An Old Story with a New Twist: Do NMDAR1 mRNA Binding Proteins Regulate Expression of the NMDAR1 Receptor in the Presence of Alcohol?

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Ann. N.Y. Acad. Sci. 1053: 311–318 (2005). © 2005 New York Academy of Sciences.doi: 10.1196/annals.1344.027

An Old Story with a New Twist: Do NMDAR1 mRNA Binding Proteins Regulate Expression of the NMDAR1 Receptor in the Presence of Alcohol?

MEENA KUMARI AND ANTJE ANJI

Department of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas 66506, USA

ABSTRACT: NMDA receptors not only play a pivotal role in normal physiolog-ical processes in the central nervous system (CNS), but have been identified asan important target of ethanol. Chronic exposure to ethanol induces a numberof adaptive processes in the CNS, including an upregulation of NMDA receptornumber and function. The increase in NMDA receptor number in response tochronic ethanol exposure both in vivo and in vitro is accompanied by an increasein NMDAR1 and NMDAR2B polypeptide levels. It is widely believed that theseadaptive changes play an important role in the development of alcohol depen-dence and withdrawal syndrome. At the molecular level, chronic ethanol expo-sure of fetal cortical neurons selectively increases expression of NMDAR1splice variants lacking exon 5 and exon 22. Chronic ethanol exposure of fetalcortical neurons also increases NMDAR1 mRNA half-life in these neurons.However, when new protein synthesis is inhibited, the half-life of NR1 mRNAin these neurons returns to control values, strongly suggesting that ethanolinduces the synthesis of protein(s) that may regulate the decay of NR1 mRNA.In recent years, it has become apparent that regulation of mRNA stability is animportant aspect of regulation of gene expression. Changes in mRNA stabilitycan be accomplished by interaction between cis-acting sequences in the 3�untranslated region (3�UTR) of mRNAs and trans-acting proteins expressed incells. Such interactions may protect RNAs from degradation by ribonucleases,thereby increasing the half-life of mRNAs.

KEYWORDS: NMDA receptors; ethanol; splice variants; RNA binding proteins;mRNA stability; fetal cortical neurons

NMDA RECEPTORS

The NMDA receptor, one of the three major subtypes of glutamate receptors,plays a key role in neuronal plasticity that is thought to underlie memory, learning,and development.1,2 Overstimulation of NMDA receptors can trigger an increase in

Address for correspondence: Meena Kumari, Department of Anatomy and Physiology, Collegeof Veterinary Medicine, Kansas State University, 231 Coles Hall, Manhattan, KS 66506. Voice:785-532-3115; fax: 785-532-4557.

[email protected]

312 ANNALS NEW YORK ACADEMY OF SCIENCES

[Ca]i, resulting in neuronal degeneration and cell death.3–6 Molecular cloning andfunctional studies have revealed the existence of multiple receptor subunits thatdiffer in anatomical distribution, properties, and regulation.7–12 The NMDA receptorforms a heteromeric complex containing combinations of the NMDAR1 (NR1),NMDAR2 (NR2), and the more recently identified NMDAR3 (NR3) subunits.13,14

The NR1 subunit is expressed ubiquitously in the brain, whereas the NR2 and NR3subunits are expressed in a more region-specific manner.7,15,16 Differential splicing

FIGURE 1. Splice variants of the NMDAR1 receptor subunit. NMDAR1 is the productof a single gene. Differential splicing leads to the expression of eight splice variants that arecharacterized by the inclusion or deletion of one 5′ insert (exon 5) and two 3′ inserts (exons21 and 22). NMDAR1 splice variants lacking exon 22 or exons 21 and 22 contain an addi-tional cassette, C2′. Inclusion of both exon 21 and exon 22 gives rise to splice variant NR1-1;exclusion of exon 21, but inclusion of exon 22, gives rise to NR1-2. Exclusion of exon 22,but inclusion of exon 21, gives rise to NR1-3; when both exons 21 and 22 are absent, theresulting splice variant is named NR1-4. All these four NR1 splice variants exist in either“b” isoform (inclusion of exon 5) (i.e., NR1-1b, NR1-2b, NR1-3b, NR1-4b) or “a” isoform(exclusion of exon 5) (i.e., NR1-1a, NR1-2a, NR1-3a, NR1-4a).

313KUMARI & ANJI: NMDA RECEPTORS AND ETHANOL

of the NR1 subunit leads to the expression of eight splice variants that are character-ized by the inclusion or deletion of one 5′ insert (exon 5) and two 3′ inserts (exons21 and 22) (FIG. 1).17 Exon 5 encodes the N1 splice cassette that lies in the extra-cellular amino-terminal domain of the NR1 subunit. Exons 21 and 22 encode thecarboxyl-terminal splice cassettes, C1 and C2, respectively, and are a part of theintracellular domain of the NR1 subunit. NR1 splice variants lacking exon 22 orexons 21 and 22 contain an additional cassette, C2′, at the carboxyl-terminal end.

Alternate splicing of the NR1 subunit facilitates the generation of NR1 subunitswith distinct pharmacological and physiological characteristics. Exon 5 (N1cassette) regulates the pharmacological properties of the receptor, while splicing ofexons 21 (C1 cassette) and 22 (C2 cassette) influences cell surface expression of theNR1 subunit.17

EFFECT OF ETHANOL ON NMDA RECEPTORS

NMDA receptors not only play a pivotal role in the normal physiological processesin the central nervous system (CNS), but have been identified as an important targetof ethanol. This realization has led to an extensive investigation into the molecularmechanisms by which ethanol affects NMDA receptors. Acute exposure to ethanolinhibits the excitatory action of glutamate at NMDA receptors, contributing to thedevelopment of alcohol intoxication. By contrast, chronic exposure to ethanol inducesa number of adaptive processes in the CNS, including an upregulation of NMDAreceptor number and function.18,19 The increase in NMDA receptor number inresponse to chronic ethanol exposure both in vivo and in vitro is accompanied by anincrease in NR1 and NR2B polypeptide levels.20–23 It is widely believed that theseadaptive changes play an important role in the development of alcohol dependenceand withdrawal syndrome.

EFFECT OF ETHANOL ON THE NR1 SUBUNIT

RT/PCR and Western blot analyses have recently demonstrated that ethanol hasa selective effect on NR1 splice variants. In mouse fetal cortical neurons, chronicethanol exposure increases mRNA levels of splice variants that lack exon 5. At thepolypeptide level, similar ethanol treatment increases expression of splice variantsthat lack exon 22 or exons 21 and 22 (NR1–3 and NR-4, respectively), but decreasesexpression of splice variants that contain exon 5. Taken together, in fetal corticalneurons, chronic exposure to ethanol increases the expression of splice variants lack-ing exon 5 and exon 22. These data suggest that chronic ethanol exposure may alterthe pharmacological profile and cell surface expression of NMDA receptors. Thisnotion is supported by the observation made by Rumbaugh and colleagues.24 Theseinvestigators showed that patch-clamp recordings of glutamate responses in humanembryonic kidney cells expressing NMDA receptors composed of NR1 splice variantslacking both exons 5 and 22, and the NR2B subunit, exhibit longer deactivationcurrents as compared to human embryonic kidney cells expressing NR1 splice variantscontaining exon 5 and lacking exon 22, and the NR2B subunit.24


Further studies in mouse fetal cortical neurons showed that chronic ethanol expo-sure increases the rate of NR2B gene transcription and, at the same time, increasesthe half-life of NR1 mRNA in fetal cortical neurons to more than 24 h (FIG. 2).25

MRNA STABILITY

Regulation of mRNA turnover or half-life is known to play an important role inregulating gene expression. Changes in mRNA abundance due to alterations inmRNA half-life ultimately affect protein expression of genes.26 mRNAs exhibit awide range of half-lives that can be correlated with their functional role in the cell.For instance, β-globin, considered a housekeeping gene, has a half-life of over 20 h,whereas the oncogene c-myc has a short half-life of about 10 min.27,28 It is nowappreciated that mRNA decay is a finely tuned process involving cis-acting sequenceelements residing primarily in the 3′UTR of mRNAs, although in some cases theymay be located in the coding region. These stability modifying sequences act inconcert with trans-acting protein factors regulating mRNA decay.29,30 To date, mostof the cis-acting sequences identified destabilize mRNAs. One such sequence

FIGURE 2. Schematic of intracellular sites of action of ethanol on NMDA receptors.Chronic exposure of fetal cortical neurons increases the rate of transcription of theNMDAR2B gene (site 1). Similar treatment increases the half-life of the NMDAR1 receptormRNA (site 2). Ethanol may also influence the interaction of trans-acting factors or RNAbinding proteins (RBP) with cis-sequences located in the 3′UTR of the NMDAR1 receptormRNA (site 3).


element is the well-characterized adenosine uridine–rich (ARE) sequence located inthe 3′UTR of some mammalian mRNAs.31 In some instances, the presence of suchan ARE sequence is sufficient to make a normally stable mRNA like β-globinunstable.32 Recent data have suggested that AREs can be variable in both length andsequence and, as more cis-acting sequences are being identified, the complexity ofthese elements is just being appreciated.33 The development of assays such as RNAelectromobility shift assays and UV cross-link analysis aided the identification ofRNA binding proteins or trans-acting proteins that target these sequences.

TRANS-ACTING PROTEINS

Data from our lab suggest that RNA binding proteins may also be importantregulators of NR1 gene expression. When fetal cortical neurons are cultured in thepresence of the protein synthesis inhibitor, cycloheximide, ethanol-induced stabili-zation of the NR1 mRNA does not occur.34 Such a requirement for protein synthesisstrongly suggests that labile protein factors are necessary for NR1 mRNA stabiliza-tion to occur (FIG. 2). Preliminary data from our laboratory further suggest that cis-acting regions may be present within the 3′UTR of NR1 mRNA. Taken together, itappears that cis-trans interactions play a role in regulating expression of the NR1subunit in the presence of ethanol. The trans-acting proteins are now known to beimportant regulators of gene expression, and a search for trans-acting factors has ledto the identification of several proteins, many of which have dual functions.26 Someof these factors are associated with mRNAs during their transport from the nucleusto the cytoplasm or to their site of translation.35–38 For instance, renin mRNA isregulated primarily at the level of mRNA turnover. In the search for proteins thatmediate the posttranscriptional effects on renin mRNA, Adams and colleagues iden-tified three RNA binding proteins that interact with and regulate the renin mRNA:hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase βsubunit (HADHB), the poly-C binding protein CP1, and HuR.39 Interestingly, intra-cellular imaging performed by the same authors revealed a distinct localization ofHADHB to the mitochondria, whereas HuR was found in the nucleus and CP1 wasdistributed throughout the cell. The exact role, however, of this differential cellularlocalization of these three RNA binding proteins remains to be elucidated. The trans-acting proteins play a role in the posttranscriptional regulation of other mRNAs aswell. The protein kinase A–induced stabilization of the lactate dehydrogenase-AmRNA is associated with an upregulation of 3′UTR binding activity of three trans-acting factors.40 Similarly, the stabilization of the tyrosine hydroxylase mRNA thatoccurs when PC12 cells are cultured under hypoxic conditions is accompanied by anincrease in formation of the RNA-protein complex.41

In the CNS, a number of genes are known to be regulated at the posttranscriptionallevel and a growing number of RNA binding proteins have been identified in theCNS. Of these, the ELAV-like Hu proteins are best characterized. ELAV-like Hu pro-teins belong to a small family of RNA binding proteins and are human homologuesof Drosophila ELAV, an RNA binding protein whose deletion results in embryoniclethal abnormal vision (ELAV) phenotype. So far, four ELAV proteins have beenidentified: HuB, HuC, HuD, and HuR.42 The expression of HuB, HuC, and HuD isrestricted to the nervous system, whereas HuR is present in most tissues.43 The Hu


proteins bind preferentially to AREs in the 3′UTR of mRNAs, are known to stabilizecertain target mRNAs, and influence translation as well as RNA transport. Recentdata have demonstrated that HuD is involved in posttranscriptional regulation of thegrowth-associated protein, GAP-43.44 Both HuD and GAP-43 are highly expressedin developing neurons and are closely associated with neuronal development andplasticity. Very recent data have solidified the role of Hu proteins in neuronal differ-entiation. HuD has now been implicated in the posttranscriptional regulation of geneexpression that occurs after spatial learning in the hippocampus of rodents. Specifi-cally, a learning-specific increase in HuD is associated with increased expression ofGAP-43 mRNA and protein levels.45,46

CONCLUSIONS

Ethanol acts on several neurotransmitter systems in the brain, and one of the majortargets for this drug is the NMDA receptor. Several laboratories around the worldhave clearly demonstrated that ethanol affects the NMDA receptor complex atmultiple levels. Work in our lab has focused on the regulation of the NR1 subunit atthe molecular level. Recent data suggest that the NR1 subunit is regulated at the post-transcriptional level (mRNA stability) in the presence of ethanol. In the last decade,it has become apparent that regulation of mRNA stability is an important regulatorof gene expression. Many cis-acting sequences and trans-acting factors have beenidentified and prompted questions regarding their physiological role. With a betterunderstanding of the emerging complexity of cis-trans interactions on mRNAs, wenot only will gain a better understanding of the regulation of gene expression, butalso will identify new potential therapeutic targets.

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An Old Story with a New Twist: Do NMDAR1 mRNA Binding Proteins Regulate Expression of the NMDAR1 Receptor in the Presence of Alcohol?