catalytic domain (green, Fig. 1A) whereas two clusters of 6 helices assemble into a contiguous 12-helix TM domain (blue) [8][9][10]. The first report of an adenylyl cyclase primary sequence in 1989 triggered a wave of studies focusing on possible functions for the TM regions beyond membrane tethering [11]. ...

Structure of adenylyl cyclase 5 in complex with Gβγ offers insights into ADCY5-related dyskinesia

Nature Structural & Molecular Biology

... Key protein targets and ligands in this article are hyperlinked to corresponding entries in the IUPHAR/BPS Guide to PHARMACOLOGY http://www.guidetopharmacology.org and are permanently archived in the Concise Guide to PHARMACOLOGY 2023/24 (Alexander, Fabbro, et al., 2023;. ...

The Concise Guide to PHARMACOLOGY 2023/24: Enzymes

... Recently, two preprints gave additional structural information on the regulation of AC8 ( Figure 1D [37]) and AC5 ( Figure 1E [38]) that correlate well with published literature. Our cryo-EM structure of full-length AC8 in complex with the stimulatory G protein Gαs, CaM, and forskolin at a resolution of 3.5 Å revealed the assembly of the ordered regions of AC8 [37]. ...

Isoform Specific Regulation of Adenylyl Cyclase 5 by Gβγ

... AC9 is a widely distributed AC isoform that is present in a variety of tissues [28]. Single-nucleotide polymorphisms in the ADCY9 gene encoding AC9 have been shown to be associated with asthma and allergy [29], and a polymorphism resulting in a M772I mutation was found to be linked Box 1. Development of new pharmacological agents targeting ACs While there are drugs that indirectly impact AC activity, there is still a lack of pharmacological agents that modulate the function of ACs in an isoform-specific manner [9,65]. The plant-derived AC activator forskolin binds to an allosteric site at the catalytic domain interface. ...

Editorial: Adenylyl cyclase isoforms as potential drug targets

... We previously demonstrated in mice that inhibition of AC1, either pharmacologically or by viral knockdown (AAV9-shRNA), was able to decrease tolerance to morphine [31]. To determine if the reduction in Adcy1 or the upregulation of neuronal KATP channel subunits, Abcc8 or Kcnj11, have further impacts on the development of morphine tolerance or withdrawal, we utilized a viral strategy. ...

Reduced activity of adenylyl cyclase 1 attenuates morphine induced hyperalgesia and inflammatory pain in mice

... The oxadiazole-based AC10065 at micromolar concentrations can suppress both ADCY1 and ADCY8 (Kaur et al., 2019). New drug screening followed by structure optimization has revealed several pyrimidinonebased compounds that show selectivity of ADCY1 over other ADCYs with an IC50 at the sub-micromolar level (Scott et al., 2022). As these newly identified ADCY1 inhibitors show moderate therapeutic efficacy in an inflammatory pain model (Scott et al., 2022), further optimization may be needed. ...

Optimization of a Pyrimidinone Series for Selective Inhibition of Ca 2+ /Calmodulin-Stimulated Adenylyl Cyclase 1 Activity for the Treatment of Chronic Pain
  • Citing Article
  • March 2022

Journal of Medicinal Chemistry

... Taking advantage of drug discovery procedures, the first step in cutting-edge strategic technologies is establishing a dedicated and customized bioassay-based screening platform that allows the rapid identification of relevant and significant bioactivity by conducting multiplexed assays (Berg, 2021). In most discovery research targeting biostimulant and biopesticide activity, screening involves only a single or a few phenotypic parameters (Murgia et al., 2022;Povero, 2020). The potential of bioactivity screens is strongly enhanced, however, by monitoring a broad spectrum of biological traits by combining targeted and untargeted phenotypic bioassays. ...

High-content phenotypic screening identifies novel chemistries that disrupt mosquito activity and development
  • Citing Article
  • January 2022

Pesticide Biochemistry and Physiology

... Many potential applications of therapeutic agents targeting ACs have been proposed, for example AC1 is a putative target for treating chronic pain, and AC5 has been put forward as a target for cardioprotection [68]. However, the suitability of ACs as drug targets is limited unless isoform-selective agents can be developed [68]. ...

Physiological Roles of Mammalian Transmembrane Adenylyl Cyclase Isoforms
  • Citing Article
  • October 2021

Physiological Reviews