Subcellular Localization Properties Research Articles

Experimental Mapping of the Canine KCNJ2 and KCNJ12 Gene Structures and Functional Analysis of the Canine KIR2.2 ion Channel

For many model organisms traditionally in use for cardiac electrophysiological studies, characterization of ion channel genes is lacking. We focused here on two genes encoding the inward rectifier current, KCNJ2 and KCNJ12, in the dog heart. A combination of RT-PCR, 5′-RACE, and 3′-RACE demonstrated the status of KCNJ2 as a two exon gene. The complete open reading frame (ORF) was located on the second exon. One transcription initiation site was mapped. Four differential transcription termination sites were found downstream of two consensus polyadenylation signals. The canine KCNJ12 gene was found to consist of three exons, with its ORF located on the third exon. One transcription initiation and one termination site were found. No alternative splicing was observed in right ventricle or brain cortex. The gene structure of canine KCNJ2 and KCNJ12 was conserved amongst other vertebrates, while current GenBank gene annotation was determined as incomplete. In silico translation of KCN12 revealed a non-conserved glycine rich stretch located near the carboxy-terminus of the KIR2.2 protein. However, no differences were observed when comparing dog with human KIR2.2 protein upon ectopic expression in COS-7 or HEK293 cells with respect to subcellular localization or electrophysiological properties.

Highly Selective Mitochondria-Targeting Amphiphilic Silicon(IV) Phthalocyanines with Axially Ligated Rhodamine B for Photodynamic Therapy

Two axially ligated rhodamine-Si(IV)-phthalocyanine (Rh-SiPc) conjugates, bearing one and two rhodamine B, were synthesized and their linear and two-photon photophysical, subcellular localization and photocytotoxic properties were studied. These Rh-SiPc conjugates exhibited an almost exclusive mitochondrial localizing property in human nasopharyngeal carcinoma (HK-1) cells and human cervical carcinoma (HeLa) cells. Strong photocytotoxic but low dark cytotoxic properties were also observed for the two Rh-SiPc conjugates toward the HK-1 cells. Using nuclei staining method and flow cytometric DNA content analysis, apoptotic cell death was induced by these conjugates upon photoactivation. This observation is consistent with their mitochondrial localization property. The observed properties of these conjugates qualify them as promising PDT agents.

NMDA receptor signaling: death or survival?

Glutamate-induced neuronal damage is mainly caused by overactivation of N-methyl-D-aspartate (NMDA) receptors. Conversely, normal physiological brain function and neuronal survival require adequate activation of NMDA receptors. Studies have revealed that NMDA receptor-induced neuronal death or survival is mediated through distinct subset of NMDA receptors triggering different intracellular signaling pathways. Here we discuss recent advances in the characterization of NMDA receptors in neuronal protection, emphasizing subunit-specific role, which contributes to temporal-spatial distribution, subcellular localization and diverse channel properties of NMDA receptors.

Balance between Fluorescence Enhancement and Association Affinity in Fluorescent Heteroditopic Indicators for Imaging Zinc Ion in Living Cells

A fluorescent heteroditopic indicator for the zinc(II) ion possesses two different zinc(II) binding sites. The sequential coordination of zinc(II) at the two sites can be transmitted into distinct fluorescence changes. In the heteroditopic ligand system that our group developed, the formations of mono- and dizinc(II) complexes along an increasing gradient of zinc(II) concentration lead to fluorescence enhancement and an emission bathochromic shift, respectively. The extents of these two changes determine the sensitivity and, ultimately, the effectiveness of the heteroditopic indicator in quantifying zinc(II) ion over a large concentration range. In this work, a strategy to increase the degree of fluorescence enhancement upon the formation of the monozinc(II) complex of a heteroditopic ligand under simulated physiological conditions is demonstrated. Fluorination of the pyridyl groups in the pentadentate N,N,N'-tris(pyridylmethyl)ethyleneamino group reduces the apparent pK(a) value of the high-affinity site, which increases the degree of fluorescence enhancement as the monozinc(II) complex is forming. However, fluorination impairs the coordination strength of the high-affinity zinc(II) binding site, which in the triply fluorinated ligand reduces the binding strength to the level of the low-affinity 2,2'-bipyridyl. The potential of the reported ligands in imaging zinc(II) ion in living cells was evaluated. The subcellular localization properties of two ligands in five organelles were characterized. Both benefits and deficiencies of these ligands were revealed, which provides directions for the near future in this line of research.

The Subcellular Distribution of Small Molecules: A Meta-Analysis

To explore the extent to which current knowledge about the organelle-targeting features of small molecules may be applicable toward controlling the accumulation and distribution of exogenous chemical agents inside cells, molecules with known subcellular localization properties (as reported in the scientific literature) were compiled into a single data set. This data set was compared to a reference data set of approved drug molecules derived from the DrugBank database, and to a reference data set of random organic molecules derived from the PubChem database. Cheminformatic analysis revealed that molecules with reported subcellular localizations were comparably diverse. However, the calculated physicochemical properties of molecules reported to accumulate in different organelles were markedly overlapping. In relation to the reference sets of DrugBank and PubChem molecules, molecules with reported subcellular localizations were biased toward larger, more complex chemical structures possessing multiple ionizable functional groups and higher lipophilicity. Stratifying molecules based on molecular weight revealed that many physicochemical properties' trends associated with specific organelles were reversed in smaller vs larger molecules. Most likely, these reversed trends are due to the different transport mechanisms determining the subcellular localization of molecules of different sizes. Molecular weight can be dramatically altered by tagging molecules with fluorophores or by incorporating organelle targeting motifs. Generally, in order to better exploit structure-localization relationships, subcellular targeting strategies would benefit from analysis of the biodistribution effects resulting from variations in the size of the molecules.

Comparative analysis of PDE10A isoforms

Phosphodiesterases (PDE) are considered to be key players in many signal transduction pathways. They degrade the second messengers cGMP and cAMP, thereby controlling their intracellular levels. So far, eleven PDE families, typically having several isoforms and splice variants are known. The PDE10 family is encoded by one gene that gives rise to at least two different isoforms in humans: the soluble PDE10A1 and the membrane associated PDE10A2. Distribution studies in human and mouse tissues showed highest expression in medium spiny neurons of the striatum and peripherally in testis. The localization of PDE10A in medium spiny neurons has led to much attention on PDE10 as a potential therapeutic target for novel antipsychotics. In neurons, membrane targeting of PDE10A2 is regulated at least in part by PKA-dependent phosphorylation of Thr16. On the other hand, little attention has been paid to PDE10A1 and the regulation of both isoforms. Here, we compared PDE10A isoforms with regard to their tissue distribution, subcellular localization and biochemical properties.

Epitope Analysis and Antibody Preparation of Human Polypeptide:N-acetylgalactosaminyltransferase-2 Protein

In order to study the subcellular localization, secondary structure, and surface properties of ppGalNAcT2, we have developed polyclonal antibodies against ricin of ppGalNAcT2. As indicated by ELISA and Western blot analysis, these antibodies exhibit good reactivity and high specificity. We also found that the prevailing epitopes of ppGalNAcT2 exist in the RICIN domain. Furthermore immunofluorescence assay has helped to localize ppGalNAcT2 to Golgi apparatus. The results suggest that ppGalNAcT2 might take part in modifying glycoproteins in the Golgi complex, thereby laying a valuable foundation for further study of ppGalNAcT2 in tumor cell lines.

Homology modeling and deletion mutants of human nicotinamide mononucleotide adenylyltransferase isozyme 2: New insights on structure and function relationship

Nicotinamide mononucleotide adenylyltransferase (NMNAT) catalyzes the formation of NAD by means of nucleophilic attack by 5'-phosphoryl of NMN on the α-phosphoryl group of ATP. Humans possess three NMNAT isozymes (NMNAT1, NMNAT2, and NMNAT3) that differ in size and sequence, gene expression pattern, subcellular localization, oligomeric state and catalytic properties. Of these, NMNAT2, the least abundant isozyme, is the only one whose much-needed crystal structure has not been solved as yet. To fill this gap, we used the crystal structures of human NMNAT1 and NMNAT3 as templates for homology-based structural modeling of NMNAT2, and the resulting raw structure was then refined by molecular dynamics simulations in a water box to obtain a model of the final folded structure. We investigated the importance of NMNAT2's central domain, which we postulated to be dispensable for catalytic activity, instead representing an isozyme-specific control domain within the overall architecture of NMNAT2. Indeed, we experimentally confirmed that removal of different-length fragments from this central domain did not compromise the enzyme's catalytic activity or the overall tridimensional structure of the active site.

CYTO 2010—Overview of the XXV ISAC Congress Proceedings Issue of Cytometry Part A

issue of Cytometry Part A presents the second occasionon which a call has been issued to encourage authors to sub-mit their best work to the Journal for publication in a specialCongress Proceedings Issue of Cytometry. The incentive forsubmission was that accepted papers would be guaranteed anoral presentation at the XXV ISAC Congress in Seattle—CYTO 2010. The manuscripts submitted for the call werereviewed via the normal Cytometry Part A review process andthen by a second level of review by the Congress OrganizingCommittee. The outcome of this strict peer-review processwas that nine papers were accepted for the Proceedings Issueof Cytometry Part A. The research articles provide excellentexamples of some of the latest trends in the field of cytometryand we highlight them here in this editorial review. The topicsof the accepted papers range from application of 12-color flowcytometry through characterization of new probes and investi-gation of cytoskeleton elements to analysis of nuclear structurefrom histopathology images.Modern polychromatic flow cytometry can providehigher resolution approaches for the dissection and identifica-tion of cell subpopulations (1,2). In this issue, the paper byAutissier et al. (page 410) describes how 12-color flow cytome-try can be used for studying the phenotypes of lymphocyte,monocyte, and dendritic cell subsets. The authors developed asingle flow cytometry panel that allows for simultaneousdetection of the lymphocyte and monocyte cell populationsand all known DC subsets. Studying these crucial subpopula-tions of the immune system in one single panel may give abroader view of the immune response during HIV infectionand can be used for monitoring AIDS pathogenesis.Polychromatic cytometry has become increasingly impor-tant in slide-based applications with the drive to increase thenumber of measurable fluorochromes for multiparametricanalysis. New fluorochromes have been introduced where thefluorescent dyes possess different optical characteristicsincluding spectral range, lifetime signatures, and improvedphotostability (3–5). The special feature of slide-based cyto-metry is that analyzed fields can be revisited, providing a basisfor the application of sequential photobleaching of fluoro-chromes next to very photostable dyes to increase the numberof parameters captured. In this issue, Wessels et al. (page 420)analyzed the newly available NorthernLights secondary anti-bodies for use in slide-based cytometry and microscopy. Theypointed out that since the NorthernLights are bright, resistantto photobleaching, stable in alcohols and xylene, these dyesare promising candidates for use with most laser- and HBO/XBA-based fluorescence microscopy techniques. It is antici-pated that combination of NorthernLights with laser scanningcytometry could provide a better methodology for analysis oftumor dissemination to bone marrow and lymph node (6).The other group of fluorescent probes aims to stain cellorganelles with high specificity. Development of fluorescent,organelle-targeted probes has been driven by an interest in dis-covering new probes that excite and emit in the visible spec-

Uptake, Subcellular Localization, Bioenergetic Effects and Antiproliferative Properties of Triphenylphosphoniumlinked Nitroxides in Breast Cancer Cells

Uptake, Subcellular Localization, Bioenergetic Effects and Antiproliferative Properties of Triphenylphosphoniumlinked Nitroxides in Breast Cancer Cells

Subcellular localization and lysophospholipase/transacylation activities of human group IVC phospholipase A2 (cPLA2γ)

cPLA2γ was identified as an ortholog of cPLA2α, which is a key enzyme in eicosanoid production. cPLA2γ was reported to be located in endoplasmic reticulum (ER) and mitochondria and to have lysophospholipase activity beside phospholipase A2 (PLA2) activity. However, subcellular localization, mechanism of membrane binding, regulation and physiological function have not been fully established. In the present study, we examined the subcellular localization and enzymatic properties of cPLA2γ with C-terminal FLAG-tag. We found that cPLA2γ was located not only in ER but also mitochondria even in the absence of the prenylation. Purified recombinant cPLA2γ catalyzed an acyltransferase reaction from one molecule of lysophosphatidylcholine (LPC) to another, forming phosphatidylcholine (PC). LPC or lysophosphatidylethanolamine acted as acyl donor and acceptor, but lysophosphatidylserine, lysophosphatidylinositol and lysophosphatidic acid (LPA) did not. PC and phosphatidylethanolamine (PE) also acted as weak acyl donors. Reaction conditions changed the balance of lysophospholipase and transacylation activities, with addition of LPA/PA, pH > 8, and elevated temperature markedly increasing transacylation activity; this suggests that lysophospholipase/transacylation activities of cPLA2γ may be regulated by various factors. As lysophospholipids are known to accumulate in ischemia heart and to induce arryhthmia, the cPLA2γ that is abundant in heart may have a protective role through clearance of lysophospholipids by its transacylation activity.

Alternatively Spliced Isoforms of TRIP8b Differentially Control h Channel Trafficking and Function

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels (h channels) are the molecular basis for the current, I(h), which contributes crucially to intrinsic neuronal excitability. The subcellular localization and biophysical properties of h channels govern their function, but the mechanisms controlling these characteristics, and especially the potential role of auxiliary subunits or other binding proteins, remain unclear. We focused on TRIP8b, an h channel-interacting protein that colocalizes with HCN1 in cortical and hippocampal pyramidal neuron dendrites, and found that it exists in multiple alternative splice variants with distinct effects on h channel trafficking and function. The developmentally regulated splice variants of TRIP8b all shared dual, C terminus-located interaction sites with HCN1. When coexpressed with HCN1 in heterologous cells individual TRIP8b isoforms similarly modulated gating of I(h), causing a hyperpolarizing shift in voltage dependence of channel activation, but differentially upregulated or downregulated I(h) current density and HCN1 surface expression. In hippocampal neurons, coexpression of TRIP8b isoforms with HCN1 produced isoform-specific changes of HCN1 localization. Interestingly, the TRIP8b isoforms most abundant in the brain are those predicted to enhance h channel surface expression. Indeed, shRNA knockdown of TRIP8b in hippocampal neurons significantly reduced native I(h). Thus, although TRIP8b exists in multiple splice isoforms, our data suggest that the predominant role of this protein in brain is to promote h channel surface expression and enhance I(h). Because I(h) expression is altered in models of several diseases, including temporal lobe epilepsy, TRIP8b may play a role in both normal neuronal function and in aberrant neuronal excitability associated with neurological disease.

Crystal structure of human carbonic anhydrase‐related protein VIII reveals the basis for catalytic silencing

Carbonic anhydrases (CAs; EC 4.2.1.1) catalyze the reversible hydration of carbon dioxide to bicarbonate and a proton.1 They are ubiquitous in prokaryotes and eukaryotes, and are encoded by four evolutionarily-unrelated gene classes (a, b, g, d). The human CA family belongs to the a class and contains 12 catalytic isozymes with different tissue distribution, subcellular localization and kinetic properties. These include the cytosolic (CA I, CA II, CA III, CA VII, CA XIII), membrane-bound (CA IV, CA IX, CA XII, CA XIV), mitochondrial (CA VA, CA VB) and secreted (CA VI) isoforms. They share an absolute requirement for a catalytic Zn ion in the active site, coordinated by an hydroxide ion and three invariant histidine residues H94CAII, H96CAII and H119CAII (human CA II numbering) that are in turn hydrogen bonded to conserved partners Q92CAII, N244CAII and E117CAII, respectively. CAs participate in physiological processes such as respiration, metabolite biosynthesis and pH regulation, and are interesting pharmaceutical targets.2 Sulphonamide-based CA inhibitors are established diuretics and anti-glaucoma drugs, and may have further therapeutic potentials in anti-obesity and anti-cancer treatment.3 The human CA family includes a subclass of three noncatalytic isoforms (CA VIII, CA X, CA XI), also known as CA-related proteins (CA-RPs), based on sequence homology with the catalytic isozymes. CA-RPs lack one or more of the essential Zn-coordinating histidines and are devoid of CO2 hydration activity.4 To date, the biological functions of CA-RPs remain undefined. The first identified CA-RP, CA VIII, replaces the Zn-coordinating H94CAII and its hydrogen-bonding partner Q92CAII with R116 and E114, respectively (human CA VIII numbering).5 CA VIII is highly expressed in the cerebellum,6 and a mouse gene deletion causes a motor coordination defect.7 Relevant to this, CA VIII has been identified as a binding partner for the inositol 1,4,5 triphosphate (IP3) receptor type 1 which is abundant in the cerebellum.8 To provide insights into the biological properties of CA-RPs, we have determined the 1.6 A crystal structure of human CA VIII (hCA VIII). This work represents the first structural characterization of a CA-RP and offers a structural basis for its catalytic silencing effect.

Subcellular localization and enzymatic properties of differentially expressed transketolase genes isolated from the desiccation tolerant resurrection plant Craterostigma plantagineum

The desiccation tolerant resurrection plant Craterostigma plantagineum encodes three classes of transketolase transcripts, which are distinguished by their gene structures and their expression patterns. One class, represented by tkt3, is constitutively expressed and two classes, represented by tkt7 and tkt10, are upregulated upon rehydration of desiccated C. plantagineum plants. The objective of this work was to characterize the differentially expressed transketolase isoforms with respect to subcellular localization and enzymatic activity. Using GFP fusion constructs and enzymatic activity assays, we demonstrate that C. plantagineum has novel forms of transketolase which localize not to the chloroplast, but mainly to the cytoplasm and which are distinct in the enzymatic properties from the transketolase enzymes active in the Calvin cycle or oxidative pentose phosphate pathway. A transketolase preparation from rehydrated leaves was able to synthesize the unusual C8 carbon sugar octulose when glucose-6-phosphate and hydroxy-pyruvate were used as acceptor and donor molecules in in vitro assays. This suggests that a transketolase catalyzed reaction is likely to be involved in the octulose biosynthesis in C. plantagineum.

Syntheses and Properties of a Series of Cationic Water-Soluble Phthalocyanines

A series of symmetrical cationic phthalocyanines (Pcs) with either Zn(II) or Si(IV) metal ions and two bulky axial ligands on the silicon complexes was synthesized in high yields. The photophysical (absorption, emission, fluorescence, and singlet oxygen quantum yields) and cellular (uptake, toxicity, and subcellular localization) properties of this series of Pcs were investigated. The Si(IV)-Pcs exist mainly as monomers in aqueous media and have higher fluorescent quantum yields in protic solvents (methanol and water) than the Zn(II)-Pcs. The presence of eight short PEG groups at the periphery of a Zn(II)-Pc significantly increases its solubility in protic solvents, but a centrally chelated silicon ion and associated bulky axial ligands were more efficient in preventing aggregation of the Pc macrocycles. The singlet oxygen quantum yields for this series of Pcs in DMSO are in the range 0.09-0.15. All Pcs were readily taken up by human HEp2 cells, and the extent of their accumulation within cells depends on their hydrophobic character. Intracellularly, all Pcs localized preferentially within the cell lysosomes. The Zn(II)-Pc 11 and Si(IV)-Pcs 12 and 14 were found to be the most phototoxic (IC50 = 2.2 microM at a 1 J cm(-2) light dose) of this series of compounds.

Sodium channel Scn1b null mice exhibit prolonged QT and RR intervals

In neurons, voltage-gated sodium channel β subunits regulate the expression levels, subcellular localization, and electrophysiological properties of sodium channel α subunits. However, the contribution of β subunits to sodium channel function in heart is poorly understood. We examined the role of β1 in cardiac excitability using Scn1b null mice. Compared to wildtype mice, electrocardiograms recorded from Scn1b null mice displayed longer RR intervals and extended QT c intervals, both before and after autonomic block. In acutely dissociated ventricular myocytes, loss of β1 expression resulted in a ∼ 1.6-fold increase in both peak and persistent sodium current while channel gating and kinetics were unaffected. Na v1.5 expression increased in null myocytes ∼ 1.3-fold. Action potential recordings in acutely dissociated ventricular myocytes showed slowed repolarization, supporting the extended QT c interval. Immunostaining of individual myocytes or ventricular sections revealed no discernable alterations in the localization of sodium channel α or β subunits, ankyrin B, ankyrin G, N-cadherin, or connexin-43. Together, these results suggest that β1 is critical for normal cardiac excitability and loss of β1 may be associated with a long QT phenotype.

The Importance of N-terminal Polycysteine and Polybasic Sequences for G14α and G16α Palmitoylation, Plasma Membrane Localization, and Signaling Function

Plasma membrane targeting of G protein alpha (Galpha) subunits is essential for competent receptor-to-G protein signaling. Many Galpha are tethered to the plasma membrane by covalent lipid modifications at their N terminus. Additionally, it is hypothesized that Gq family members (Gqalpha,G11alpha,G14alpha, and G16alpha) in particular utilize a polybasic sequence of amino acids in their N terminus to promote membrane attachment and protein palmitoylation. However, this hypothesis has not been tested, and nothing is known about other mechanisms that control subcellular localization and signaling properties of G14alpha and G16alpha. Here we report critical biochemical factors that mediate membrane attachment and signaling function of G14alpha and G16alpha. We find that G14alpha and G16alpha are palmitoylated at distinct polycysteine sequences in their N termini and that the polycysteine sequence along with the adjacent polybasic region are both important for G16alpha-mediated signaling at the plasma membrane. Surprisingly, the isolated N termini of G14alpha and G16alpha expressed as peptides fused to enhanced green fluorescent protein each exhibit differential requirements for palmitoylation and membrane targeting; individual cysteine residues, but not the polybasic regions, determine lipid modification and subcellular localization. However, full-length G16alpha, more so than G14alpha, displays a functional dependence on single cysteines for membrane localization and activity, and its full signaling potential depends on the integrity of the polybasic sequence. Together, these findings indicate that G14alpha and G16alpha are palmitoylated at distinct polycysteine sequences, and that the adjacent polybasic domain is not required for Galpha palmitoylation but is important for localization and functional activity of heterotrimeric G proteins.

Mechanism of Diacylglycerol-induced Membrane Targeting and Activation of Protein Kinase Cθ

Protein kinase C (PKC) is a novel PKC that plays a key role in T lymphocyte activation. PKC has been shown to be specifically recruited to the immunological synapse in response to T cell receptor activation. To understand the basis of its unique subcellular localization properties, we investigated the mechanism of in vitro and cellular sn-1,2-diacylglycerol (DAG)-mediated membrane binding of PKC. PKC showed phosphatidylserine selectivity in membrane binding and kinase action, which contributes to its translocation to the phosphatidylserine-rich plasma membrane in HEK293 cells. Unlike any other PKCs characterized so far, the isolated C1B domain of PKC had much higher affinity for DAG-containing membranes than the C1A domain. Also, the mutational analysis indicates that the C1B domain plays a predominant role in the DAG-induced membrane binding and activation of PKC. Furthermore, the Ca(2+)-independent C2 domain of PKC has significant affinity for anionic membranes, and the truncation of the C2 domain greatly enhanced the membrane affinity and enzyme activity of PKC. In addition, membrane binding properties of Y90E and Y90F mutants indicate that phosphorylation of Tyr(90) of the C2 domain enhances the affinity of PKC for model and cell membranes. Collectively, these results show that PKC has a unique membrane binding and activation mechanism that may account for its subcellular targeting properties.

Distinct roles of TIR and non-TIR regions in the subcellular localization and signaling properties of MyD88

MyD88 is a cytoplasmic adaptor protein that is critical for Toll-like receptor (TLR) signaling. The subcellular localization of MyD88 is characterized as large condensed forms in the cytoplasm. The mechanism and significance of this localization with respect to the signaling function, however, are currently unknown. Here, we demonstrate that MyD88 localization depends on the entire non-TIR region and that the correct cellular targeting of MyD88 is indispensable for its signaling function. The Toll-interleukin I receptor-resistance (TIR) domain does not determine the subcellular localization, but it mediates interaction with specific TLRs. These findings reveal distinct roles for the TIR and non-TIR regions in the subcellular localization and signaling properties of MyD88.

Functional genetic analysis of mutations implicated in a human speech and language disorder

Mutations in the FOXP2 gene cause a severe communication disorder involving speech deficits (developmental verbal dyspraxia), accompanied by wide-ranging impairments in expressive and receptive language. The protein encoded by FOXP2 belongs to a divergent subgroup of forkhead-box transcription factors, with a distinctive DNA-binding domain and motifs that mediate hetero- and homodimerization. Here we report the first direct functional genetic investigation of missense and nonsense mutations in FOXP2 using human cell-lines, including a well-established neuronal model system. We focused on three unusual FOXP2 coding variants, uniquely identified in cases of verbal dyspraxia, assessing expression, subcellular localization, DNA-binding and transactivation properties. Analysis of the R553H forkhead-box substitution, found in all affected members of a large three-generation family, indicated that it severely affects FOXP2 function, chiefly by disrupting nuclear localization and DNA-binding properties. The R328X truncation mutation, segregating with speech/language disorder in a second family, yields an unstable, predominantly cytoplasmic product that lacks transactivation capacity. A third coding variant (Q17L) observed in a single affected child did not have any detectable functional effect in the present study. In addition, we used the same systems to explore the properties of different isoforms of FOXP2, resulting from alternative splicing in human brain. Notably, one such isoform, FOXP2.10+, contains dimerization domains, but no DNA-binding domain, and displayed increased cytoplasmic localization, coupled with aggresome formation. We hypothesize that expression of alternative isoforms of FOXP2 may provide mechanisms for post-translational regulation of transcription factor function.