Biomedical subjects

Danxin Wang

Publications and source records attributed to Danxin Wang.

13 recordsLinked to original sources

PubMed2006

Searching for polymorphisms that affect gene expression and mRNA processing: example ABCB1 (MDR1).

Cis-acting genetic variations can affect the amount and structure of mRNA/protein. Genomic surveys indicate that polymorphisms affecting transcription and mRNA processing, including splicing and turnover, may account for the main share of genetic factors in human phenotypic variability; however, most of these polymorphisms remain yet to be discovered. We use allelic expression imbalance (AEI) as a quantitative phenotype in the search for functional cis-acting polymorphisms in many genes, including ABCB1 (multidrug resistance 1 gene, MDR1, Pgp). Previous studies have shown that ABCB1 activity correlates with a synonymous polymorphism, C3435T; however, the functional polymorphism and molecular mechanism underlying this clinical association remained unknown. Analysis of allele-specific expression in liver autopsy samples and in vitro expression experiments showed that C3435T represents a main functional polymorphism, accounting for 1.5- to 2-fold changes in mRNA levels. The mechanism appears to involve increased mRNA turnover, probably as a result of different folding structures calculated for mRNA with the Mfold program. Other examples of the successful application of AEI analysis for studying functional polymorphism include 5-HTT (serotonin transporter, SLC6A4) and OPRM1 (mu opioid receptor). AEI is therefore a powerful approach for detecting cis-acting polymorphisms affecting gene expression and mRNA processing.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2006

Calmodulin binding to peptides derived from the i3 loop of muscarinic receptors.

PURPOSE: This study was conducted to identify and characterize the structural requirements of a calmodulin-binding motif identified in the third intracellular (i3) loop of muscarinic acetylcholine receptors (M1-M5), a region important for G protein coupling. METHODS: GST fusion proteins and synthetic peptides derived from the hM1 i3 loop were tested for binding to CaM using a cross-linking gel shift assay and a dansyl-CaM fluorescence assay. Mutagenesis studies further characterized the structural requirements for the interaction and identified critical residues. RESULTS: 28-Mer peptides from the C terminus of i3, representing the putative calmodulin domains of M1, M2, and M3, were found capable of interacting with CaM. In addition, smaller peptides defined a 5-amino-acid sequence essential for calmodulin binding. Studies performed with M1 peptides derived from GST fusion proteins, representing larger portions of the i3 C terminus, suggested the presence of a second adjacent CaM binding site. Mutagenesis studies identified two mutants that are unable to bind CaM: a point mutation, E360A, and a deletion mutant, delta232-358. CONCLUSION: Calmodulin can bind to an M1 region implicated in G protein coupling. This indicates an important role for CaM in the regulation of muscarinic signal transduction.

Amino Acid Sequence↗

PubMed2006

Highly variable mRNA expression and splicing of L-type voltage-dependent calcium channel alpha subunit 1C in human heart tissues.

OBJECTIVES: The voltage-dependent L-type calcium channel alpha-subunit 1c (Cav1.2, CACNA1C) undergoes extensive mRNA splicing, leading to numerous isoforms with different functions. L-type calcium channel blockers are used in the treatment of hypertension and arrhythmias, but response varies between individuals. We have studied the interindividual variability in mRNA expression and splicing of CACNA1C, in 65 heart tissue samples, taken from heart transplant recipients. METHODS: Splice variants were measured quantitatively by polymerase chain reaction in 12 splicing loci of CACNA1C mRNA. To search for functional cis-acting polymorphisms, we determined allelic expression ratios for total CACNA1C mRNA and several splice variants using marker single nucleotide polymorphisms in exon 4 and exon 30. RESULTS: Total CACNA1C mRNA levels varied approximately 50-fold. Substantial splicing occurred in six loci generating two or more splice variants, some with known functional differences. Splice patterns varied broadly between individuals. Two heart tissues expressed predominantly the dihydropyridine-sensitive smooth muscle isoform of CACNA1C (containing exon 8), rather than the cardiac isoform (containing exon 8a). Lack of significant allelic expression imbalance, observed with total mRNA and several splice variants, argued against CACNA1C polymorphisms as a cause of variability. Taken together, highly variable splicing can cause profound phenotypic variations of CACNA1C function, potentially associated with disease susceptibility and response to L-type calcium channel blockers.

Alleles↗

PubMed2005

Calmodulin interaction with peptides from G-protein coupled receptors measured with S-Tag labeling.

We have developed a quantitative assay of calmodulin (CaM) binding to S-Tag labeled peptides derived from G-protein coupled receptor (GPCR) sequences. CaM binding of peptides derived from the third intracellular loop (i3) of mu opioid receptor (MOR) was confirmed and the CaM-binding motif refined. A MORi3 peptide with a Lys > Ala substitution--shown to reduce CaM-binding of intact MOR--bound fivefold less avidly than the wild-type peptide. Screening peptides derived from i3 loops of other GPCR families confirmed 5HT1A, and identified muscarinic receptor 3, and melanocortin receptor 1, as proteins carrying CaM-binding domains. The use of S-Tag labeling can serve for rapid screening of putative CaM-binding domains in GPCRs.

Binding Sites↗

PubMed2005

Allelic expression imbalance of human mu opioid receptor (OPRM1) caused by variant A118G.

As a primary target for opioid drugs and peptides, the mu opioid receptor (OPRM1) plays a key role in pain perception and addiction. Genetic variants of OPRM1 have been implicated in predisposition to drug addiction, in particular the single nucleotide polymorphism A118G, leading to an N40D substitution, with an allele frequency of 10-32%, and uncertain functions. We have measured allele-specific mRNA expression of OPRM1 in human autopsy brain tissues, using A118G as a marker. In 8 heterozygous samples measured, the A118 mRNA allele was 1.5-2.5-fold more abundant than the G118 allele. Transfection into Chinese hamster ovary cells of a cDNA representing only the coding region of OPRM1, carrying adenosine, guanosine, cytidine, and thymidine in position 118, resulted in 1.5-fold lower mRNA levels only for OPRM1-G118, and more than 10-fold lower OPRM1 protein levels, measured by Western blotting and receptor binding assay. After transfection and inhibition of transcription with actinomycin D, analysis of mRNA turnover failed to reveal differences in mRNA stability between A118 and G118 alleles, indicating a defect in transcription or mRNA maturation. These results indicate that OPRM1-G118 is a functional variant with deleterious effects on both mRNA and protein yield. Clarifying the functional relevance of polymorphisms associated with susceptibility to a complex disorder such as drug addiction provides a foundation for clinical association studies.

Alleles↗

PubMed2005

Opioid receptor homo- and heterodimerization in living cells by quantitative bioluminescence resonance energy transfer.

Opioid receptors have been shown to dimerize or oligomerize among themselves and each other, affecting their functional properties. This study used bioluminescence resonance energy transfer (BRET) between the mu, delta, and kappa opioid receptors to study opioid receptor aggregation in transfected human embryonic kidney 293 cells. Titration of receptor levels indicated that all three opioid receptors have a similar affinity to form homo- or hetero-oligomers in combination with any other opioid receptor type. In contrast, none of the opioid receptors formed detectable oligomers with the muscarinic M2 receptor, indicating that interactions among opioid receptors are selective. The formation of opioid receptor dimers, rather than higher order oligomers, is supported by binding kinetics in competition experiments between labeled and unlabeled receptors. Opioid receptor dimerization occurred at physiological temperatures upon receptor biosynthesis, before trafficking to the plasma membrane. Moreover, using BRET, coimmunoprecipitation, receptor binding, and G protein coupling, we demonstrate for the first time functional mu opioid receptor-kappa opioid receptor heterodimerization. These combined results demonstrate that opioid receptors can undergo homo- and heterodimerization, a process with potential implications for opioid physiology and pharmacology.

Animals↗

PubMed2005

In vivo characterization of 6beta-naltrexol, an opioid ligand with less inverse agonist activity compared with naltrexone and naloxone in opioid-dependent mice.

The mu-opioid receptor displays basal signaling activity, which seems to be enhanced by exposure to opioid agonists. This study assesses the in vivo pharmacology of the putative "neutral" antagonist 6beta-naltrexol in comparison to other ligands with varying efficacy, such as naloxone, an inverse agonist in the opioid-dependent state. ICR mice were used to generate full antagonist dose-response curves for naloxone, naltrexone, nalbuphine, and 6beta-naltrexol in blocking acute antinociceptive effects of morphine and precipitating opioid withdrawal in models of physical dependence. 6beta-Naltrexol was roughly equipotent to naloxone and between 4.5- and 10-fold less potent than naltrexone in blocking morphine-induced antinociception and locomotor activity, showing that 6beta-naltrexol enters the central nervous system. In contrast to naloxone and naltrexone, 6beta-naltrexol precipitated only minimal withdrawal at high doses in an acute dependence model and was approximately 77- and 30-fold less potent than naltrexone and naloxone, respectively, in precipitating withdrawal in a chronic dependence model. 6beta-Naltrexol reduced the inverse agonist effects of naloxone in vitro and in vivo, as expected for a neutral antagonist. Therefore, the pharmacological effects of 6beta-naltrexol differ markedly from those of naloxone and naltrexone in the opioid-dependent state. A reduction of withdrawal effects associated with neutral mu-opioid receptor antagonists may offer advantages in treating opioid overdose and addiction.

Animals↗

PubMed2005

Multidrug resistance polypeptide 1 (MDR1, ABCB1) variant 3435C>T affects mRNA stability.

OBJECTIVES: ABCB1 (multidrug resistance 1 polypeptide, MDR1, Pgp) is a multispecific efflux transporter of drugs and xenobiotics. Among numerous polymorphisms in human ABCB1, the synonymous SNP 3435C > T has been associated with decreased mRNA and protein levels, via unknown mechanisms. METHODS: To search for cis-acting polymorphism affecting transcription or mRNA processing, we used 3435C > T as a marker single nucleotide polymorphism (SNP), for measuring differences in allelic mRNA expression. Ratios of allelic abundance in genomic DNA and mRNA (after conversion to cDNA) were measured quantitatively with a primer extension assay, in human liver samples. RESULTS: mRNA expression of the 3435C allele was significantly higher than that of the 3435T allele (3435C/3435T ratios ranging from 1.06-1.61). Cotransfection of equal amounts of ABCB1 expression plasmids containing 3435C or 3435T also revealed higher 3435C mRNA expression. Increasing 3435C/3435T ratios after cessation of transcription indicated that the 3435C > T substitution decreases mRNA stability. 3435C > T is in strong linkage disequilibrium with two other coding SNPs (1236C > T and 2677G > T) forming two abundant haplotypes (ABCB1*1 and ABCB1*13). Transfection of all possible combinations of these three SNPs demonstrated that only 3435T is associated with lower mRNA levels. Calculations of mRNA folding, using Mfold, suggested an effect on mRNA secondary structure. CONCLUSIONS: the abundant 3435C > T SNP appears to be a main factor in allelic variation of ABCB1 mRNA expression in the liver, by changing mRNA stability.

ATP-Binding Cassette Transporters↗

PubMed2004

Polymorphisms affecting gene regulation and mRNA processing: broad implications for pharmacogenetics.

Functional polymorphisms that alter gene expression and mRNA processing appear to play a critical role in shaping human phenotypic variability. Intensive studies on polymorphisms affecting drug response have revealed multiple modes of altered gene function, frequently involving cis-acting regulatory sequence variants. Experimental and in silico methods have advanced the search for such polymorphisms, but considerable challenges remain. Here, a survey of polymorphisms in drug-related genes indicates that: (a) a substantial proportion of genetic variability still remains unaccounted for; (b) a majority of these genes harbors known regulatory polymorphisms; and (c) a portion of polymorphisms affect splicing and mRNA turnover. Pharmacogenetic optimiziation of individual drug therapy may require a complete understanding of all functional sequence variants in key genes. This review surveys known noncoding polymorphisms in genes encoding cytochrome P450s and other drug-metabolizing enzymes, drug transporters, and drug targets and receptors. Current methods and challenges associated with the identification and characterization of functional polymorphisms are also discussed.

Animals↗

PubMed2004

Basal opioid receptor activity, neutral antagonists, and therapeutic opportunities.

The mu opioid receptor (MOR, OPRM)--the principal receptor involved in narcotic addiction--has been shown to display basal (spontaneous, constitutive) signaling activity. Interaction with other signaling proteins, such as calmodulin, regulates basal MOR activity. Providing a mechanism for long-lasting regulation, basal MOR activity potentially plays a key role in addiction, in combination with gene regulation and synaptic remodeling. Recent results support a link to physical dependence--one of the main manifestations of addiction to drugs of abuse. The prototypical opioid antagonists, naloxone and naltrexone, were shown to act as inverse agonists in the morphine-dependent state (i.e., they suppress basal MOR signaling) and thereby appear to elicit or contribute to precipitated withdrawal. This affords the opportunity to explore therapeutic applications for neutral antagonists (blocking agonists at MOR without affecting basal activity) with reduced adverse effects. Neutral antagonists are promising drug candidates in the treatment of addiction and overdose, and of peripheral adverse effects of narcotic analgesics.

Animals↗

PubMed2003

Basal signaling activity of mu opioid receptor in mouse brain: role in narcotic dependence.

Narcotic analgesics cause addiction by poorly understood mechanisms, involving mu opoid receptor (MOR). Previous cell culture studies have demonstrated significant basal, spontaneous MOR signaling activity, but its relevance to narcotic addiction remained unclear. In this study, we tested basal MOR-signaling activity in brain tissue from untreated and morphine-pretreated mice, in comparison to antagonist-induced withdrawal in morphine-dependent mice. Using guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTP gamma S) binding and adenylyl cyclase activity assay in brain homogenates, we demonstrated that morphine pretreatment of mice enhanced basal MOR signaling in mouse brain homogenates and, moreover, caused persistent changes in the effects of naloxone and naltrexone, antagonists that elicit severe withdrawal in dependent subjects. Naloxone and naltrexone suppressed basal [(35)S]GTP gamma S binding (acting as "inverse agonists") only after morphine pretreatment, but not in drug-naive animals. Moreover, naloxone and naltrexone stimulated adenylyl cyclase activity in striatum homogenates only after morphine pretreatment, by reversing the inhibitory effects of basal MOR activity. After cessation of morphine treatment, the time course of inverse naloxone effects on basal MOR signaling was similar to the time course of naltrexone-stimulated narcotic withdrawal over several days. The neutral antagonist 6 beta-naltrexol blocked MOR activation without affecting basal signaling (G protein coupling and adenylyl cyclase regulation) and also elicited substantially less severe withdrawal. These results demonstrate long-lasting regulation of basal MOR signaling as a potential factor in narcotic dependence.

Adenylyl Cyclases↗

PubMed2002

Serum response factor activation by muscarinic receptors via RhoA. Novel pathway specific to M1 subtype involving calmodulin, calcineurin, and Pyk2.

The muscarinic cholinergic receptor (mAChR) subtypes share high sequence similarity except in their third intracellular loop and COOH terminus, domains thought to be involved in signal transduction. Subtypes M1, M3, and M5 couple mainly through Galpha(q/11), and M2 and M4 couple mainly through Galpha(i/o). Whether subtypes within each of these two groups differ in their signaling pathways remains to be resolved. This study focused on nuclear signaling pathways leading to activation of the transcription factor, serum response factor (SRF). Genes encoding M1, M2, and M3 were co-expressed in Jurkat T lymphocytes with a reporter gene driven by a mutant serum response element, SRE.L, which responds to SRF activation. We show that only M1 mAChR activated SRF through a pathway involving the small GTPase RhoA, with no response observed for M2 and M3. Transfection of GTPase-deficient Galpha subunits (GalphaQL; constitutively active form) demonstrated that SRF was activated by Galpha(13)QL but only marginally by Galpha(q)QL and Galpha(12)QL in Jurkat cells. Yet transfection of regulator of G protein-signaling protein, RGS2 and RGS4, which inhibit Galpha(q/11) activity, indicated that Galpha(q/11) and Ca(2+) mobilization were required for SRF activation by M1. Calmodulin inhibitors suppressed the M1 and the Galpha(13)QL pathways, acting both upstream and downstream of RhoA. However, calcineurin inhibitors and the tyrosine kinase inhibitor genistein selectively suppressed SRF activation by M1, but not by Galpha(13)QL, indicating the presence of separate pathways. The calmodulin-dependent tyrosine kinase Pyk2 was also activated by M1 but not M3, and Pyk2 appears also to play a role in M1-SRF activation, as judged by experiments with two dominant-negative Pyk2 mutants. These results reveal a novel calmodulin-dependent RhoA-SRF signaling pathway unique to the M1 mAChR subtype.

Calcineurin↗

PubMed2002

Differential effects of mu-opioid receptor ligands on Ca(2+) signaling.

Activation of mu-opioid receptors (MORs) transfected into human embryonic kidney 293 cells, caused a multiphasic increase in cytosolic free Ca(2+) levels (Ca(2+)i). The first Ca(2+)i maximum (peak 1) between 5 and 7 s depended on the presence of extracellular Ca(2+) (Ca(2+)e). The second phase peaking at approximately 15 s (peak 2) was independent of Ca(2+)e and thus represents Ca(2+) release from intracellular stores. A decrease in temperature from 37 to 25 degrees C also caused reduction of peak 1 but not peak 2, suggesting that the two responses arise from mechanistically distinct pathways. A delayed Ca(2+)e-dependent third response phase is thought to represent capacitative Ca(2+)e influx evoked after release of Ca(2+) from internal stores. Agonists and antagonists of two major classes of opioid ligands, oxymorphinans (morphine and naloxone) and oripavines (etorphine and diprenorphine), had differential effects on Ca(2+) currents. Although morphine activated both phases with equal potency, etorphine was 20-fold less potent at stimulating peak 1 over peak 2. Similarly, the antagonists, naloxone and diprenorphine, blocked the Ca(2+) response to each agonist with greatly varying potencies. Specifically, concomitant injection of diprenorphine failed to affect peak 1 (thought to represent rapid Ca(2+)e influx) stimulated by morphine while fully blocking peak 2 (intracellular Ca(2+) release). However, diprenorphine potently inhibited peak 1 as well when added to the cells before morphine, indicating limited or slow access of diprenorphine to these morphine binding sites. The existence of multiple, functionally distinct binding site conformations could account for these findings. In conclusion, different opioid ligands can differentially affect Ca(2+) response patterns resulting from MOR activation.

Analgesics, Opioid↗