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1.
Gioannini Theresa L. Onoprishvili Irma Hiller Jacob M. Simon Eric J. 《Neurochemical research》1999,24(1):37-42
We have investigated the role of cysteine residues in a highly purified opioid receptor protein (ORP) by examining the effect of -SH reagents on the binding of opioid ligands. Treatment of ORP, which is devoid of additional proteins, eliminates complications that arise from reaction of -SH reagents with other components, such as G proteins. Reagents tested include N-ethylmaleimide, 5,5-dithiobis(2-nitrobenzoic) acid, and two derivatives of methanethiosulfonate. Specific opioid binding was inactivated by micromolar concentrations of all -SH reagents tested. Agonist binding ([3H]DAMGO) was much more sensitive to inactivation than antagonist binding ([3H]bremazocine). Prebinding ORP with 100 nM naloxone protected antagonist and agonist binding from inactivation by -SH reagents. The results of these experiments strongly suggest that at least one, and possibly more, reactive cysteine residue(s) is present on the opioid receptor protein molecule, positioned near the ligand binding site and accessible to -SH reagents. 相似文献
2.
Jarkko J. Lackman Piia M. H. Markkanen Mireille Hogue Michel Bouvier Ulla E. Pet?j?-Repo 《The Journal of biological chemistry》2014,289(25):17830-17842
Quality control (QC) in the endoplasmic reticulum (ER) scrutinizes newly synthesized proteins and directs them either to ER export or ER-associated degradation (ERAD). Here, we demonstrate that the human δ-opioid receptor (hδOR) is subjected to ERQC in both N-glycan-dependent and -independent manners. This was shown by investigating the biosynthesis and trafficking of wild-type and non-N-glycosylated F27C variants in metabolic pulse-chase assays coupled with flow cytometry and cell surface biotinylation. Both QC mechanisms distinguished the minute one-amino acid difference between the variants, targeting a large fraction of hδOR-Cys27 to ERAD. However, the N-glycan-independent QC was unable to compensate the N-glycan-dependent pathway, and some incompletely folded non-N-glycosylated hδOR-Cys27 reached the cell surface in conformation incompatible with ligand binding. The turnover of receptors associating with the molecular chaperone calnexin (CNX) was significantly slower for the hδOR-Cys27, pointing to an important role of CNX in the hδOR N-glycan-dependent QC. This was further supported by the fact that inhibiting the co-translational interaction of hδOR-Cys27 precursors with CNX led to their ERAD. Opioid receptor pharmacological chaperones released the CNX-bound receptors to ER export and, furthermore, were able to rescue the Cys27 variant from polyubiquitination and retrotranslocation to the cytosol whether carrying N-glycans or not. Taken together, the hδOR appears to rely primarily on the CNX-mediated N-glycan-dependent QC that has the capacity to assist in folding, whereas the N-glycan-independent mechanism constitutes an alternative, although less accurate, system for directing misfolded/incompletely folded receptors to ERAD, possibly in altered cellular conditions. 相似文献
3.
Karen Wu Chuansheng Chen Robert K. Moyzis Ellen Greenberger Zhaoxia Yu 《Human nature (Hawthorne, N.Y.)》2016,27(3):244-260
We examined an understudied but potentially important source of romantic attraction—genetics—using a speed-dating paradigm. The mu opioid receptor (OPRM1) polymorphism A118G (rs1799971) and the serotonin receptor (HTR2A) polymorphism ?1438 A/G (rs6311) were studied because they have been implicated in social affiliation. Guided by the social role theory of mate selection and prior genetic evidence, we examined these polymorphisms’ gender-specific associations with speed-dating success (i.e., date offers, mate desirability). A total of 262 single Asian Americans went on speed-dates with members of the opposite gender and completed interaction questionnaires about their partners. Consistent with our prediction, significant gender-by-genotype interactions were found for speed-dating success. Specifically, the minor variant of A118G (G-allele), which has been linked to submissiveness/social sensitivity, predicted greater speed-dating success for women, whereas the minor variant of ?1438 A/G (G-allele), which has been linked to leadership/social dominance, predicted greater speed-dating success for men. For both polymorphisms, reverse “dampening” effects of minor variants were found for opposite-gender counterparts. These results support previous research on the importance of the opioid and serotonergic systems in social affiliation, indicating that their influence extends to dating success, with opposite, yet gender-norm consistent, effects for men and women. 相似文献
4.
Chronic morphine causes the mu opioid receptor (MOR) to switch its coupling from Gi/o to Gs, resulting in excitatory signaling via both Gαs and its Gβγ dimer. Ultra-low-dose naloxone (NLX) prevents this switch and attenuates opioid tolerance and dependence. This protective effect is mediated via a high-affinity interaction of NLX to a pentapeptide region in c-terminal filamin A (FLNA), a scaffolding protein interacting with MOR. In organotypic striatal slice cultures, we now show that acute morphine induces a dose-dependent Go-to-Gs coupling switch at 5 and 15 min that resolves by 1 hr. The acute Gs coupling induced by 100 µM morphine was completely prevented by co-treatment with 100 pM NLX, (+)NLX, or naltrexone (NTX), or their pentapeptide binding site (FLNA2561–2565), which we show can act as a decoy for MOR or bind to FLNA itself. All of these co-treatments presumably prevent the MOR–FLNA interaction. Since ultra-low-dose NTX also attenuates the addictive properties of opioids, we assessed striatal cAMP production and CREB phosphorylation at S133. Correlating with the Gs coupling, acute morphine induced elevated cAMP levels and a several-fold increase in pS133CREB that were also completely blocked by NLX, NTX or the FLNA pentapeptide. We propose that acute, robust stimulation of MOR causes an interaction with FLNA that allows an initially transient MOR–Gs coupling, which recovers with receptor recycling but persists when MOR stimulation is repeated or prolonged. The complete prevention of this acute, morphine-induced MOR–Gs coupling by 100 pM NLX/NTX or 10 µM pentapeptide segment of FLNA further elucidates both MOR signaling and the mechanism of action of ultra-low-dose NLX or NTX in attenuating opioid tolerance, dependence and addictive potential. 相似文献
5.
Masaaki Yoshdcawa Takashi Yoshimura Hideo Chiba 《Bioscience, biotechnology, and biochemistry》2013,77(12):3185-3187
A bacterium that assimilates 2,3-dichloro-1-propanol was isolated from soil by enrichment culture. The strain was identified as Pseudomonas sp. by the taxonomic studies. The strain converted 2,3-dichloro-1-propanol to 3-chloro-1,2-propanediol, releasing chloride ion. The conversion was stereospecific because the residual 2,3-dichloro-1-propanol and formed 3-chloro-1,2-propanediol gave optical rotation. The resting cells converted various halohydrins to the dehalogenated alcohols, and cell-free extracts had strong epoxyhydrolase activity. These results indicated that the strain assimilated 2,3-dichloro-1-propanol via 3-chloro-1,2-propanediol, glycidol, and glycerol. The possibility to manufacture optically active 2,3-dichloro-1-propanol is discussed. 相似文献
6.
Matthew P. Rowan Sonya M. Bierbower Michael A. Eskander Kalina Szteyn Elaine D. Por Ruben Gomez Nicholas Veldhuis Nigel W. Bunnett Nathaniel A. Jeske 《PloS one》2014,9(4)
The transient receptor potential family V1 channel (TRPV1) is activated by multiple stimuli, including capsaicin, acid, endovanilloids, and heat (>42C). Post-translational modifications to TRPV1 result in dynamic changes to the sensitivity of receptor activation. We have previously demonstrated that β-arrestin2 actively participates in a scaffolding mechanism to inhibit TRPV1 phosphorylation, thereby reducing TRPV1 sensitivity. In this study, we evaluated the effect of β-arrestin2 sequestration by G-protein coupled receptors (GPCRs) on thermal and chemical activation of TRPV1. Here we report that activation of mu opioid receptor by either morphine or DAMGO results in β-arrestin2 recruitment to mu opioid receptor in sensory neurons, while activation by herkinorin does not. Furthermore, treatment of sensory neurons with morphine or DAMGO stimulates β-arrestin2 dissociation from TRPV1 and increased sensitivity of the receptor. Conversely, herkinorin treatment has no effect on TRPV1 sensitivity. Additional behavioral studies indicate that GPCR-driven β-arrestin2 sequestration plays an important peripheral role in the development of thermal sensitivity. Taken together, the reported data identify a novel cross-talk mechanism between GPCRs and TRPV1 that may contribute to multiple clinical conditions. 相似文献
7.
Yong Chen Chongguang Chen Evangelia Kotsikorou Diane L. Lynch Patricia H. Reggio Lee-Yuan Liu-Chen 《The Journal of biological chemistry》2009,284(3):1673-1685
We demonstrated previously that the protein GEC1 (glandular epithelial cell
1) bound to the human κ opioid receptor (hKOPR) and promoted cell
surface expression of the receptor by facilitating its trafficking along the
secretory pathway. Here we showed that three hKOPR residues
(Phe345, Pro346, and Met350) and seven GEC1
residues (Tyr49, Val51, Leu55,
Thr56, Val57, Phe60, and Ile64)
are indispensable for the interaction. Modeling studies revealed that the
interaction was mediated via direct contacts between the kinked hydrophobic
fragment in hKOPR C-tail and the curved hydrophobic surface in GEC1 around the
S2 β-strand. Intramolecular Leu44-Tyr109
interaction in GEC1 was important, likely by maintaining its structural
integrity. Microtubule binding mediated by the GEC1 N-terminal domain was
essential for the GEC1 effect. Expression of GEC1 also increased cell surface
levels of the GluR1 subunit and the prostaglandin EP3.f receptor, which have
FPXXM and FPXM sequences, respectively. With its widespread
distribution in the nervous system and its predominantly hydrophobic
interactions, GEC1 may have chaperone-like effects for many cell surface
proteins along the biosynthesis pathway.κ opioid receptor
(KOPR)2 is one of the
three major types of opioid receptors mediating effects of opioid drugs and
endogenous opioid peptides. Stimulation of KOPR generates many effects in
vivo, for example antinociception (especially for visceral chemical pain,
antipruritis, and water diuresis
(1). The KOPR agonist
nalfurafine (TRK-820) is used clinically in Sweden for the treatment of uremic
pruritus in kidney dialysis patients
(2). Because KOPR agonists
produce profound sedative effects, it has been proposed that KOPR agonists may
be useful in treating mania, antagonists as anti-depressants, and partial
agonists for the management of mania depression
(3). KOPR antagonists may also
be useful for curbing cocaine craving and as anti-anxiety drugs
(4,
5).KOPR, a member of the rhodopsin subfamily of the seven-transmembrane
receptor superfamily, is coupled preferentially to pertussis toxin-sensitive G
proteins, namely Gi/o proteins
(6). KOPR has been found to
interact with several non-G protein-binding partners, such as
Na+,H+-exchanger regulatory
factor-1/ezrin-radixin-moesin-binding phosphoprotein-50 and the δ opioid
receptor. These interactions have influence on signal transduction and
trafficking of the receptor
(7–9).
By yeast two-hybrid (Y2H) assay using the hKOPR C-tail to screen a human brain
cDNA library, we identified GEC1, also named GABAA
receptor-associated protein like 1 (GABARAPL1), to be a binding partner of
hKOPR (10).GEC1 cDNA was first cloned as an early estrogen-regulated mRNA from guinea
pig endometrial glandular epithelial cells by Pellerin et al.
(11). Subsequently, it was
cloned from other species, including human and house mouse
(12). Interestingly, the amino
acid sequences of GEC1 are completely conserved among all these species except
orangutan, in which Arg99 substitutes for His99.
Northern blot and immunoblotting analyses revealed that it has widespread
tissue distribution
(12–14).
In particular, GEC1 was found to be abundant in the central nervous system and
expressed throughout the rat brain
(14,
15). This wide tissue
distribution and the high sequence identity across species strongly suggest
that GEC1 has important biological functions in mammalian cells.Based on sequence similarity, GEC1 is classified as a member of
microtubule-associated proteins (MAPs), which also include GABAA
receptor-associated protein (GABARAP), Golgi-associated ATPase enhancer of 16
kDa (GATE16), GABARAP-like 3 (GABARAPL3), light chain 3 (LC3) of MAP 1A/1B,
and the yeast autophagy protein 8 (Atg8)
(12,
13). Among these homologues,
GEC1 share the highest identity with GABARAPL3 (93%), followed by GABARAP
(86%), GATE16 (61%), Atg8 (55%), and LC3 (∼30%).A growing body of evidence shows that this protein family is closely
related to two distinct biological functions. Studies mainly on GABARAP,
GATE16, and GEC1 indicate that they promote intracellular protein trafficking
by enhancing vesicle fusion
(10,
16–21).
In addition, they facilitate degradation of proteins and intracellular
organelles via autophagy-related pathways, which is bolstered largely by
research on Atg8 and LC3 (22,
23).We previously reported that GEC1 interacted with the hKOPR C-tail and
enhanced cell surface levels of hKOPR stably expressed in CHO cells. GEC1
expression enhances hKOPR expression through facilitating its anterograde
trafficking along the protein biosynthesis pathway without affecting
degradation of the receptor
(10). This represented the
first biological function reported for GEC1. Mansuy et al.
(24) demonstrated that GEC1
interacted with tubulin and promoted microtubule bundling in vitro,
and that green fluorescence protein-tagged GEC1 was localized in the
perinuclear vesicles with a scattered pattern. Our electron microscopic
studies in the rat brain showed that GEC1 was associated with ER, Golgi
apparatus, endosome-like vesicles, and plasma membranes and scattered in
cytoplasm in neurons (14). In
addition, N-ethylmaleimide-sensitive factor, a protein critical for
intracellular membrane-trafficking events, binds directly to GEC1
(10).In this study, we employed Y2H techniques to determine the amino acid
residues in both GEC1 and hKOPR C-tail involved in the interaction. Further
studies were then carried out in mammalian cells to examine if elimination of
the interaction affected the effect of GEC1 on hKOPR expression. In addition,
we generated a molecular model of GEC1 based on the x-ray crystal structure of
GABARAP and found that the residues involved in hKOPR binding formed
hydrophobic patches on the exterior surface of GEC1. Moreover, we found that
the cytosolic tail of AMPA receptor subunit GluR1 has the same FPXXM
motif as that found in the hKOPR C-tail to be involved in GEC1 binding and
that GEC1 expression up-regulated GluR1. 相似文献
8.
9.
Harold H Bach IV Yee M Wong Abhishek Tripathi Amanda M Nevins Richard L Gamelli Brian F Volkman Kenneth L Byron Matthias Majetschak 《Molecular medicine (Cambridge, Mass.)》2014,20(1):435-447
Chemokine (C-X-C motif) receptor (CXCR) 4 and atypical chemokine receptor (ACKR) 3 ligands have been reported to modulate cardiovascular function in various disease models. The underlying mechanisms, however, remain unknown. Thus, it was the aim of the present study to determine how pharmacological modulation of CXCR4 and ACKR3 regulate cardiovascular function. In vivo administration of TC14012, a CXCR4 antagonist and ACKR3 agonist, caused cardiovascular collapse in normal animals. During the cardiovascular stress response to hemorrhagic shock, ubiquitin, a CXCR4 agonist, stabilized blood pressure, whereas coactivation of CXCR4 and ACKR3 with CXC chemokine ligand 12 (CXCL12), or blockade of CXCR4 with AMD3100 showed opposite effects. While CXCR4 and ACKR3 ligands did not affect myocardial function, they selectively altered vascular reactivity upon α1-adrenergic receptor (AR) activation in pressure myography experiments. CXCR4 activation with ubiquitin enhanced α1-AR-mediated vasoconstriction, whereas ACKR3 activation with various natural and synthetic ligands antagonized α1-AR-mediated vasoconstriction. The opposing effects of CXCR4 and ACKR3 activation by CXCL12 could be dissected pharmacologically. CXCR4 and ACKR3 ligands did not affect vasoconstriction upon activation of voltage-operated Ca2+ channels or endothelin receptors. Effects of CXCR4 and ACKR3 agonists on vascular α1-AR responsiveness were independent of the endothelium. These findings suggest that CXCR4 and ACKR3 modulate α1-AR reactivity in vascular smooth muscle and regulate hemodynamics in normal and pathological conditions. Our observations point toward CXCR4 and ACKR3 as new pharmacological targets to control vasoreactivity and blood pressure. 相似文献
10.
Jin Xu Zhigang Lu Mingming Xu Grace C. Rossi Benjamin Kest Amanda R. Waxman Gavril W. Pasternak Ying-Xian Pan 《PloS one》2014,9(10)
The µ opioid receptor gene, OPRM1, undergoes extensive alternative pre-mRNA splicing in rodents and humans, with dozens of alternatively spliced variants of the OPRM1 gene. The present studies establish a SYBR green quantitative PCR (qPCR) assay to more accurately quantify mouse OPRM1 splice variant mRNAs. Using these qPCR assays, we examined the expression of OPRM1 splice variant mRNAs in selected brain regions of four inbred mouse strains displaying differences in µ opioid-induced tolerance and physical dependence: C56BL/6J, 129P3/J, SJL/J and SWR/J. The complete mRNA expression profiles of the OPRM1 splice variants reveal marked differences of the variant mRNA expression among the brain regions in each mouse strain, suggesting region-specific alternative splicing of the OPRM1 gene. The expression of many variants was also strain-specific, implying a genetic influence on OPRM1 alternative splicing. The expression levels of a number of the variant mRNAs in certain brain regions appear to correlate with strain sensitivities to morphine analgesia, tolerance and physical dependence in four mouse strains. 相似文献
11.
12.
Docking methodology aims to predict the experimental binding modes and affinities of small molecules within the binding site of particular receptor targets and is currently used as a standard computational tool in drug design for lead compound optimisation and in virtual screening studies to find novel biologically active molecules. The basic tools of a docking methodology include a search algorithm and an energy scoring function for generating and evaluating ligand poses. In this review, we present the search algorithms and scoring functions most commonly used in current molecular docking methods that focus on protein–ligand applications. We summarise the main topics and recent computational and methodological advances in protein–ligand docking. Protein flexibility, multiple ligand binding modes and the free-energy landscape profile for binding affinity prediction are important and interconnected challenges to be overcome by further methodological developments in the docking field. 相似文献
13.
14.
Unzicker C Erberich H Moldrich G Woldt H Bulla J Mechoulam R Ehrenreich H Sirén AL 《Neurochemical research》2005,30(10):1305-1309
Endothelin (ETB)-receptors mediate anti-apoptotic actions. Lack of functional ETB-receptors leads to increased neuronal apoptosis
in the hippocampus. The increased apoptosis must be compensated by other mechanisms, however, as ETB-deficient rats display
normal overall brain morphology. To illuminate on brain plasticity in ETB-receptor deficiency, we studied the expression and
function of another neuroprotective system, the cannabinoid CB1-receptors, in ETB-deficient hippocampus. We show that CB1
expression in hippocampus increases postnatally in all rats but that the increase in CB1-receptor expression is significantly
higher in ETB-deficient compared to wildtype littermates. Neuronal apoptosis decreases during brain maturation but remains
on a significantly higher level in the ETB-deficient compared to wildtype dentate. When investigating survival of hippocampal
neurons in culture, we found significant protection against hypoxia-induced cell death with CB1-analogs (noladin, (9-tetrahydrocannabinol)
only in ETB-deficient neurons. We suggest that CB1-receptor upregulation in the ETB-mutant hippocampus reflects an attempt
to compensate for the lack of ETB-receptors.
Special issue dedicated to Dr. Bernd Hamprecht 相似文献
15.
Characteristics of Non-opioid β-Endorphin Receptor 总被引:4,自引:0,他引:4
Navolotskaya EV Kovalitskaya YA Zolotarev YA Kolobov AA Kampe-Nemm EA Malkova NV Yurovsky VV Lipkin VM 《Biochemistry. Biokhimii?a》2004,69(4):394-400
Tritium-labeled selective agonist of non-opioid beta-endorphin receptor, the decapeptide immunorphine ([3H]SLTCLVKGFY) with specific activity of 24 Ci/mmol has been prepared. By its use, non-opioid beta-endorphin receptors were revealed and characterized on mouse peritoneal macrophages and rat myocardium, spleen, adrenal, and brain membranes. The non-opioid beta-endorphin receptor of macrophages has in addition to immunorphine (Kd of the [3H]immunorphine-receptor complex was 2.4 +/- 0.1 nM) and beta-endorphin (Ki of the [3H]immunorphine specific binding was 2.9 +/- 0.2 nM) a high affinity for Fc-fragment of human IgG1, pentarphine (VKGFY), cyclopentarphine [cyclo(VKGFY)], and [Pro3]pentarphine (VKPFY) (Ki values were 0.0060 +/- 0.0004, 2.7 +/- 0.2, 2.6 +/- 0.2, and 2.8 +/- 0.2 nM, respectively) and is insensitive to naloxone and [Met5]enkephalin (Ki > 100 microM). Treatment of macrophages with trypsin resulted in the loss of their ability for the specific binding of [3H]immunorphine. Values of the specific binding of 8.4 nM [3H]immunorphine to rat adrenal, spleen, myocardium, and brain membranes were determined to be 1146.0 +/- 44.7, 698.6 +/- 28.1, 279.1 +/- 15.4, and 172.2 +/- 1.8 fmol/mg protein, respectively. Unlabeled beta-endorphin, pentarphine, [Pro3]pentarphine, cyclopentarphine, cyclodipentarphine [cyclo(VKGFYVKGFY)], and Fc-fragment of IgG1 inhibited the binding of [3H]immunorphine to membranes from these organs. No specific binding of [3H]immunorphine to rat liver, lung, kidney, and intestine membranes was found. 相似文献
16.
17.
18.
19.
Lee YS Agnes RS Cain JP Kulkarni V Cai M Salibay C Ciano K Petrov R Mayorov A Vagner J Trivedi D Davis P Ma SW Lai J Porreca F Vardanyan R Hruby VJ 《Biopolymers》2008,90(3):433-438
We have identified compound 1 as a novel ligand for opioid and melanocortin (MC) receptors, which is derived from the overlapping of a well known structure for the delta opioid receptor, 2,6-dimethyltyrosine (Dmt)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), and a small molecule for the MC receptor, Tic-DPhe(p-Cl)-piperidin-4-yl-N-phenyl-propionamide. Ligand 1 showed that there is an overlapping pharmacophore between opioid and MC receptors through the Tic residue. The ligand displayed high biological activities at the delta opioid receptor (Ki = 0.38 nM in binding assay, EC(50) = 0.48 nM in GTP-gamma-S binding assay, IC(50) = 74 nM in MVD) as an agonist instead of an antagonist and showed selective binding affinity (IC(50) = 2.3 muM) at the MC-3 receptor rather than at the MC-5 receptor. A study of the structure-activity relationships demonstrated that the residues in positions 2, 3, and the C-terminus act as a pharmacophore for the MC receptors, and the residues in positions 1 and 2 act as a pharmacophore for the opioid receptors. Thus, this structural construct can be used to prepare chimeric structures with adjacent or overlapping pharmacophores for opioid and MC receptors. 相似文献
20.
Glucose metabolism is under the cooperative regulation of both insulin receptor (IR) and β2-adrenergic receptor (β2AR), which represent the receptor tyrosine kinases (RTKs) and seven transmembrane receptors (7TMRs), respectively. Studies demonstrating cross-talk between these two receptors and their endogenous coexpression have suggested their possible interactions. To evaluate the effect of IR and prospective heteromerization on β2AR properties, we showed that IR coexpression had no effect on the ligand binding properties of β2AR; however, IR reduced β2AR surface expression and accelerated its internalization. Additionally, both receptors displayed a similar distribution pattern with a high degree of colocalization. To test the possible direct interaction between β2AR and IR, we employed quantitative BRET2 saturation and competition assays. Saturation assay data suggested constitutive β2AR and IR homo- and heteromerization. Calculated acceptor/donor (AD50) values as a measure of the relative affinity for homo- and heteromer formation differed among the heteromers that could not be explained by a simple dimer model. In heterologous competition assays, a transient increase in the BRET2 signal with a subsequent hyperbolical decrease was observed, suggesting higher-order heteromer formation. To complement the BRET2 data, we employed the informational spectrum method (ISM), a virtual spectroscopy method to investigate protein-protein interactions. Computational peptide scanning of β2AR and IR identified intracellular domains encompassing residues at the end of the 7th TM domain and C-terminal tail of β2AR and a cytoplasmic part of the IR β chain as prospective interaction domains. ISM further suggested a high probability of heteromer formation and homodimers as basic units engaged in heteromerization. In summary, our data suggest direct interaction and higher-order β2AR:IR oligomer formation, likely comprising heteromers of homodimers. 相似文献