WO1998049271A1

WO1998049271A1 - Human neuronal cell line

Info

Publication number: WO1998049271A1
Application number: PCT/US1998/007224
Authority: WO
Inventors: Marke Cockett; Andrew Wood; Myles Fennell
Original assignee: American Home Products Corp
Current assignee: Wyeth LLC
Priority date: 1997-04-25
Filing date: 1998-04-13
Publication date: 1998-11-05
Anticipated expiration: 1999-10-25
Also published as: AU6897498A

Abstract

NTera 2/D1 (NT2) precursor cells were transfected with human β2 adrenergic receptor under the control of a neuron-specific synapsin-1 promoter. Stably transfected precursor cells did not display elevated expression of the β2 adrenergic receptor and no change was seen in the receptor coupled generation of cAMP. Upon differentiation with RA, the transfected cells displayed elevated cAMP production induced by adrenergic receptor agonists. Expression of the β2 adrenergic receptor in the neuronal NT2 cells resulted in a significantly enhanced level of neuronal differentiation compared to the untransfected NT2 cell as evidenced by the neuronal marker MAP2.

Description

Human Neuronal Cell Line

Field of the Invention The present invention relates to immortalized cell lines. More particularly, the present invention relates to immortalized human testicular teratocarcinoma cell lines and cell lines derived therefrom.

Background

Mature mammalian neurons are a terminally differentiated phenotype and are incapable of undergoing cell division. Thus continuously dividing cell lines with certain neuronal characteristics have proven very useful in the study of the nervous system. Such lines allow for the manipulation of homogeneous populations through gene transfer to yield novel derivatives expressing foreign gene products. This has led to the development of a large variety of neuronal cell lines, some of which have been useful for ceil biological, biochemical and molecular biological studies. The utility of these cell lines is primarily related to how closely their characteristics mimic that of post-mitotic neurons in vivo and also the level of difficulty involved in generating large numbers of pure neurons. Dividing neuronal cell lines usually do not possess the phenotypic properties of terminally differentiated neurons. Many of these cell lines elaborate a phenotype similar to immature neurons and neuronal precursor cells, however, since these cells are rapidly dividing they are useful for biochemical and cell transfection assays. Many neuronal cell types of the central and peripheral nervous systems fall into this category (e.g., neuroblastomas, pheochromocytomas and medulloblastomas). Other cell lines displaying some of the characteristics of post-mitotic neurons have a very slow doubling time and are therefore not amenable to many experimental manipulations (Ronnet, 1 990). It is possible to isolate neuronal precursor cells from the developing nervous system, which can be cultured for a limited period of time as a cell line before maturing to a more neuronal phenotype (Gensburger, 1 987). This approach has been very useful in the elucidation of mechanisms involved in the development of a neuronal phenotype. An ideal cell line in which to study neuronal differentiation and still be amenable to large scale biochemical and molecular biological manipulation would be a cell line that grows as a homogeneous, rapidly dividing stem cell population. Upon treatment with an appropriate stimulus this cell type would then differentiate into a population of pure post-mitotic neurons displaying properties the same as primary post-mitotic neurons in culture.

It has been demonstrated that embryonal teratocarcinoma cells are able to satisfy some of the above criteria. These cells consist of undifferentiated multipotent cells which have the ability to differentiate into several cell types under certain conditions (usually involving treatment with retinoic acid (RA)). This process is thought to resemble the actual commitment to different phenotypes found in vivo. The phenotype of these cells often resemble neurons, glia, muscle and endothelial cells at various developmental stages. This heterogeneity has often limited there usefulness in studying any one phenotype. NTera 2/D1 (NT2) described in U.S. patent 51 75103 by Lee et al. is a human teratocarcinoma cell line derived from a lung metastisis of a testicular teratocarcinoma which when treated with RA yields a population of post- mitotic neurons which can be purified from other cell types present in the culture. Prior studies with this cell line have demonstrated that NT2 cells of both the stem cell and neuronal phenotype can be transfected with an expression plasmid for an exogenous gene product, i.e. beta- galactosidase. In certain circumstances it would be useful to restrict expression of an exogenous gene product exclusively to the differentiated neuronal phenotype. An exogenous gene of interest to study in the NT2 cell line is the β2 adrenergic receptor, since previous studies have indicated that this gene plays an important role in the development of the CNS. The adenylate cyclase signaling pathway has also been shown to play an important trophic role in neuronal development.

Neurotrophic factors are essential for the development and maintenance of a mature neuronal phenotype. Two of these factors, basic fibroblast growth factor (bFGF) and nerve growth factor (NGF) have been shown to be upregulated as a result of b-adrenergic receptor activation in the rat central nervous system (Walicke, 1 988; Whittemore and Seiger, 1 987). Expression of NGF is restricted primarily to neurons whereas bFGF is restricted to glial ceils (Follesa and Mocchetti, 1 993;

Hayes et al., 1 995). There is strong evidence that this gene expression is mediated through the transcription factor c-fos, which enhances the expression of a family of genes that contain a seven-base consensus promoter sequence known as AP-1 (Chiu et al., 1 988) . Cyclic AMP plays a significant role in the terminal differentiation of several cell types. In the development of noradrenergic neurons the cAMP analogue dibutyryl cAMP has been shown to increase the differentiation of the noradrenergic phenotype (Sklair-Tavron and Segal, 1 993). It has also been demonstrated that human prostate carcinoma cells undergo neuroendocrine terminal differentiation in response to cAMP (Bang et al., 1 994).

Thus, it would be desirable to provide an NT2 cell line capable of restricting β2 adrenergic receptor expression to differentiated neuronal cells in order to promote the differentiation of this phenotype in preference to any other. Summary of the Invention

Thus, it is the object to provide an improved NT2 cell line, wherein said cell line is stably transfected with a gene encoding the human β2 adrenergic receptor expressed exclusively in the neurons to facilitate functional studies in the NT2 cell line. Expression of the β2 adrenergic receptor is under the control of a neuronal-specific regulatory element in the differentiated NT2 cell line. This cell line has the identifying characteristics of ATCC CRL-1 2356.

Another object of the present invention is to provide a method for producing a stable population of post-mitotic human neurons expressing β2 adrenergic receptor comprising transfecting at least one plasmid comprising a neuron-specific promoter operabiy linked to the human β2 adrenergic receptor into cultured NT2 cells; culturing the transfected NT2 cells with retinoic acid to obtain a multi-layer culture; dispersing the cultured cells; and culturing the dispersed cell with cytosine arabinoside, flurodeoxyuridine and uridine to yield highly differentiated post-mitotic cells expressing the β2 adrenergic receptor. Brief Description of the Drawings

FIGURE 1 a: Generation of cAMP in untransfected and transfected precursor NT2 cells following a 1 5 minute stimulation with 100 nM isoproterenol. Breakdown of cAMP was blocked by IBMX (3-isobutyl-1 - methylxanthine). Results are represented as pmol per mg protein. Errors are standard error of the mean; n = 6

FIGURE 1 b: Generation of cAMP in untransfected and transfected neuronal NT2 cells following a 1 5 minute stimulation with 100 nM isoproterenol. Breakdown of cAMP was blocked by IBMX. Results are represented as pmol per mg protein. Errors are standard error of the mean; n = 6.

FIGURE 2a: Immunocytochemical results demonstrating the increase in differentiation in neuronal NT2 cell expressing the β-adrenergic receptor as compared to untransfected NT2 cells. Photographs show an increased level of expression of neuronal marker MAP-2 in transfected differentiated NT2-N cells versus the untransfected.

FIGURE 2b: Counts of cells displaying positive staining for MAP-2 in untransfected cells and cells transfected with the human β2 adrenergic receptor. Description of the Invention

A human teratocarcinoma cell line (NTera 2/DI or NT2 cells) was transfected with a plasmid comprising a neuronally restricted promoter operably linked to β-adrenergic receptor, followed by treatment with retinoic acid (RA) to yield highly differentiated pure cultures of neuronal cells (hereinafter NT2-N cells).

Functional expression of human β-adrenergic receptors exclusively in NT2-N cells was achieved by operably linking cDNA encoding the receptor to a promoter region of a neuronal specific protein. Neuronal specific promoters include, but are not limited to, the synapsin I promoter, the synapsin II promote , tyrosine hydroxylase promoter, dopamine β-hydroxylase promoter, neuron-specific enolase promoter, hypoxanthine phosphoribosyltransferase promoter, low affinity NGF receptor promoter, and choline acetyl transferase promoter (Bejanin et al., 1 992; Carroll et al., 1 995; Chin and Greengard, 1 994; Foss-Petter et al., 1 990; Harrington et al., 1 987; Mercer et al., 1 991 ; Patei et al., 1 986). Preferred neuronal specific proteins are the synapsins. The synapsins are a family of phosphoproteins that selectively bind the small synaptic vesicles in the presynaptic nerve terminal. Human synapsin I was functionally analyzed to identify control elements directing neuron specific expression of synapsin I (Benfenati et al., 1 989). Previous studies indicate that the proximal region of the synapsin I promoter is sufficient for directing neuron-specific gene expression. This proximal region is highly conserved between rodent and human. The truncated rat synapsin- 1 promoter, containing positive regulatory elements, has previously been used to positively regulate the expression of chloramphenicol acetyltransferase (CAT) in PC1 2 cells which display a neuronal phenotype (Howland et al., 1991 ). In the present invention, the rat synapsin I promoter was used to restrict the expression of the human β2 adrenergic receptor to the neuronal differentiated phenotype of NT2 cells. Synapsin-I regulation resulted in an increased level of β-2 adrenergic receptor expression in the neuronal phenotype and an unexpected increase in the level of neuronal differentiation of NT2 cells.

Untransfected and transfected precursor NT-2 cells display the same level of β receptor expression as demonstrated by the level of cAMP production in Example 4 and Figure 1 a and the measurement of receptor binding sites in Example 6 and Table 1 . Upon differentiation with RA the transfected cells displayed elevated β2 adrenergic receptor levels (Table 1 ) and an elevated coupling to cAMP production (Figure 1 b). The level of β1 adrenergic receptors remained unchanged between transfected and untransfected NT2 precursors and neurons. Expression of the β2 adrenergic receptor in the neuronal NT2 cells resulted in an enhanced level of neuronal differentiation compared to the untransfected NT2 cells. The extent of neuronal differentiation of untransfected and transfected cells was assessed by immunocytochemistry using an antibody for the neuronal cytoskeletal protein, microtubular-associated protein (MAP-2) as a marker (Figure 2a) and by counting the number of MAP-2 positive cells at various stages of differentiation in wild type and transfected cultures (Example 5 and Figure 2b). The earliest time point at which MAP-2 staining was detectable in both untransfected and transfected cells was 10 days. At this point short processes were visible, with some staining also within the cell body. A further ten days of continuous treatment with RA resulted in an increase in the number of MAP-2 positive cells in the transfected cell. The length of the cell processes increased more rapidly in the transfected cells compared to the untransfected cells up to 20 days of retinoic acid treatment when numerous MAP-2 positive cells with long processes were visible. There was no apparent increase in the MAP-2 staining between 20 and 25 days of retinoic acid treatment in either population of cells. Due to the increased proportion of neurons from the cultures expressing the β2 adrenergic receptor it was significantly easier to produce large numbers of relatively pure neuronal cultures compared to the untransfected cells using the previously published protocol (Andrews et al., 1 984). Thus, the present invention provides a method for producing a stable population of post-mitotic human neurons expressing β-adrenergic receptors.

To examine the validity of directed expression of the β receptor to the neurons, NT2 precursor cells derived from the same source used to generate the transfected cell lines were cloned out so that colonies were cultured from a single cell. There was no apparent difference in the ability of any of these clones to differentiate to a neuronal phenotype in response to retinoic acid. Thus, it appears that the enhanced differentiation seen with the cells expressing the β2 adrenergic receptor is not due to any change induced by the cloning procedure.

To demonstrate the restricted expression of the β receptor in neurons, the levels of cAMP production and the b receptor density in precursor and post-mitotic cells were examined (Example 4 and 6). Undifferentiated transfected NT2 displayed a functional cAMP response to the β receptor agonist isoproteronol similar to that of untransfected NT2 precursors with a 1 5 minute stimulation (Figure 1 a). The amount of cAMP generated was determined using a scintillation proximity assay. When the cells were differentiated with RA and purified to yield pure neuronal cultures the untransfected NT2-N cells had a similar response to isoproteronol as was seen with the precursor cells. However, neurons expressing the β2 adrenergic receptor controlled by the synapsin promoter displayed an elevated cAMP response under the same conditions (Figure 1 b). Undifferentiated wild type and transfected precursors display the same density of β1 and β2 receptors. However, upon differentiation with retinoic to a neuronal phenotype there was an elevation in the β2 adrenergic receptor density in transfected cells compared with wild type, whereas the β1 receptor density was unchanged (Example 6 and Table 1 ). The promoter controlling β2 adrenergic receptor expression was inactive in the non-neuronal precursor cells, as demonstrated by the fact that there was no difference in the cAMP accumulation in response to the adrenergic receptor agonist isoproterenol in the untransfected or transfected precursors. Upon RA treatment, the rate of neuronal differentiation and the proportion of cells staining for MAP-2 was greater in the transfected or transfected cell line as compared to the untransfected cell line.

In accordance with the present invention, the truncated synapsin promoter elevated β2 adrenergic receptor expression only when the cells have developed a neuronal phenotype. If all cells showing initial signs of neuronal development continued to become mature neurons, it would be expected that there would be an equal number of neurons in both untransfected and transfected cultures. At the earliest point of detectable MAP-2 staining there was an equal number of MAP-2 positive cells in both wild type and transfected cells. However, at later time points the transfected line displayed a greater number of cells developing into mature neurons (Table 1 ). Therefore the overexpression of the β2 adrenergic receptor at a point when the developing neurons may not be fully committed to a neuronal phenotype appears to result in this phenotype, either through the enhanced survival of neuronal precursors in the culture or by preventing a population of precursors which would otherwise display more plasticity from developing into other phenotypes. Adrenergic stimulation of developing neurons in the developing CNS has been shown to have an important trophic effect, acting through cAMP, the transcription factor c-fos and ornithine decarboxylase, an enzyme important for the production of polyamines, which are obligatory for differentiation (Wagner et al., 1 994).

The ability to induce β-adrenergic receptor expression in the NT2-N human cell line is significant for several reasons. First, it provides an opportunity to study the effect of adrenergic receptor gene expression on neuronal differentiation in a model system that is both clonal and neuronal. Second, this line is amenable to molecular biological manipulations such as stable transfection and subcloning. Third, because of the human origin of the receptor and the cells and because the precursor cells are capable of rapid proliferation, the present invention provides an unlimited homogenous population of cells for the study of the β2 adrenergic receptor in relation to human neurological disorders. Thus, in accordance with the present invention, the effects of a stimulation by an agonist may be observed by measuring the cellular response of cell expressing the β-receptor. For example, cAMP levels were measured as in Example 4. Results shown in Figure 1 demonstrate that the population of adrenergic receptors is much the same in both the untransfected and transfected NT2 precursor cells since there is no significant difference in the amount of cAMP generated by isoproterenol. Thus, the truncated synapsin promoter is not driving significant levels of β2 adrenergic receptor expression in non-neuronal cells. In untransfected NT2 neurons the level of cAMP generation stimulated by isoproterenol was similar to that seen in precursor cells. However, in the transfected neurons expressing the β2 adrenergic receptor regulated by the synapsin promoter there was an elevated level of cAMP produced in response to isoproterenol. These observations are further evidence that the truncated synapsin promoter is only inducing gene expression after the cells have begun their differentiation to a neuronal phenotype.

During the differentiation process no additional adrenergic receptor agonists were added to the cell culture medium. It is possible that adrenergic receptor agonists were present in the fetal bovine serum used to supplement the growth medium, or that agonists were released by the cells during the course of the differentiation. Another possible explanation is that the receptors are able to maintain an activated state in the absence of ligand. It has been demonstrated that the β2 adrenergic receptor exists in two states in the absence of ligand, one of which is active (Bond et al., 1 995). It may be that this basal receptor activity, with an increased number of receptors is responsible for the enhanced neuronal differentiation seen in the transfected cultures. The overexpression of the human β2 adrenergic receptor under the control of the neuronally restricted synapsin promoter resulted in an enhancement in the proportion of NT2 precursor cells differentiating to a neuronal phenotype in response to retinoic acid. Thus, in accordance with the present invention, there is provided an immortalized neuronal NT2 cell line for functionally studying the effect of the human β2 adrenergic receptor in human neurons.

This study provides an insight into the importance of neurotransmitter signaling mechanisms in providing developmental and trophic support in the developing CNS. It also provides a useful mechanism for enhancing the yield of neurons from cultures of NT2 cells, which often requires an extensive purification procedure for a low yield of purified neurons.

Indeed, the transfection of β2 adrenergic receptor with other constructs into NT2 cells that are then induced to differentiate into stable, post-mitotic neurons, may be useful as a novel delivery system for bioactive molecules in human neurodegenerative disorders.

The present invention also provides methods for producing a stable population of post-mitotic human neurons expressing exogenous β-receptor gene products comprising transfecting one or more plasmids into cultured undifferentiated human teratocarcinoma cells; culturing said undifferentiated human teratocarcinoma cells with retinoic acid to obtain a heterogeneous culture; dispersing said cultured cells; and culturing said dispersed cells with a mitotic inhibitor or a combination of mitotic inhibitors. It is contemplated that a single selected expression vector may be transfected and expressed in the stable population of post-mitotic cells.

The present invention is further illustrated by the following examples, which are not intended to be limiting in any way. Examples Example 1 : Cell Culture

NT2 precursor cells were maintained in OPTIMEM with Glutamax-1 (GIBCO, Gaithersburg, MD), supplemented with 5 % fetal bovine serum (GIBCO) and 0.5% penicillin/streptomycin (GIBCO, Gaithersburg, MD)as previously described (Andrews, 1 984). For neuronal differentiation, cells were plated at a density of 2.66x10⁴ /cm² and treated with growth medium containing retinoic acid (RA) ( 10^"5M) (Sigma, St. Louis, MO.) every two days for a 21 -25 day period. After this period of differentiation, the cells were separated by trypsinisation followed by repeated pipetting with a 10 ml tissue culture pipette to break up clumps of cells. The cells then went through two differential adhesion procedures in tissue culture flasks (Falcon) in order to separate neuronal and non- neuronal cells. After this differential adhesion the cells were plated into either a multi-well plate or flask coated with poly-D-lysine (0.01 %; Sigma) and laminin (5μg/ml; Collaborative Bioscience). This final plating medium was OptiMEM described above supplemented with cytosine b-D- arabinofuranoside ( 1 /M; Sigma), 5'-fluoro 2'-deoxyuridine ( 10 /M; Sigma) and uridine ( 10μM; Sigma). Cells were allowed to mature for 10 days in a low oxygen tension incubator (9% 0₂) before being used.

Example 2: Construction of β-Adrenerqic Receptor Expression Vector

Human β2 adrenergic receptor cDNA was inserted into a mammalian expression vector pWE1 (Cockett et al., 1 997) between the unique Xbal and BamHI restriction endonuclease cleavage sites. The hCMV promoter was removed by digestion with Mlul and Hindlll, and the resulting DNA was religated with adaptors of the sequence 5'- CGCGTGACGA-3' and 5'-AGCTTCGTCA-3' to generate a promoterless vector. A 459bp Avail to BamHI rat Synapsin I gene promoter (Howland et al., 1 991 ) was then inserted into the unique Hindlll site using adaptors of the sequence 5'-GACGGCGAAGCTTCGCC-3' and 5'- GATCCAAGCTTG-3' to generate the vector pWE-Synβ2. Example 3: Transfection of IMT2 Cells with pWE-Svnβ2.

Plasmid pWE-Synβ2 (40μg) was linearised using Pvu-1 . Approximately 10⁷ NT2 precursor cells were resuspended in 1 ml ice cold PBS in an electroporation cuvette (BioRad). Plasmid was added to the cells and left on ice for 1 minute before electroporation (BioRad Gene Pulsar) (single pulse, 960 μF, 200mV) . Following electroporation cells were left on ice for 5 minutes and then diluted in 30 ml growth medium with dialyzed serum followed by two further 1 /2 dilutions. Cells were then plated in 96 well plates. Selection was started the day after transfection by the addition of 2x GPT (xanthine-guanine phospororibosyltransferse) selection medium as previously described (Kriegler, 1 990). Colonies were removed from the plates after 3-4 weeks and expanded through larger wells and flasks. Example 4: cAMP Response in Transfected and Untransfected Cells Transfected and untransfected neuronal cells were plated at a density of 1 x10 /cm² 10 days prior to the assay in medium containing dialyzed serum. Cells were allowed to equilibrate in Krebs buffer for 1 5 in prior to treatment with isoproteronol and all stimulations were for 1 5 min. Breakdown of cAMP was prevented by the addition of 3-isobytyl-1 - methylxanthine (IBMX) (Research Biochemicals International, Natick, MA). Reactions were terminated by the addition of perchloric acid (0.03M final concentration). Protein content was determined by adding an equal volume of 1 M NaOH to control wells and using BioRad protein assay to determine protein content, all results were corrected for protein. The amount of cAMP generated was determined using an ¹²⁵l scintillation proximity assay (Amersham International) (non-acetylation assay). Results are shown in Figure 1 . Precursor NT2 ceils transfected with pWE-Synβ2 displayed a functional cAMP response to the β receptor agonist isoproteronol similar to that of untransfected NT2 precursors with a 1 5 minute stimulation with isoproteronol. The amount of cAMP generated was determined using a scintillation proximity assay described above (Figure 1 (a)). When the cells were differentiated with RA and purified to yield pure neuronal cultures the untransfected NT2 cells had a similar response to isoproteronol as was seen with the precursor cells, however neurons expressing the β2 adrenergic receptor controlled by the synapsin promoter displayed an elevated cAMP response under the same conditions (Figure Kb)). Example 5: Immunocytochemical Staining of MAP-2 in Transfected Cells

The extent of neuronal differentiation of untransfected and transfected cells was assessed by immunocytochemistry using an antibody for the neuronal cytoskeletal protein MAP-2 (Figure 2).

Transfected and untransfected NT2 cells were grown on poly-D-lysine (0.01 %) and laminin (5μg/ml) coated coverslips (25mm diameter) in the presence of retinoic acid ( 10μM) for different lengths of time. Cells were fixed with 4% paraformaldehyde for 1 hr at room temperature, then washed with PBS/glycine (10mM). Cells were blocked with 0.3% Triton X-100 (Sigma) and 2% horse serum in PBS for 1 hr. Cells were probed with primary antibody (MAP-2, mouse monoclonal (clone AP20)) (Boehringer Mannheim) at a concentration of 5μg/ml in 0.3% Triton X- 100, 1 % BSA in PBS for 2 hr at room temperature, followed by probing with secondary antibody (Vector Labs) at a concentration of 5μg/ml in 0.3% Triton X-100, 3% BSA in PBS for 1 hr at room temperature. This was followed by treatment with fluorescein/ strepavidin (5μg/ml) in PBS for 30 min at room temperature. Cells were washed three times with PBS for 5 min between each of these steps. Coverslips were mounted on slides with Prolong antifade (Molecular Probes) and photographs were taken on a Zeiss 100 Axiovert on Provia 1 600 slide film. Results are shown in Figure 2.

The earliest time point at which MAP-2 staining was detected in both untransfected and transfected cells was 10 days. At this point short processes were visible, with some staining also within the cell body.

After a further ten days continued retinoic acid treatment, the number of MAP-2 positive cells and the length of the cell processes increased more rapidly in the transfected cells compared to the untransfected cells up to 20 days of retinoic acid treatment when numerous MAP-2 positive cells with long processes were visible. There was no apparent increase in the MAP-2 staining between 20 and 25 days of retinoic acid treatment in either population of cells. Due to the increased proportion of neurons from the cultures expressing the β-2 adrenergic receptor it was significantly easier to produce large numbers of relatively pure neuronal cultures compared to the untransfected cells using the previously published protocol (Andrews et al., 1 984).

Example 6: Measurement of specific β1 and β2 adrenoceptor binding sites in IMT-2 clones Twenty-five μl of [^ 25|]_Cy_an0pj_nc|₀|₀| ( - 35-40 pM final concentration) was incubated with 400 μl of NT-2 clone membrane preparations in the absence (total binding) or presence (non-specific binding) of 25 μl of a β1 antagonist ( 1μM metoprolol) or aβ-2 antagonist ( 1μM ICI 1 1 8551 ) . Incubations were performed at room-temperature for 1 20 min. in 96-well microtitre plates. Final incubation volume was 0.45 ml and buffer medium was 75 mM Tris-HCI, 25mM MgCI2, pH 7.4. Following incubation, membrane-bound and free radioiigand were separated by rapid vacuum filtration (Tomtec cell harvester) over pre- soaked (0.5% polyethyleneimine, PEI) Whatman GF/B filter strips, and washed through with three 2 ml volumes of ice-cold 75 mM Tris-HCI, 25mM MgCI2, (pH 7.4). Radioactivity was measured using a Beckman liquid scintillation counter. Specific binding was calculated as the difference between total and non-specific binding. Table

[ |]cyanopindolol specific binding (fmoles/mg protein ± SEM)

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Claims

What is claimed is:

1 . An immortalized human teratocarcinoma human central nervous system neuronal cell line having the identifying characteristics of ATCC CRL-1 2356.

2. An immortalized human teratocarcinoma human central nervous system neuronal cell line obtainable from, and having the identifying characteristics of, ATCC CRL-1 2356.

3. An immortalized human teratocarcinoma neuronal cell line, said cell transfected with a gene encoding a human β2 adrenergic receptor and said cells displaying an enhanced level of neuronal differentiation upon treatment with retinoic acid compared to untransfected cells.

4. The cell line of claim 3, wherein said cell line originates from the Ntera2/D 1 cell line.

5. The cell line of claim 3, wherein said gene encoding a human β2 adrenergic receptor is operably linked to a neuronal-specific protein promoter.

6. The cell line of claim 5, wherein said promoter is from synapsin.

7. The cell line of claim 6, wherein said synapsin is synapsin I.

8. A method of screening compounds which bind to a β2 adrenergic receptor subtype comprising contacting a plurality of cells of claim 5, detecting which compounds bind to the receptor in said cells so as to identify compounds which bond to the β2 adrenergic receptor subtype.