Introduction

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-receptors are defined as nonopiate, nondopaminergic, and nonphencyclidine binding sites that interact with several psychoactive agents including benzomorphans, haloperidol, and phencyclidine (Walker et al., 1990; Ferris et al., 1991; Su, 1991). These binding sites are, however, heterogeneous, consisting of several subtypes that can be differentiated by pharmacological and biochemical characteristics. One of these subtypes, namely -R1, has recently been cloned from guinea pig liver (Hanner et al., 1996). Subsequently, we have cloned the human (Kekuda et al., 1996), rat (Seth et al., 1998), and mouse (Seth et al., 1997) homologs of the receptor and also deduced the structure, exon-intron organization, and chromosomal location of the human (Prasad et al., 1998) and murine (Seth et al., 1997) gene coding for the receptor. Interest in -receptors in immune cells stems from the findings that -specific ligands have profound effects on immune function. -receptor ligands induce inhibition of proliferative response to mitogens on lymphocytes both in vivo and in vitro (Carr et al., 1992; Casellas et al., 1994). Furthermore, Liu et al. (1995), using a large number of  ligands, have shown that a high degree of correlation exists between drug binding potency at -receptors and the ability of these drugs to modulate splenocyte proliferation. The ability of several  ligands to inhibit mitogen-induced human T lymphocyte proliferation in vitro is comparable to that of cyclosporin A (Casellas et al., 1994). Studies on the cellular mechanism of  ligand-induced immunosuppression have shown that these ligands interfere with the production of proinflammatory cytokines IL-1, IL-6, and TNF- (Derocq et al., 1995) and also inhibit experimental acute graft-versus-host disease by blocking the production of IFN- by Th1 CD4⁺ T cells (Carayon et al., 1995). These effects that the  ligands have on the function of immune cells are similar to those of the anti-inflammatory cytokine IL-10, which is produced by Th2 CD4⁺ T cells and macrophages. Recent studies have in fact demonstrated that  ligands enhance the production of endotoxin-induced IL-10 in vivo (Bourrie et al., 1995). Taken collectively, these studies provide strong evidence for an important role of -receptors in the function of immune cells, especially lymphocytes.

The presence of -receptors in immune cells was first reported by Su et al. (1988) in guinea pig spleen and by Wolfe et al. (1988) in human peripheral blood leukocytes. Subsequently, T-enriched lymphocytes and B-enriched lymphocytes isolated from mouse spleen were used to characterize these receptors (Carr et al., 1991; Garza et al., 1993). Even though all of these studies have unequivocally demonstrated the presence of -receptors in immune cells, the biochemical and pharmacological profiles of these receptors differ markedly, indicating species- and cell type-dependent differential expression of various subtypes of -receptors in immune cells. Because the -receptor subtypes exhibit profound differences in affinity and selectivity toward  ligands, including the putative endogenous ligands such as progesterone, molecular identification of the receptor subtypes that are expressed in different cell types of the immune system is needed to further our current understanding of the role of  ligands and -receptors in immune function. The present investigation was undertaken to characterize, using pharmacological and molecular biological approaches, the -receptor that is expressed in the Jurkat cell, a human T cell line. This cell line is CD4-positive and has the ability to produce IL-2 and IFN- in response to stimuli, a characteristic of Th1 CD4⁺ T cells (Mohagheghpour et al., 1984). The results of this investigation show that Jurkat cells functionally express the -R1 subtype. In addition, we have demonstrated in this investigation that -R1 cDNA cloned from the Jurkat cells can be functionally expressed in an heterologous system, and we have obtained direct evidence that the cloned receptor interacts with progesterone, a putative endogenous ligand with well documented immunomodulating properties. We have also obtained evidence for the existence of an alternatively spliced form of -R1, which exhibits markedly reduced ligand binding capacity.

	Experimental Procedures

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Materials. [³H]Haloperidol (sp. radioactivity, 15 Ci/mmol), [ring-1,3-³H]-(+)-pentazocine (sp. radioactivity, 31.4 Ci/mmol), [³H]-(+)-1-propyl-3-(3-hydroxyphenyl)piperidine [(+)-PPP; sp. radioactivity, 92.4 Ci/mmol], and [1,2-³H]progesterone (sp. radioactivity, 47.5 Ci/mmol) were purchased from DuPont-NEN (Boston, MA). The unlabeled  ligands haloperidol, (+)-pentazocine, (+)-PPP, 1,3-di(2-tolyl) guanidine (DTG), clorgyline, dextromethorphan, spiperone, (+)-allylnormetazocine, ()-N-(3-phenyl-1-propyl)-1-phenyl-2-aminopropane [()-PPAP], and carbetapentane were purchased from Research Biochemicals, Inc. (Natick, MA). The cell lines Jurkat (clone E6-1), HeLa, and JAR cells were obtained from the American Type Culture Collection (Rockville, MD). MCF-7 cells were kindly given by Dr. J.A. Moscow (National Cancer Institute, Bethesda, MD). Cell culture media were obtained from Life Technologies, Inc. (Gaithersburg, MD) and fetal bovine serum was obtained from Atlanta Biologicals (Atlanta, GA).

Culture of Jurkat Cells and Preparation of Cell Membranes. Jurkat cells were grown in 225-cm² culture flasks, using Roswell Park Memorial Institute-1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. The cells were collected by centrifugation and suspended in 5 mM K₂HPO₄/KH₂PO₄ buffer (pH 7.5). The suspension was homogenized using the Ultra-Turrax Tissuemizer (Tekmar Company, Cincinnati, OH). The resulting homogenate was centrifuged at 60,000g for 30 min, and the membrane pellets were suspended in 5 mM K₂HPO₄/KH₂PO₄ buffer (pH 7.5) at a protein concentration of 5 mg/ml. The membrane suspensions were stored in small aliquots in liquid nitrogen until use. HeLa, JAR, and MCF-7 cells were cultured as previously described (Kekuda et al., 1996; Seth et al., 1997, 1998).

Binding Assays. Binding of different radiolabeled ligands to Jurkat cell membranes was measured as described previously (Ramamoorthy et al., 1995). Membranes (250 µg protein) were incubated with ligands in 200 µl of 5 mM K₂HPO₄/KH₂PO₄ buffer (pH 7.5) for 3 h at room temperature. Binding was terminated by the addition of ice-cold binding buffer, followed by rapid filtration of the mixture on a Whatman GF/F glass fiber filter (pore size, 0.7 µm) that had been presoaked in 0.3% polyethyleneimine. The filter was washed three times with 5 ml of ice-cold binding buffer. Radioactivity associated with the filter was determined by liquid scintillation spectrometry. Nonspecific binding was determined under similar conditions, but in the presence of 10 µM unlabeled haloperidol. This value was subtracted from total binding to calculate specific binding. Nonspecific binding was less than 15% of total binding at saturating concentrations of haloperidol. Use of 10 µM clorgyline or carbetapentane instead of unlabeled haloperidol yielded similar values for nonspecific binding. Stock solutions of competitive inhibitors were prepared either in dimethyl sulfoxide (haloperidol, DTG, ()-PPAP, and spiperone) or in binding buffer. When stock solutions made in dimethyl sulfoxide were used, an equal concentration (final concentration of dimethyl sulfoxide during binding assay was 1%) of the solvent was included in the assays to provide appropriate controls.

Northern Blot Analysis. Poly(A)⁺ RNA was isolated from Jurkat and JAR cells using the FastTrack mRNA isolation kit (Invitrogen, San Diego, CA). The human -R1 (h-R1) cDNA (Kekuda et al., 1996) was radiolabeled with [-³²P]-dCTP by random priming using the Ready-to-go oligo-labeling kit (Pharmacia, Piscataway, NJ). Poly(A)⁺ RNA samples were size-fractionated on a denaturing formaldehyde-agarose gel and probed with [³²P]cDNA under high-stringency conditions (Kekuda et al., 1996).

Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Subcloning and Sequencing of RT-PCR Products. RT-PCR using poly(A)⁺ RNA isolated from Jurkat cells was done with -R1-specific primers. The upstream (sense) primer was 5'-CGTCCTCGAGCCGGCTCCCTCCT-3', which corresponds to nucleotide positions 5 to 17 of the h-R1 cDNA and contains an added nucleotide sequence (underlined) to introduce an XhoI site. The downstream (antisense) primer was 5'-CCCGCTCTAGACCATCCGCAGGT-3', which corresponds to nucleotide positions 740 to 762 of the h-R1 cDNA. Nucleotide substitutions were present in this primer in three places, nucleotides 752, 754, and 755, to introduce an XbaI site (underlined). These restriction sites were introduced for directional cloning of the RT-PCR products in pBluescript vector under the control of T7 promoter. These two primers encompass the complete protein-coding region of the h-R1 cDNA (nucleotide positions 48 to 719 with the termination codon). The RT-PCR products were genecleaned and, after digestion with XhoI and XbaI, were ligated into pBluescript for nucleotide sequencing and functional expression. Sequencing was done using an automated DNA sequencer (Perkin-Elmer Cetus Instruments, Eden Prairie, MN).

Vaccinia Virus Expression and Measurement of -Receptor Ligand Binding. This was done using the procedure described previously (Kekuda et al., 1996; Seth et al., 1997, 1998). HeLa and MCF-7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Subconfluent cultures grown in 24-well culture plates were first infected with a recombinant vaccinia virus VTF 7-3, which carries the gene for T7 RNA polymerase as a part of its genome. This enables HeLa and MCF-7 cells to express T7 RNA polymerase. After the infection, the cells were transfected with either pBluescript vector alone or pBluescript-cDNA construct. In the constructs, the cDNA inserts were present in such an orientation that the transcription of the cDNAs was under the control of T7 promoter present in the vector. Transfection was mediated by lipofection. The virus-encoded T7 RNA polymerase catalyzes the transcription of the cDNA inserts in the constructs, allowing transient expression of the cDNA-encoded proteins in the cells. After 10 to 12 h after transfection, the medium from each well was removed and replaced with 300 µl of 5 mM K₂HPO₄/KH₂PO₄ buffer (pH 7.5). The culture plate was kept at 80°C for 2 h to freeze the cells. The frozen cells were then thawed and homogenized by passing through a 25-gauge needle several times. The homogenate was used in ligand binding assays. The procedure for binding assays was similar to that described previously for Jurkat cell membranes, except that the final concentration of dimethyl sulfoxide in the assay mixture was kept at 2% in experiments dealing with progesterone binding. This was necessary to keep the steroid in solution.

Data Analysis. The kinetic parameters of equilibrium binding, namely K_d (apparent dissociation constant) and B_max (maximal binding capacity) were calculated by linear as well as nonlinear regression methods using the Fig. P (version 6.0) computer program (BioSoft, Cambridge, United Kingdom). Inhibition constants (K_i) were calculated from the IC₅₀ values (i.e., the concentration of unlabeled test compound that was needed to cause 50% inhibition of the specific binding of radiolabeled ligand) according to the method of Cheng and Prusoff (1973). Experiments were performed in triplicate and each experiment was repeated two or three times. Data are presented as means ± S.E. (n = 6-9).

	Results

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Characteristics of [³H]haloperidol Binding to Jurkat Cell Membranes. To characterize the  binding sites in the Jurkat cell line, we first used [³H]haloperidol as the ligand. Haloperidol is a high-affinity ligand to both type 1 and type 2 -receptors (Walker et al., 1990; Ferris et al., 1991; Su, 1991). The binding assays were done using Jurkat cell membranes in the presence of [³H]haloperidol (10 nM) alone to determine the total binding, and in the presence of [³H]haloperidol (10 nM) and unlabeled haloperidol (10 µM) to determine the nonspecific binding. The total binding increased with time and the binding was approximately at equilibrium between 2 and 6 h of incubation. The nonspecific binding did not show time-dependent increase and this component was always found to be ~15% of total binding under equilibrium binding conditions.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Saturation kinetics of [3H]haloperidol binding to Jurkat cell membranes. Binding assays were done by incubating Jurkat cell membranes (250 µg protein) with 0.5 to 10 nM [3H]haloperidol for 2.5 h. Nonspecific binding was determined under identical assay conditions but in the presence of 10 µM unlabeled haloperidol. A, total binding () and nonspecific binding () at varying concentrations of [3H]haloperidol. B, Scatchard plot, describing the relationship between specific binding and specific binding/free haloperidol concentration.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Dose-response relationship for the inhibition of [3H]haloperidol binding to Jurkat cell membranes by various  ligands. Binding assays were done by incubating Jurkat cell membranes (250 µg protein) with 10 nM [3H]haloperidol in the presence of increasing concentrations of various  ligands. The control binding measured in the absence of inhibitors was taken as 100% which was 0.96 ± 0.02 pmol/mg of protein. , haloperidol; , clorgyline; , ()-PPAP; , carbetapentane; , (+)-PPP; , DTG; , (+)-allylnormetazocine; , spiperone; , dextromethorphan.

View this table: [in this window] [in a new window]   TABLE 1 IC50 and Ki values for various  ligands to inhibit [3H]haloperidol binding to Jurkat cell membranes

Molecular Identity of the  Binding Site in Jurkat Cells. The -R1 has been cloned from several animal species (Hanner et al., 1996; Kekuda et al., 1996; Seth et al., 1997, 1998). In humans, the size of the -R1 mRNA is about 1.7 kb (Kekuda et al., 1996; Prasad et al., 1998). We investigated, using molecular biological approaches, whether the -R1 is expressed in Jurkat cells. We first carried out Northern blot hybridization of size-fractionated poly(A)⁺ RNA isolated from Jurkat cells with the h-R1 cDNA as the probe. Poly(A)⁺RNA from JAR human placental choriocarcinoma cell line was used as a positive control because the h-R1 cDNA was isolated from a JAR cell cDNA library. As shown in Fig. 3, Jurkat cells as well as JAR cells contain a 1.7-kb mRNA species that specifically hybridizes to the h-R1 cDNA probe.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Northern blot analysis of poly(A)+ RNA from Jurkat cells and JAR cells using h-R1 cDNA as the probe.

View larger version (29K): [in this window] [in a new window]   Fig. 4.   A, comparison of amino acid sequence of the RT-PCR products obtained from Jurkat cell mRNA (clone A and clone B). B, generation of -R1 and -R1A by alternative splicing.

Ligand-Binding Characterization of the Jurkat Cell -R1 and -R1A cDNAs. The -R1 cDNA (clone A) was expressed in HeLa cells using the vaccinia virus expression technique. The ligand-binding function of the cDNA was assessed by comparing [³H]haloperidol binding to cell membranes between HeLa cells transfected with empty pSPORT vector and HeLa cells transfected with pSPORT--R1 cDNA construct (Fig. 5). [³H]Haloperidol binding was 2 to 3 times higher in cDNA-transfected cells than in vector-transfected cells, demonstrating that the Jurkat cell -R1 cDNA is functionally active in terms of  ligand binding.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Time course of [3H]haloperidol binding to membranes derived from HeLa cells transfected with empty vector (), -R1 cDNA (), or -R1A cDNA (). The cDNAs were expressed in HeLa cells by vaccinia virus expression system. Cells transfected with empty vector served as control. Binding assays were done with cell homogenates using 5 nM [3H]haloperidol.

View larger version (10K): [in this window] [in a new window]   Fig. 6.   Binding of [3H]-(+)-PPP and [3H]-(+)-pentazocine to membranes derived from HeLa cells transfected with empty vector (pSPORT), -R1 cDNA, or -R1A cDNA. The cDNAs were expressed using the vaccinia virus expression technique. Binding assays were done with cell homogenates using an incubation period of 2.5 h (equilibrium binding) and 5 nM ligand concentration.

View this table: [in this window] [in a new window]   TABLE 2 Expression of -R1 and -R1A cDNAs in MCF-7 cells as assessed by [3H]-(+)-pentazocine binding

View larger version (10K): [in this window] [in a new window]   Fig. 7.   Saturation kinetics of [3H]haloperidol binding to membranes derived from HeLa cells transfected with empty pSPORT vector () or -R1 cDNA (). The cDNA was expressed by the vaccinia virus expression technique. Binding assays were done with cell homogenates using [3H]haloperidol in the concentration range of 0.1 to 5 nM. Results are given as Scatchard plots describing the relationship between ligand binding (B) and ligand binding/free ligand concentration (B/F). Inset: Data for -R1 cDNA-specific binding, which were obtained by subtracting the binding in empty vector-transfected cells from the binding in -R1 cDNA-transfected cells. B is given in pmol/106 cells and F is given in nM.

Interaction of Progesterone with the Cloned Jurkat -R1. Progesterone is believed to be an endogenous ligand to -R1. However, the direct binding of this steroid has not been demonstrated with any of the cloned -R1 (guinea pig, rat, mouse, or human). Therefore, we investigated the binding of progesterone to Jurkat cell -R1 using HeLa cells expressing the Jurkat -R1 cDNA. We first measured the binding of [³H]progesterone in control HeLa cells transfected with empty pSPORT vector and in HeLa cells transfected with the Jurkat cell -R1 cDNA (Table 3). There was almost a 2-fold increase in the binding in cDNA-transfected cells compared with control cells. The cDNA-specific [³H]progesterone binding was inhibited >90% by the  ligands haloperidol, clorgyline, carbetapentane, and (+)-PPP, indicating that the observed increase is due to specific binding to the cDNA-encoded -receptor. R5020 is a progesterone analog that is an inhibitor of progesterone binding to the progesterone receptor. This compound did not inhibit [³H]progesterone binding in HeLa cells induced by the cDNA. This rules out the participation of the classical progesterone receptor in the observed increase in [³H]progesterone binding in cDNA-transfected cells.

View this table: [in this window] [in a new window]   TABLE 3 Inhibition of -R1 cDNA-induced [3H]Progesterone binding by  ligands

View larger version (12K): [in this window] [in a new window]   Fig. 8.   Saturation kinetics of [3H]progesterone binding induced by -R1 cDNA. HeLa cells were transfected with either empty pSPORT vector or pSPORT--R1 cDNA. Vaccinia virus expression technique was used to express the cDNA functionally. Cell homogenates were used for [3H]progesterone binding at varying concentrations of progesterone in the range of 10 to 250 nM. The cDNA-specific binding, which was obtained by subtracting the binding in empty vector-transfected cells from the binding in cDNA-transfected cells, was used for data analysis. Inset: Scatchard plot describing the relationship between progesterone binding (B) and progesterone binding/progesterone concentration (B/l).

View larger version (8K): [in this window] [in a new window]   Fig. 9.   Kinetics of inhibition of [3H]haloperidol binding by progesterone in control HeLa cells transfected with empty vector (A) and in HeLa cells transfected with -R1 cDNA (B). Vaccinia virus expression technique was used to express the cDNA functionally. Haloperidol binding to cell homogenates was measured with varying concentrations of [3H]haloperidol in the range of 0.5 to 7.5 nM in the absence () or presence () of 200 nM progesterone. Data are given as Scatchard plots, describing the relationship between haloperidol binding and haloperidol binding/free haloperidol concentration. Values for binding are given in pmol/106 cells and values for free haloperidol concentration are given in nM.

View larger version (14K): [in this window] [in a new window]   Fig. 10.   Dose-response relationship for the inhibition of [3H]haloperidol binding to the cloned -R1 by (+)-pentazocine () and progesterone (). HeLa cells were transfected with either empty pSPORT vector or pSPORT--R1 cDNA. Vaccinia virus expression technique was used to express the cDNA functionally. Cell homogenates were used for the binding of [3H]haloperidol (3 nM) in the presence of increasing concentrations of (+)-pentazocine and progesterone. The cDNA-specific binding, which was obtained by subtracting the binding in empty vector-transfected cells from the binding in cDNA-transfected cells, was used for data analysis. The results are given as a percentage of control binding measured in the absence of (+)-pentazocine and progesterone.

View larger version (11K): [in this window] [in a new window]   Fig. 11.   Saturation kinetics of [3H]-(+)-pentazocine binding (A) and inhibition of [3H]-(+)-pentazocine binding by progesterone (B). Binding measurements were made in cell homogenates derived from HeLa cells transfected with either empty pSPORT vector or pSPORT--R1 cDNA. Only the cDNA-specific binding was used for data analysis. Saturation binding was analyzed over a (+)-pentazocine concentration of 3 to 100 nM (A). In inhibition studies with progesterone, the concentration of [3H]-(+)-pentazocine was 3 nM.

	Discussion

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There is increasing evidence that suggests potent immunomodulatory effects of -receptor ligands (Carr et al., 1992; Casellas et al., 1994; Liu et al., 1995; Derocq et al., 1995; Carayon et al., 1995; Bourrie et al., 1995). Because some of these ligands have been shown to elevate the circulating levels of anti-inflammatory cytokines and at the same time to suppress the circulating levels of proinflammatory cytokines,  ligands may prove to be potentially useful therapeutic agents in the treatment of various immune disorders. -receptors, which are the targets for these ligands, have thus far been studied in immune cells only by ligand binding assays to characterize the pharmacological profiles of these receptors (Su et al., 1988; Wolfe et al., 1988; Carr et al., 1991; Garza et al., 1993). Very little is known about the molecular nature of these receptors. The present investigation was undertaken to study the -receptors in immune cells at molecular level.

We have established in this investigation that Jurkat cells express the recently cloned -R1. This was done by Northern analysis and RT-PCR, which unequivocally showed the presence of -R1 mRNA in these cells. The RT-PCR yielded a functionally active -R1 cDNA as assessed by binding of  ligands. Using this cDNA, we have now investigated the ligand-binding characteristics of the h-R1 for the first time by using heterologous expression systems. These studies have shown that the -R1 cDNA, cloned from the Jurkat cells, induces  ligand binding activity when expressed in HeLa cells and MCF-7 cells. HeLa cells are known to possess endogenous -R1 activity, whereas MCF-7 cells lack -R1 activity. The fact that the -R1 cDNA can be functionally expressed in both of these cells indicates that the cDNA-induced  ligand binding activity is unrelated to the endogenous -R1. The cDNA-induced ligand binding was demonstrated with more than one ligand: haloperidol, (+)-PPP, and (+)-pentazocine. Haloperidol can interact with several subtypes of -receptors, whereas (+)-pentazocine is believed to be a specific ligand for the -R1. This suggests that the cDNA cloned from the Jurkat cells codes for the -R1. When functionally assessed in HeLa cells, the cDNA induces a single saturable binding site that exhibits K_d values of 1.9 ± 0.3 nM for haloperidol and 12 ± 2 nM for (+)-pentazocine. When assessed in native Jurkat cell membranes, haloperidol was found to bind to a single saturable binding site with a K_d of 3.9 ± 0.3 nM. Thus, the affinity for haloperidol for the native Jurkat cell binding site and for the cDNA-induced binding site was comparable.

The time course of [³H]haloperidol binding to either native Jurkat cell membranes or HeLa cell membranes expressing the cloned -R1 showed that the binding reached equilibrium only with incubation periods of >2 h. Similar observations were made when haloperidol binding to human placental brush-border membranes was studied (Ramamoorthy et al., 1995). A survey of the literature on the binding of haloperidol to membrane preparations shows that a 90- to 120-min incubation period has been used in a majority of the studies. -R1 is membrane-bound and is associated with the plasma membrane as well as with intracellular membranes. The cloned receptor possesses a single putative transmembrane domain. The exact membrane topology of the receptor is not known; neither is the location of the ligand-binding site. Because membrane preparations exist as vesicles to a significant extent, the slow reaching of equilibrium binding suggests that the binding site may not be readily accessible to the ligand in these membrane vesicles. This is, however, only speculative because the exact topology of the ligand-binding site has not yet been elucidated.

Interaction with progesterone is a characteristic that is unique to the -R1 because other subtypes of -receptor do not bind this steroid. We have shown in this study that the cloned Jurkat cell -R1 binds progesterone. This is the first demonstration of the direct interaction of progesterone with a cloned -R1. The cDNA-induced progesterone binding was inhibitable by various  ligands but not by R5020, a steroid analog that antagonizes the binding of progesterone to the classical progesterone receptor. We have also shown that progesterone was a competitive inhibitor of the cDNA-induced haloperidol binding. The interaction of progesterone with the cloned Jurkat cell -R1 was of high affinity as suggested by the K_d value (~80 nM). Because progesterone is thought to be an endogenous ligand for the -R1, the high-affinity interaction between the cloned -R1 and the steroid as demonstrated in this study is physiologically relevant. Progesterone concentrations in human plasma are in the range of 30 to 40 nM (Johansson, 1969), a value that is close to the observed K_d value. In women, the circulating levels of progesterone vary dramatically under various physiological conditions such as menstrual cycle and pregnancy. The levels are known to be as high as 500 nM in late pregnancy (Johansson, 1969). It is therefore likely that, with a K_d value of ~80 nM, the fractional occupancy of the receptor with progesterone fluctuates markedly in women depending on the physiological status. This would mean that the biological effects of progesterone that are mediated by the interaction of this steroid with the -R1 may vary in magnitude in women under different physiological conditions, determined by the circulating levels of progesterone. Progesterone has been shown to be active in anti-inflammatory tests (Siiteri et al., 1977) and a direct comparison between the relative affinities of various steroids for the -R1 and the relative potencies of these steroids in anti-inflammatory tests has suggested that the immunomodulatory effects of progesterone are likely to be produced through the -receptor (Siiteri et al., 1977; Su et al., 1988). These observations are especially relevant to pregnancy, when the circulating levels of progesterone are high enough to saturate the -R1 in immune cells, because it is possible that the immunosuppressive function of progesterone may play a significant role in the maternal immunotolerance of the placental allograft.

Another significant aspect of the present study was the demonstration of the existence of an alternatively spliced form of the -R1. This receptor variant, -R1A, arises as a result of deletion of the third exon. This causes the deletion of 31 amino acids. This alternatively spliced receptor variant is inactive as assessed by the binding of haloperidol, (+)-PPP, and (+)-pentazocine. We have not yet studied the expression of the alternatively spliced variant in normal tissues. The inactive nature of this variant implies that the generation of this variant in immune cells is likely to have profound consequences in immune function. Furthermore, because the -R1 is expressed in other tissues as well, especially in the nervous system, the consequences of the production of the inactive variant would involve multiple organs. Therefore, the evidence provided in this study for the occurrence of an inactive splice variant of the -R1 may have clinical significance.