bluuebunny/crossref_metadata_2025_split huggingface dataset:
Heat Shock Protein 90α, Assisted by Co‐Chaperones p23 and Cdc37, Promotes Opioid Anti‐Nociception in the Brain via Promoting ERK MAPK Signaling
Authors: Lei Wei , Duron David I. , Stine Carrie A. , Blagg Brian , Streicher John M. 
Published: June 2021

Reducing opioid side effects like constipation and addiction has been a strong focus for improving opioid therapy in treating acute and chronic pain. One such approach includes manipulation of mu opioid receptor (MOR) signaling cascades, such as βarrestin2. However, the MOR signaling complex and mechanisms of signaling regulation are poorly understood. In our earlier work, we identified Heat shock protein 90 (Hsp90) as a novel MOR signaling regulator, which could be a target for improving the therapeutic profile of opioids. We found that intracerebroventricular (icv) 17‐AAG, a well‐established Hsp90 inhibitor, strongly blocked or abolished morphine anti‐nociception in paw incision and HIV neuropathic pain in mice. The current study was designed to identify molecular mechanisms of this regulation, including which isoform(s) and/or co‐chaperone(s) are involved in the regulatory effect of Hsp90 on opioid signaling. Male and female CD‐1 mice were treated with selective inhibitors for different Hsp90 isoforms (KU‐A, KUNB106, KUNG65) and co‐chaperones (KU177, gedunin, celastrol) via icv injection. We found that an Hsp90α selective inhibitor blocked morphine anti‐nociception by ~ 80% in the paw incision model, however, inhibitors for Hsp90β and Grp94 had no impact on morphine anti‐nociception. Knockdown of Hsp90α in the brain by CRISPR‐Cas9 also showed strong inhibition of morphine anti‐nociception in paw incision, confirming the results. Further, inhibition of the co‐chaperones p23 and Cdc37, but not Aha1, showed similar effects as Hsp90α inhibition in the paw incision model. To determine the molecular mechanism of these activities, we found that Hsp90α inhibitor treatment blocked ERK and JNK MAPK activation induced by the selective MOR agonist DAMGO without changing the expression of Hsp70 in mouse periaqueductal grey (PAG). Treatment with the ERK MAPK inhibitor U0126 recapitulated our results of Hsp90 inhibition on paw incision, HIV neuropathic, and tail flick pain, suggesting that ERK MAPK regulation is the means by which Hsp90α regulates morphine anti‐nociception. Identification of the isoforms and co‐chaperones involved in Hsp90 regulation of ERK and opioid anti‐nociception may permit more selective manipulation of the MOR signaling machinery to improve opioid efficacy and reduce side effects.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Glial Glutamate Transporter Activator, LDN‐212320, Attenuates Formalin‐Induced Pain Behavior in Mice
Authors: Alotaibi Ghallab , Rahman Shafiqur 
Published: June 2021

Previous studies have shown that glial glutamate transporter‐1 (GLT‐1) in the hippocampus and anterior cingulate cortex (ACC) are critically involved in pain processing and modulation in the central nervous system. The objective of the present study was to investigate the role of GLT‐1 activator, 3‐[[(2‐Methylphenyl) methyl]thio]‐6‐(2‐pyridinyl)‐pyridazine (LDN‐212320), in formalin‐induced pain behavior in mice. Animal received 10 μl injection of formalin (2.5%) into the planter surface of the hind paw and the duration of licking/biting responses was measured. In addition, GLT‐1 expression and extracellular signal‐regulated kinase phosphorylation (pERK) were measured in the hippocampus and ACC using Western blot analysis. The LDN‐212320 (10 or 20 mg/kg, i.p) significantly attenuated formalin‐induced nociceptive behavior. These antinociceptive effects of LDN‐212320 were reversed by systemic administration of dihydrokianic acid (10 mg/kg, i.p), a GLT‐1 antagonist. Moreover, the LDN‐212320 (10 or 20 mg/kg, i.p) increased GLT‐1 expressions in the hippocampus and ACC. Formalin induced‐ERK phosphorylation, a molecular marker of nociception, was significantly reduced by LDN‐212320 (20 mg/kg, i.p) in the hippocampus and ACC. Overall, these results suggest that LDN‐212320 plays a critical role as antinociceptive agent by upregulating GLT‐1 expression in the hippocampus and ACC. Therefore, LDN‐212320 could be a novel therapeutic drug candidate for nociceptive pain.Support or Funding InformationSupported in part by grant from Saudi Arabian Cultural Mission, USAThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Analysis of Antinociception Produced by Positive Allosteric Modulators of the Mu‐Opioid Receptor
Authors: Kandasamy Ram , Traynor John R. 
Published: June 2021

Traditional opioids (e.g., morphine) provide analgesia by acting at the orthosteric site on the mu‐opioid receptor (MOR); however, G protein‐coupled receptor function can also be controlled by compounds acting at a separate (allosteric) site on the receptor. We have identified two compounds, BMS‐986122 and BMS‐986187, that bind to the allosteric site on the MOR and enhance the affinity and potency of MOR agonists in vitro. Thus, we hypothesized that BMS‐986122 and BMS‐986187 will produce antinociception by enhancing endogenous and exogenous opioid activity in vivo. Male and female mice were administered BMS‐986122, BMS‐986187, or vehicle via intraperitoneal injections (10 mg/kg; i.p.). Antinociception was assessed using the hot plate and warm water tail withdrawal tests. D,L‐methadone (10 mg/kg) produced antinociception on the tail withdrawal and hot plate tests. Co‐administration of BMS‐986187 did not enhance D,L‐methadone (3.2 mg/kg) antinociception on either test. However, a 30 min pretreatment of BMS‐986187 (10 mg/kg, i.p.) enhanced antinociception produced by an active dose of D,L‐methadone (10 mg/kg), but not an inactive dose (3.2 mg/kg), on the hot plate test. Intraperitoneal injection of both BMS‐986122 and BMS‐986187 alone produced near‐maximal antinociception on the tail withdrawal test for 2 hours that is likely caused by enhancement of endogenous opioid activity. Our data suggest complex interactions underlying antinociception produced by allosteric modulators of MOR that depend on several factors including dose, timing, and orthosteric agonist (methadone vs. endogenous opioid). Further, these data suggest that PAM‐mediated opioid antinociception affect pain processing at spinal and supraspinal levels.Support or Funding InformationRK was supported by T32 DA007268. Funding provided by R37 DA039997 (to JRT).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ligand‐specific patterns of PTH1R and arrestin3 internalization and trafficking define a novel form of ligand “bias”
Authors: Lee Mi‐Hye , Strungs Erik G. , Luttrell Louis M. 
Published: June 2021

The type 1 parathyroid hormone receptor (PTH1R) couples to Gs, Gq/11, and Gi/o family heterotrimeric G proteins and binds arrestins, which mediate receptor desensitization and arrestin‐dependent signaling. It is known that ligand structure can “bias” PTH1R signaling by changing the efficiency with which it couples to these downstream effectors. Here, we characterize the effect of ligand structure on the signaling, internalization, and intracellular trafficking of human PTH1R in a common HEK293 cell background using a panel of four structurally distinct ligands. Two ligands, hPTH(1‐34) and hPTH(1‐31), behave as conventional PTH1R agonists, activating both Gs‐cAMP and Gq/11‐Ca2+ signaling; while [Trp1]‐PTHrP(1‐36) is a Gs‐cAMP selective agonist; and [D‐Trp12 Tyr34]‐bPTH(7‐34) is a Gs‐cAMP selective inverse agonist. All four promote arrestin‐dependent ERK1/2 activation, with [Trp1]‐PTHrP(1‐36) and [D‐Trp12 Tyr34]‐bPTH(7‐34) exhibiting the greatest degree of arrestin‐dependence. To investigate the effect of ligand structure on PTH1R internalization and trafficking, we employed a “bystander BRET” approach in which the BRET pair consisted of a Renilla luciferase‐tagged Ras‐like small GTPase (Rab) marking a specific intracellular membrane compartment (Rab 1, Rab2, Rab5, Rab7, Rab9 or Rab11) and PTH1R‐YFP or arrestin3‐YFP. Three ligands, hPTH(1‐34), hPTH(1‐31), and [Trp1]‐PTHrP(1‐36) promoted PTH1R‐arrestin3 binding and clathrin‐dependent internalization, while [D‐Trp12 Tyr34]‐bPTH(7‐34), which fails to stabilize a PTH1R‐arrestin3 complex, did not promote internalization. In response to hPTH(1‐34) or hPTH(1‐31), PTH1R transited sequentially through early (Rab5) and late (Rab7/9) endosomes, before distributing between the slow recycling (Rab1/2) trans‐Golgi/ER network and rapid recycling (Rab11) endosome pool. Arrestin3 trafficking paralleled the receptor, consistent with the known role of arrestins in promoting slow PTH1R recycling and degradation. In contrast, PTH1R stimulated with [Trp1]‐PTHrP(1‐36) did not accumulate in Rab5 or Rab7/9 positive endosomes, instead transiting rapidly into the Rab11 and Rab2 positive pools. Notably, arrestin3 did not accompany the PTH1R, indicating that rapid dissociation of the PTH1R‐arrestin complex changed the pattern of PTH1R sorting in favor of receptor recycling. These results suggest that ligand structure, in particular the rate of ligand‐receptor‐arrestin complex dissociation, can “bias” not only the effector coupling profile of a G protein‐coupled receptor, but also it subsequent intracellular trafficking and rate of degradation/resensitization.Support or Funding InformationNIH grants R01 DK055524 (LML), GM095497 (LML); the MUSC FLIPRTETRA Core S10 RR027777 (LML); the Research Service of the Ralph H. Johnson VA Medical Center, Charleston, SC.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Homologous Regulation of Mu Opioid Receptor Recycling by G Beta‐gamma
Authors: Kunselman Jennifer , Zajac Amanda , Weinberg Zara , Puthenveedu Manojkumar 
Published: June 2021

The mu opioid receptor (mOR) is the primary target for opioid analgesics that are widely used and abused. Opioid receptors are removed from the cell surface after activation, causing down regulation of responses. Receptor recycling to the surface, which allows recovery of cellular responses, determines acute tolerance to opioids, and might have long‐term effects on signaling. Whether and how mOR recycling is acutely regulated by homologous signals is not clear. Here we used total internal reflection fluorescence microscopy (TIR‐FM) to directly visualize and quantify individual receptor recycling events, to test whether mOR recycling is regulated by opioid signaling downstream of the receptor itself. Attenuation of continued signaling, by removing the agonist, reduced the frequency of recycling events, indicating that opioid signaling increases the rate of mOR recycling. This homologous regulation was reduced in cells treated with the G protein inhibitor pertussis toxin, but not a Protein Kinase A inhibitor. Further, inhibition of G beta‐gamma (Gbg), Phospholipase C, or Protein Kinase C (PKC) abolished the decrease in recycling caused by agonist removal, while activation of Gbg or PKC caused an increase in recycling even in the absence of agonist. This suggests that mOR recycling is regulated by PKC activation through Gbg. Importantly, the homologous regulation was abolished by mutating the serine 363 residue on mOR, a PKC phosphorylation site. Our results support a model where Gbg activation of PLC/PKC and phosphorylation of mOR site S363 is required for the homeostatic regulation of mOR recycling. This represents a novel mechanism for homologous regulation of receptor recycling, which could provide control points for regulation by different opioid drugs.Support or Funding InformationSupported by the NIH Cellular and Molecular Biology Training Grant T32‐GM007315This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Novel paradigms governing β1‐adrenergic receptor trafficking in primary adult rat cardiac myocytes
Authors: Nooh Mohammed Mostafa , Mancarella Salvatore M. , Bahouth Suleiman W. 
Published: June 2021

Background and PurposeThe β1‐adrenergic receptor (β1‐AR) is a major cardiac G protein‐coupled receptor (GPCR), which mediates cardiac actions of catecholamines and is involved in genesis and treatment of numerous cardiovascular disorders. Catecholamines induce the internalization of the β1‐AR into endosomes and their removal promotes the recycling of endosomal β1‐AR back to the plasma membrane (PM).Experimental ApproachTrafficking of the β1‐AR and its regulation was determined by confocal microscopy in primary adult rat ventricular myocytes (ARVM), which are terminally differentiated myocytes with unique structures such as t‐tubules and contractile sarcomeres.Key ResultsIn unstimulated ARVM, the fluorescently labeled wild type (WT) β1‐AR was expressed on the external membranes (the sarcolemma) of cardiomyocytes. Exposing ARVM to isoprenaline promoted the translocation of surface β1‐AR into small (~225–250 nm) regularly spaced internal punctate structures that overlapped with puncta stained by Di‐8‐ANEPPS, a t‐tubule‐specific dye. Replacing the β‐agonist with a β‐blocker such as alprenolol, induced the recycling of the WT β1‐AR from t‐tubules back to the PM. This step was dependent on two previously identified β1‐AR barcodes, namely the type‐1 PDZ binding motif (PBM) and Ser312 of the β1‐AR, which is phosphorylated by the PBM anchored PKA‐AKAP5‐SAP97 complex.Conclusions and ImplicationsWe present data to show that translocation of the β1‐AR from t‐tubules back to the PM in ARVM was mediated by a novel sorting mechanism. These findings might explain unique aspects of cardiac β1‐AR signaling under normal or pathological conditions.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural Insights into Phospholipase Cɛ Activity
Authors: Garland‐Kuntz Elisabeth Erin , Vago Frank S. , Van Camp Michelle M. , Sieng Monita , Blaine Arryn T. , Jiang Wen , Lyon Angeline M. 
Published: June 2021

Phospholipase C (PLC) enzymes hydrolyze phosphatidylinositol lipids to produce second messengers, including inositol‐1,4,5‐triphosphate (IP3) and diacylgycerol (DAG), which increase intracellular calcium and activate protein kinase C (PKC), respectively. PLCɛ contributes to cardiac hypertrophy and contractility, as well as to oncogenic and inflammatory signaling pathways downstream of receptor tyrosine kinases and G protein‐coupled receptors. PLCɛ shares a conserved core with other PLC enzymes, but the roles of individual domains in activity and membrane binding have not been established. Using biochemical assays, small‐angle X‐ray scattering (SAXS), and electron microscopy (EM), we have found that the PLCɛ PH domain is necessary for basal lipase activity, but is dispensable for stability. Importantly, we provide the first structural insights into domain organization of PLCɛ, and reveal that the PH domain is conformationally heterogeneous in solution. Comparisons of the PLCɛ solution structure to that of the closely related PLCβ enzyme reveal that, in contrast to its crystal structure, the PLCβ PH domain is also mobile in solution. We also show the dynamic nature of the PH domain in these enzymes is functionally important, and may contribute to their regulation. These findings provide critical new insights into the solution structures of these signaling enzymes and their roles in cardiovascular disease and cancer.Support or Funding InformationAmerican Heart Association Scientist Development Grant 16SDG29920017; Purdue Center for Cancer Research; Showalter Foundation; all to A.M.LThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cellular Compensation for Chronic Decreases in Cyclic AMP Concentration: Gαs‐Deficient Dendritic Cells as a Model and Implications for Allergic Disorder Therapeutics
Authors: Chinn Amy M. , Lee Jihyung , Raz Eyal , Insel Paul A. 
Published: June 2021

Dendritic cells (DCs), the main antigen‐presenting cells, induce type II helper T cell (Th2) immune responses, but the mechanism by which this occurs is not fully elucidated. Mice that have a CD11c (DC)‐specific deletion of the Gnas gene (CD11cΔGnas), which encodes the α subunit of the heterotrimeric (αβγ) G protein Gαs, display very low basal cyclic AMP (cAMP) concentrations and an allergic phenotype; the decrease in cAMP and protein kinase A (PKA) activation biases DCs to provoke a Th2 response ex vivo and in vivo (Lee et al., PNAS 112: 1529–34 [2015]). In order to assess how cells compensate for a chronic decrease in cAMP/PKA signaling, we compared the mRNA expression of cAMP pathway components in WT and CD11cΔGnas bone marrow‐derived DCs (BMDCs). Using quantitative real‐time PCR (qPCR), we found that BMDCs from CD11cΔGnas mice have decreased expression of numerous cyclic nucleotide phosphodiesterases (PDEs) that degrade cAMP including PDE4B and 4D, but no overall increase in Gαi proteins or the cAMP transporter MRP4/ABCC4. Treatment of the mouse dendritic cell line DC2.4 with a cell‐permeable PKA‐specific cAMP analog and the Gαs agonist prostaglandin E2 increased PDE4B and 4D expression >2‐fold, indicating the dynamic changes in PDE4B and 4D expression are in response to cellular cAMP concentration. We also assessed the expression of G protein‐coupled receptors (GPCRs; assayed by qPCR‐based Taqman GPCR arrays), including those that regulate cAMP formation. We found a greater number and larger increase in expression for GPCRs that couple to Gαi (rather than other Gα proteins) in CD11cΔGnas BMDCs compared to BMDCs from WT mice. Additionally, expression of multiple PKA subunits (RIIα, RIIβ) decreased >50% in CD11cΔGnas BMDCs. A‐kinase anchor protein (AKAP)6 and AKAP3 have an increase and decrease, respectively, in those BMDCs. Together, these data indicate that DCs with lower cAMP levels have altered expression of both “upstream” cAMP signaling components (GPCRs) and “downstream” components that regulate cAMP levels (PDE isoforms) and actions (PKA subunits, AKAPs). Such changes in expression of GPCRs and post‐G protein components likely reflect withdrawal of “basal”, tonic effects of cAMP, may contribute to the greater Th2 responses and allergic asthma of CD11cΔGnas mice, and may be novel therapeutic targets for allergic asthma.Support or Funding InformationSupported by NIH grants R56AI110505 and T32GM007752.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular Mechanism of Rap1A‐Dependent Activation of PLCɛ
Authors: Sieng Monita , Garland‐Kuntz Elisabeth E. , Lyon Angeline M. 
Published: June 2021

Phospholipase C (PLC) enzymes hydrolyze phosphatidylinositol lipids to produce diacylglycerol (DAG) and inositol phosphates, to activate protein kinase C (PKC) and downstream signaling pathways, including cell growth and survival. The PLCɛ subfamily has emerged as a key player in cardiovascular health, where it is required for maximum contractility. However, prolonged activation of PLCɛ results in cardiac hypertrophy and heart failure through its ability to regulate hypertrophic gene expression. This process is regulated by the small GTPase Rap1A, which is activated downstream of β‐adrenergic receptors. Rap1A binds to the C‐terminal Ras association (RA) domain of PLCɛ, simultaneously translocating the complex to the perinuclear region and activating PLCɛ. However, the molecular mechanism of this process is not known. We seek to characterize the interactions between Rap1A and PLCɛ using structural and functional studies to map the Rap1A binding site on PLCɛ and determine whether activation results in conformational changes that release autoinhibition and/or increase membrane association. These studies provide the first molecular details of the Rap1A‐dependent activation of PLCɛ and opens the door to the development of new therapeutic strategies for treating cardiac hypertrophy.Support or Funding InformationAmerican Heart Association Scientist Development Grant (16SDG29920017) to A.M.L.; Purdue Center for Cancer ResearchThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanistic Study of a Novel Thienyl Napthalenesulfonate Molecule that Selectively Inhibits Melanoma Cells with Constitutively‐Active ERK1/2
Authors: Martinez Ramon , Huang Weiliang , Centola Garrick , Samadani Ramin , Chen Lijia , Scheenstra Jacob , Fletcher Steven , Kane Maureen , Mackerell Alexander , Shapiro Paul 
Published: June 2021

The uncontrolled proliferation of cancer cells is often a result of constitutive activation of the extracellular signal‐regulated kinase (ERK 1/2) signaling pathway. Current trends in the design of kinase inhibitors for the ERK1/2 pathway employ the use of ATP‐competitive scaffolds, targeting the catalytic core of the proteins. As an alternative, we used computer‐aided drug design (CADD) to identify a novel thienyl napthalenesulfonate compound that selectively inhibits ERK2 at a binding site used by F‐site containing substrates, such as members of the Fos family of proteins. This compound has been shown to selectively inhibit the growth of melanoma cells that have constitutively active ERK1/2 via mutations in upstream activators BRaf or NRas. Proteomic and transcriptomic analysis of drug‐treated lysates showed inhibition in the expression of important downstream F‐site containing substrates of ERK 1/2, such as c‐Fos, Fra‐1, and c‐Myc. In addition, increased expression of the protein Nrf2, which is a regulator of pathways generating reactive oxygen species (ROS), suggests that this compound may be involved with impeding cell growth in melanoma cells via upregulation of oxidative stress pathways. When treating cells with known inhibitors of ROS, such as α‐tocopherol or N‐acetyl cysteine, partial to complete cellular proliferation is restored, further supporting implication of oxidative stress in the compound function. In this work, we attempt to further characterize and validate the regulatory mechanisms and apoptotic effect this novel compound is having on cancer cells, and how it can serve as a promising alternate line of therapy to traditional ATP‐competitive kinase inhibitors.Support or Funding InformationThis work was supported in part by an NIGMS Initiative for Maximizing Student Development Grant (2‐R25‐GM55036) and by an NIGMS Institutional Training Grant (T32 GM066706).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.