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At least 793 records · Page 44Linked to original sources
PubMed2001

Agonist-induced internalization and mitogen-activated protein kinase activation of the human prostaglandin EP4 receptor.

We examined the pathway of prostaglandin E(2) (PGE(2))-induced internalization of the prostaglandin EP4 receptor in HEK 293 cells. Co-expression of dominant negative beta-arrestin (319-418) or dynamin I (K44A) with the EP4 receptor reduced internalization. The activated receptor co-localized with GFP-arrestin 2 and GFP-arrestin 3, confirming the requirement for beta-arrestins in internalization. Inhibition of clathrin-coated vesicle-mediated internalization resulted in inhibition of sequestration, whereas inhibition of caveola-mediated internalization had no effect. PGE(2) stimulation of the EP4 receptor resulted in rapid mitogen-activated protein (MAP) kinase activation. Examination of an internalization-resistant mutant and co-expression of mutant accessory proteins with EP4 revealed that MAP kinase activation proceeds independently of internalization.

Acetic Acid↗
PubMed1999

Modulation of gene expression following long-term synaptic depression in the striatum.

A number of behavioural and cellular studies have suggested that activity-dependent synaptic plasticity associated with learning and memory may lead to the expression of various genes whose protein products can play a critical role in memory acquisition and consolidation. Long-term potentiation (LTP) and long-term depression (LTD) represent two forms of synaptic plasticity which have been widely studied by electrophysiological techniques. However, the molecular mechanisms at target gene involved in the generation of long term depression remain to be determined. To elucidate the molecular mechanism underlying activity dependent synaptic remodeling in striatal long term depression, we used the mRNA differential display technology to isolate genes that are induced or modulated by high frequency stimulation of the corticostriatal pathway in a rat brain slice preparation. We have differentially displayed, by means of reverse transcriptase-polymerase chain reaction, mRNA species isolated from striatal slices in which long term depression was induced by tetanic stimuli as well as from slices stimulated at low frequency. We then compared radio-labeled RT-PCR banding patterns to isolate cDNAs that are differentially expressed. Three independent cDNAs were isolated and identified whose mRNA level were enhanced by tetanic stimulation inducing long term depression. We provide evidence that two of these genes encode proteins involved in synaptic vesicle trafficking (dynamin I and amphiphysin II). Moreover, expression of tissue plasminogen activator (t-PA) gene was also increased following striatal long term depression. Our data suggest that a complex pattern of genes acting at presynaptic level and extracellularly may be involved in LTD-associated synaptic remodeling.

Animals↗
PubMed1999

RACK1, a protein kinase C anchoring protein, coordinates the binding of activated protein kinase C and select pleckstrin homology domains in vitro.

The pleckstrin homology (PH) domain, identified in numerous signaling proteins including the beta-adrenergic receptor kinase (betaARK), was found to bind to various phospholipids as well as the beta subunit of heterotrimeric G proteins (Gbeta) [Touhara, K., et al. (1994) J. Biol. Chem. 269, 10217-10220]. Several PH domain-containing proteins are also substrates of protein kinase C (PKC). Because RACK1, an anchoring protein for activated PKC, is homologous to Gbeta (both contain seven repeats of the WD-40 motif), we determined (i) whether a direct interaction between various PH domains and RACK1 occurs and (ii) the effect of PKC on this interaction. We found that recombinant PH domains of several proteins exhibited differential binding to RACK1. Activated PKC and the PH domain of beta-spectrin or dynamin-1 concomitantly bound to RACK1. Although PH domains bind acidic phospholipids, the interaction between various PH domains and RACK1 was not dependent on the phospholipid activators of PKC, phosphatidylserine and 1, 2-diacylglycerol. Binding of these PH domains to RACK1 was also not affected by either inositol 1,4,5-triphosphate (IP(3)) or phosphatidylinositol 4,5-bisphosphate (PIP(2)). Our in vitro data suggest that RACK1 binds selective PH domains, and that PKC regulates this interaction. We propose that, in vivo, RACK1 may colocalize the kinase with its PH domain-containing substrates.

Animals↗
PubMed2006

WNK4 kinase regulates surface expression of the human sodium chloride cotransporter in mammalian cells.

Pseudohypoaldosteronism type II (PHA II) is caused by mutations of two members of WNK ((with no lysine (k)) kinase family. WNK4 wild type (WT) has been shown to inhibit the activity and surface expression of sodium chloride cotransporter (NCC) when expressed in Xenopus oocytes. Here, we have studied NCC protein processing in mammalian cells in the presence or absence of WNK4 WT and its mutants, E562K and R1185C, by surface biotinylation, Western blot, co-immunoprecipitation (Co-IP) and immunostaining. WNK4 WT significantly reduced NCC surface expression in Cos-7 cells (58.9+/-6.8% vs 100% in control, P<0.001, n=6), whereas its mutant E562K has no significant effect on NCC surface expression (92.9+/-5.3% vs 100%, P=NS, n=6). Another mutant R1185C still partially reduces surface expression of NCC (76.2+/-11.8% vs 100%, P<0.05, n=6). The reduction of NCC surface expression by WNK4 WT (62.9+/-3.3% of control group) is not altered by WT dynamin ((61.8+/-3.7% (P=NS)) or its mutant K44A ((65.4+/-14.1% (P=NS)). A Co-IP study showed that both WNK4 WT and WNK4 E562K interact with NCC. Furthermore, a proton pump inhibitor, bafilomycin A1, partially reverses the inhibitory effect of WNK4 WT on NCC expression. Our data suggest that WNK4 WT significantly inhibits NCC surface expression, which is not owing to an increase in clathrin-mediated endocytosis of NCC, but likely results from enhanced degradation of NCC through a lysosomal pathway.

Animals↗
PubMed2001

Detection of differentially expressed genes in human colon carcinoma cells treated with a selective COX-2 inhibitor.

Numerous reports suggest that use of nonsteroidal anti-inflammatory drugs (NSAIDs) decrease mortality from colorectal cancer. To better understand all of the mechanisms underlying this effect, the global pattern of gene expression in colon carcinoma cells following treatment with NS-398, a selective cyclo-oxygenase-2 inhibitor was evaluated. We utilized suppression subtractive hybridization combined with differential screening to identify genes whose expression was affected following treatment. Among the subtracted cDNA fragments confirmed as differentially expressed, there were two which are known to be involved in the regulation of cell adhesion (human FAT and proto-cadherin-7). We identified two other genes whose levels were decreased and these are known to be involved in the regulation of cell proliferation (cyclin K and p-100). We identified additional genes which are involved in different signaling pathways which regulate programmed cell death (Dynamin 2, Pdcd4 and LIP.1). These results provide evidence that some of the effects of NS-398 on carcinoma cells may be due to modulation of genes which regulate programmed cell death, cell proliferation and cell-cell communication. Additional studies are underway to determine the biological function of the novel genes that were identified.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Developmental expression of amphiphysin in the retinotectal system of the chick: from mRNA to protein.

The role of amphiphysin in clathrin-mediated endocytosis of synaptic vesicles is well established. However, it is still uncertain if the protein is also involved in developmental mechanisms, e.g. axon outgrowth and synapse formation. To investigate the developmental changes in the expression of amphiphysin we used the retinotectal system of the chick, a highly ordered and easily accessible primary neuronal pathway. Reverse transcription polymerase chain reaction (RT-PCR) of total RNA from chick retina and tectum revealed first transcripts for amphiphysin, dynamin and synaptotagmin at embryonic day 5 (E5) for both regions. Surprisingly, Western blots of the retina revealed an increase of protein expression for amphiphysin only after E11 in the retina and the tectum. Immunofluorescence for amphiphysin was not detectable before E10 in the developing chick retina, while other presynaptic proteins like synaptotagmin showed already intense signals in the inner and outer plexiform layers. Subsequently, amphiphysin immunoreactivity follows the expression of synaptotagmin and synaptic vesicle protein 2 (SV2) as seen in the retina and the tectum, and exhibits the same staining as the other proteins in the mature chick brain. Ultrastructural data revealed for the first time that amphiphysin is not only limited to conventional synapses but is also abundant in retinal ribbon terminals. Taken together our data reveal that: (i) there is a developmental delay between mRNA transcription and protein expression for key proteins involved in endocytosis; (ii) amphiphysin gets upregulated after synapse formation; and (iii) amphiphysin is present in the synaptic vesicle cycle in retinal ribbon synapses.

Aging↗
PubMed2002

Phosphatidylinositol 4,5-bisphosphate stimulates vesicle formation from liposomes by brain cytosol.

As a step toward the elucidation of mechanisms in vesicle budding, a cell-free assay that measures cytosol-induced vesicle generation from liposomes was established. This assay then was used to explore the role of phosphoinositides in vesicle formation. Liposomes incubated with brain cytosol in the presence of ATP and GTP massively generated small vesicles, as assessed both quantitatively and qualitatively by a dynamic light-scattering assay. Both ATP and GTP were required. Vesicle formation was inhibited greatly by the immunodepletion of dynamin 1 from the cytosol, indicating a major contribution of this GTPase in this reaction and suggesting that it mimics endocytic vesicle fission. Increasing the concentration of l-alpha-phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] but not of l-alpha-phosphatidylinositol 4-monophosphate or l-alpha-phosphatidylinositol in the lipid membranes enhanced vesicle formation. Lipid analysis revealed rapid degradation of PtdIns(4,5)P2 to l-alpha-phosphatidylinositol during the incubation with the reaction reaching a maximum within 5 sec, whereas vesicle formation proceeded with a longer time course. PtdIns(4,5)P2 degradation was independent of vesicle formation and occurred also in the presence of guanosine 5'-O-(thiotriphosphate), where few vesicle formations occurred. These results suggest that PtdIns(4,5)P2 plays a critical role in the early step of vesicle formation, possibly in the recruitment of coats and fission factors to membranes.

Adenosine Triphosphate↗
PubMed1997

A central role for beta-arrestins and clathrin-coated vesicle-mediated endocytosis in beta2-adrenergic receptor resensitization. Differential regulation of receptor resensitization in two distinct cell types.

G protein-coupled receptor (GPCR) sequestration to endosomes is proposed to be the mechanism by which G protein-coupled receptor kinase (GRK)-phosphorylated receptors are dephosphorylated and resensitized. The identification of beta-arrestins as GPCR trafficking molecules suggested that beta-arrestins might represent critical determinants for GPCR resensitization. Therefore, we tested whether beta2-adrenergic receptor (beta2AR) resensitization was dependent upon beta-arrestins and an intact clathrin-coated vesicle endocytic pathway. The overexpression of either the beta-arrestin 1-V53D dominant negative inhibitor of beta2AR sequestration or dynamin I-K44A to block clathrin-coated vesicle-mediated endocytosis impaired both beta2AR dephosphorylation and resensitization. In contrast, resensitization of a sequestration-impaired beta2AR mutant (Y326A) was reestablished following the overexpression of either GRK2 or beta-arrestin 1. Moreover, beta2ARs did not resensitize in COS-7 cells as the consequence of impaired sequestration and dephosphorylation. However, beta2AR resensitization was restored in these cells following the overexpression of beta-arrestin 2. These findings demonstrate, using both loss and gain of function paradigms, that beta2AR dephosphorylation and resensitization are dependent upon an intact sequestration pathway. These studies also indicate that beta-arrestins play an integral role in regulating not only the desensitization and intracellular trafficking of GPCRs but their ability to resensitize. beta-Arrestin expression levels appear to underlie cell type-specific differences in the regulation of GPCR resensitization.

Adenylyl Cyclases↗
PubMed1999

Trafficking of the HIV coreceptor CXCR4. Role of arrestins and identification of residues in the c-terminal tail that mediate receptor internalization.

The G protein-coupled chemokine receptor CXCR4 serves as the primary coreceptor for entry of T-cell tropic human immunodeficiency virus. CXCR4 undergoes tonic internalization as well as internalization in response to stimulation with phorbol esters and ligand (SDF-1alpha). We investigated the trafficking of this receptor, and we attempted to define the residues of CXCR4 that were critical for receptor internalization. In both COS-1 and HEK-293 cells transiently overexpressing CXCR4, SDF-1alpha and phorbol esters (PMA) promoted rapid internalization of cell surface receptors as assessed by both enzyme-linked immunosorbent assay and immunofluorescence analysis. Expression of GRK2 and/or arrestins promoted modest additional CXCR4 internalization in response to both PMA and SDF. Both PMA- and SDF-mediated CXCR4 internalization was inhibited by coexpression of dominant negative mutants of dynamin-1 and arrestin-3. Arrestin was also recruited to the plasma membrane and appeared to colocalize with internalized receptors in response to SDF but not PMA. We then evaluated the ability of CXCR4 receptors containing mutations of serines and threonines, as well as a dileucine motif, within the C-terminal tail to be internalized and phosphorylated in response to either PMA or SDF-1alpha. This analysis showed that multiple residues within the CXCR4 C-terminal tail appear to mediate both PMA- and SDF-1alpha-mediated receptor internalization. The ability of coexpressed GRK2 and arrestins to promote internalization of the CXCR4 mutants revealed distinct differences between respective mutants and suggested that the integrity of the dileucine motif (Ile-328 and Leu-329) and serines 324, 325, 338, and 339 are critical for receptor internalization.

Amino Acid Sequence↗
PubMed2001

Phosphorylation of a synaptic vesicle-associated protein by an inositol hexakisphosphate-regulated protein kinase.

Despite the fact that inositol hexakisphosphate (InsP(6)) is the most abundant inositol metabolite in cells, its cellular function has remained an enigma. In the present study, we present the first evidence of a protein kinase identified in rat cerebral cortex/hippocampus that is activated by InsP(6). The substrate for the InsP(6)-regulated protein kinase was found to be the synaptic vesicle-associated protein, pacsin/syndapin I. This brain-specific protein, which is highly enriched at nerve terminals, is proposed to act as a molecular link coupling components of the synaptic vesicle endocytic machinery to the cytoskeleton. We show here that the association between pacsin/syndapin I and dynamin I can be increased by InsP(6)-dependent phosphorylation of pacsin/syndapin I. These data provide a model by which InsP(6)-dependent phosphorylation regulates synaptic vesicle recycling by increasing the interaction between endocytic proteins at the synapse.

Amino Acid Sequence↗
PubMed2002

Transfer of M2 muscarinic acetylcholine receptors to clathrin-derived early endosomes following clathrin-independent endocytosis.

Upon agonist stimulation, many G protein-coupled receptors such as beta(2)-adrenergic receptors are internalized via beta-arrestin- and clathrin-dependent mechanisms, whereas others, like M(2) muscarinic acetylcholine receptors (mAChRs), are internalized by clathrin- and arrestin-independent mechanisms. To gain further insight into the mechanisms that regulate M(2) mAChR endocytosis, we investigated the post-endocytic trafficking of M(2) mAChRs in HeLa cells and the role of the ADP-ribosylation factor 6 (Arf6) GTPase in regulating M(2) mAChR internalization. Here, we report that M(2) mAChRs are rapidly internalized by a clathrin-independent pathway that is inhibited up to 50% by expression of either GTPase-defective Arf6 Q67L or an upstream Arf6 activator, Galpha(q) Q209L. In contrast, M(2) mAChR internalization was not affected by expression of dominant-negative dynamin 2 K44A, which is a known inhibitor of clathrin-dependent endocytosis. Nevertheless, M(2) mAChRs, which are initially internalized in structures that lack clathrin-dependent endosomal markers, quickly localize to endosomes that contain the clathrin-dependent, early endosomal markers early endosome autoantigen-1, transferrin receptor, and GTPase-defective Rab5 Q79L, which is known to swell early endosomal compartments. These results suggest that M(2) mAChRs initially internalize via an Arf6-associated, clathrin-independent pathway but then quickly merge with the clathrin endocytic pathway at the level of early endosomes.

ADP-Ribosylation Factor 6↗
PubMed1999

Division versus fusion: Dnm1p and Fzo1p antagonistically regulate mitochondrial shape.

In yeast, mitochondrial division and fusion are highly regulated during growth, mating and sporulation, yet the mechanisms controlling these activities are unknown. Using a novel screen, we isolated mutants in which mitochondria lose their normal structure, and instead form a large network of interconnected tubules. These mutants, which appear defective in mitochondrial division, all carried mutations in DNM1, a dynamin-related protein that localizes to mitochondria. We also isolated mutants containing numerous mitochondrial fragments. These mutants were defective in FZO1, a gene previously shown to be required for mitochondrial fusion. Surprisingly, we found that in dnm1 fzo1 double mutants, normal mitochondrial shape is restored. Induction of Dnm1p expression in dnm1 fzo1 cells caused rapid fragmentation of mitochondria. We propose that dnm1 mutants are defective in the mitochondrial division, an activity antagonistic to fusion. Our results thus suggest that mitochondrial shape is normally controlled by a balance between division and fusion which requires Dnm1p and Fzo1p, respectively.

Cell Division↗
PubMed2000

Clathrin-mediated endocytosis of MUC1 is modulated by its glycosylation state.

MUC1 is a mucin-like type 1 transmembrane protein associated with the apical surface of epithelial cells. In human tumors of epithelial origin MUC1 is overexpressed in an underglycosylated form with truncated O-glycans and accumulates in intracellular compartments. To understand the basis for this altered subcellular localization, we compared the synthesis and trafficking of various glycosylated forms of MUC1 in normal (Chinese hamster ovary) cells and glycosylation-defective (ldlD) cells that lack the epimerase to make UDP-Gal/GalNAc from UDP-Glc/GlcNAc. Although the MUC1 synthesized in ldlD cells was rapidly degraded, addition of GalNAc alone to the culture media resulted in stabilization and near normal surface expression of MUC1 with truncated but sialylated O-glycans. Interestingly, the initial rate of endocytosis of this underglycosylated MUC1 was stimulated by twofold compared with fully glycosylated MUC1. However, the half-lives of the two forms were not different, indicating that trafficking to lysosomes was not affected. Both the normal and stimulated internalization of MUC1 could be blocked by hypertonic media, a hallmark of clathrin-mediated endocytosis. MUC1 endocytosis was also blocked by expression of a dominant-negative mutant of dynamin-1 (K44A), and MUC1 was observed in both clathrin-coated pits and vesicles by immunoelectron microscopy of ultrathin cryosections. Our data suggest that the subcellular redistribution of MUC1 in tumor cells could be a direct result of altered endocytic trafficking induced by its aberrant glycosylation; potential models are discussed. These results also implicate a new role for O-glycans on mucin-like membrane proteins entering the endocytic pathway through clathrin-coated pits.

Animals↗
PubMed2002

Retrieval of human cytomegalovirus glycoprotein B from cell surface is not required for virus envelopment in astrocytoma cells.

Human cytomegalovirus (HCMV) is a prototypic member of the betaherpesvirus family. The HCMV virion is composed of a large DNA genome encapsidated within a nucleocapsid, which is wrapped within an inner proteinaceous tegument and an outer lipid envelope containing viral glycoproteins. Although genome encapsidation clearly occurs in the nucleus, the subsequent steps in the virion assembly process are unclear. HCMV glycoprotein B (gB) is a major component of the virion envelope that plays a critical role in virus entry and is essential for the production of infectious virus progeny. The aim of our present study was to identify the secretory compartment to which HCMV gB was localized and to investigate the role of endocytosis in mediating gB localization and HCMV biogenesis. We show that HCMV gB is localized to the trans-Golgi network (TGN) in HCMV-infected cells and that gB contains all of the trafficking information necessary for TGN localization. Endocytosis of gB was shown to play a role in mediating TGN localization of gB and in targeting of the protein to the site of virus envelopment. However, inhibition of endocytosis with a dominant-negative dynamin I molecule did not affect the production of infectious virus. These observations indicate that, although endocytosis is involved in the trafficking of gB to the site of glycoprotein accumulation in the TGN, endocytosis of gB is not required for the production of infectious HCMV.

Astrocytoma↗
PubMed2006

Differential distributions of the Ca2+ -dependent activator protein for secretion family proteins (CAPS2 and CAPS1) in the mouse brain.

The Ca(2+)-dependent activator protein for secretion (CAPS/Cadps) family consists of two members, CAPS1 and CAPS2, and plays an important role in secretory granule exocytosis. It has been shown that CAPS1 regulates catecholamine release from neuroendocrine cells, whereas CAPS2 is involved in the release of two neurotrophins, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), from parallel fibers of cerebellar granule cells. Although both CAPS proteins are expressed predominantly in the brain, their cellular and regional distributions in the brain are largely unknown. In this study we analyzed the immunohistochemical distributions of the CAPS family proteins in the mouse brain. In most areas of the embryonic nervous system CAPS1 and CAPS2 proteins were complementarily expressed. In the postnatal brain, CAPS1 was widespread at different levels. On the other hand, CAPS2 was localized to distinct cell types and fibers of various brain regions, including the olfactory bulb, cerebrum, hippocampal formation, thalamus, mesencephalic tegmentum, cerebellum, medulla, and spinal cord, except for some regions that overlapped with CAPS1. These CAPS2 cellular distribution patterns had the marked feature of coinciding with those of BDNF in various brain regions. Immunolabels for CAPS2 were also colocalized with those for some proteins related to exocytosis (VAMP and SNAP-25) and endocytosis (Dynamin I) in the cell soma and processes of the mesencephalic tegmentum and cerebellum, suggesting that these proteins might be involved in the dynamics of CAPS2-associated vesicles, although their colocalization on vesicles remains elusive. These results demonstrate that the CAPS family proteins are involved in the secretion of different secretory substances in developing and postnatal brains, and that CAPS2 is probably involved in BDNF secretion in many brain areas.

Animals↗
PubMed2006

Differential roles of two major brain structures, mushroom bodies and central complex, for Drosophila male courtship behavior.

Drosophila male courtship is a complex and robust behavior, the potential for which is genetically built into specific neural circuits in the central nervous system. Previous studies using male-female mosaics and the flies with defects in particular brain structures implicated the critical central regions involved in male courtship behavior. However, their acute physiological roles in courtship regulation still largely remain unknown. Using the temperature-sensitive Dynamin mutation, shibire(ts1), here we demonstrate the significance of two major brain structures, the mushroom bodies and the central complex, in experience-independent aspects of male courtship. We show that blocking of synaptic transmission in the mushroom body intrinsic neurons significantly delays courtship initiation and reduces the courtship activity by shortening the courtship bout length when virgin females are used as a sexual target. Interestingly, however, the same treatment affects neither initiation nor maintenance of courtship toward young males that release courtship-stimulating pheromones different from those of virgin females. In contrast, blocking of synaptic transmission in a central complex substructure, the fan-shaped body, slightly but significantly reduces courtship activity toward both virgin females and young males with little effect on courtship initiation. Taken together, our results indicate that the neuronal activity in the mushroom bodies plays an important role in responding to female-specific sex pheromones that stimulate initiation and maintenance of male courtship behavior, whereas the fan-shaped body neurons are involved in maintenance of male courtship regardless of the nature of courtship-stimulating cues.

Animals↗
PubMed2006

Proteins differentially expressed in response to nicotine in five rat brain regions: identification using a 2-DE/MS-based proteomics approach.

To determine protein expression patterns within the central nervous system (CNS) in response to nicotine, 2-DE/MS was performed on samples from five brain regions of rats that had received nicotine bitartrate by osmotic minipump infusion at a dose of 3.15 mg/kg/day for 7 days. After spot matching and statistical analysis, 41 spots in the amygdala, 49 in the nucleus accumbens (NA), 46 in the prefrontal cortex (PFC), 36 in the striatum, and 28 in the ventral tegmental area (VTA) showed significant differences in the nicotine-treated compared with control samples. Using MALDI-TOF MS peptide fingerprinting, 14 proteins in the amygdala, 11 in the NA, 19 in the PFC, 13 in the striatum, and 19 in the VTA were identified. Several proteins (e.g. dynamin 1, laminin receptors, aldolase A, enolase 1 alpha, Hsc70-ps1, and N-ethylmaleimide-sensitive fusion protein) were differentially expressed in multiple brain regions. By gene ontology analysis, these differentially expressed proteins were grouped into biological process categories, such as energy metabolism, synaptic function, and oxidative stress metabolism. These data, in combination with microarray analysis of mRNA transcripts, have the potential to identify the CNS gene products that show coordinated changes in expression at both the RNA and protein levels in response to nicotine.

Animals↗
PubMed2003

Chronic opioid antagonist treatment selectively regulates trafficking and signaling proteins in mouse spinal cord.

Chronic opioid antagonist treatment produces functional supersensitivity and mu-opioid receptor (muOR) upregulation. Studies suggest a role for G-protein receptor kinases (GRKs) and dynamin (DYN), but not signaling proteins (e.g., G(ialpha2)), in regulation of muOR density following opioid treatment. Therefore, this study examined muOR density, agonist potency, and the abundance and gene expression of GRK-2, DYN-2, and G(ialpha2) in mouse spinal cord after opioid antagonist treatment. Mice were implanted with a 15 mg naltrexone (NTX) or placebo pellet and 8 days later pellets were removed. At 24 and 192 h following NTX treatment, mice were tested for spinal DAMGO analgesia. Other mice were sacrificed at 0 or 192 h following NTX treatment and G(ialpha2), GRK-2, and DYN-2 protein and mRNA levels determined. [(3)H] DAMGO binding studies were also conducted. Immediately following NTX treatment (0 h), muOR density was increased (+ approximately 135%), while 192 h following NTX treatment muOR density was unchanged. NTX increased DAMGO analgesic potency (3.1-fold) 24 h following NTX treatment, while there was no effect at 192 h. NTX decreased protein and mRNA abundance of GRK-2 (-32%; -48%) and DYN-2 (-25%; -29%) in spinal cord at 0 h. At 192 h following 8-day NTX treatment, GRK-2 protein and mRNA were at control levels, while DYN-2 protein remained decreased (-31%) even though DYN-2 mRNA had returned to control levels. G(ialpha2) was unaffected by NTX treatment. These data suggest that opioid antagonist-induced mu-receptor upregulation is mediated by changes in abundance and gene expression of proteins implicated in receptor trafficking, which may decrease constitutive receptor cycling.

Animals↗