INTRODUCTION

Endothelins are a family of peptide hormones having profound cardiovascular, mitogenic, and potential neuroregulatory functions. In mammals, the ET¹ peptide family is composed of three members, ET-1, ET-2, and ET-3, that are encoded by three separate genes, which are differentially expressed in the tissues of the periphery and central nervous system (for reviews, see Refs. 1 and 2). Mammalian ETs share high sequence homology and structural similarity with a family of 21 amino acid peptide toxins from the snake Atractaspis engaddensis, the sarafotoxins (3).

Two major subtypes of ET receptors (ET_A and ET_B) (4) have been identified based on the rank order potency of ET-1, ET-2, ET-3, and S6c (5, 6). ET_A receptor is defined by high and equal affinity for ET-1 and ET-2, approximately 70-100-fold lower affinity for ET-3, and a 1000-fold lower affinity for S6c. In contrast, the ET_B receptor subtype displays equal high affinities for all ET-related peptides. Two additional receptors have been cloned and characterized from Xenopus melanophores (ET_C) and heart (ET_AX) (7, 8). While ET_C receptors display high affinity for ET-3 compared to ET-1, ET_AX receptors displayed extremely weak affinity for BQ123 as well as S6c (ET_A- and ET_B-selective ligands, respectively). Receptors for ET are differentially expressed in a wide variety of tissues and cell types (9, 10). ETs and sarafotoxins bind to a common receptor and initiate a common signal transduction pathway, principally a G-protein-mediated activation of phospholipase C and subsequent inositol triphosphate-mediated increase in Ca²⁺ levels (1, 11).

ET mediates a number of physiological effects including vasoconstriction mitogenesis, and induction of c-fos transcription (12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23). These diverse and complex physiological effects mediated by ET in conjunction with the molecular heterogeneity and differential tissue distribution of the ET-related peptides and their receptors underscores the importance of utilizing molecular biological approaches to dissect the components of ET physiology. Several laboratories have postulated the presence of additional ET receptors to account for the diverse biochemical and physiological activities of various ETs (24). This hypothesis has been supported by binding as well as functional studies. We have previously reported the cloning, functional characterization, and regulation of the human ET_A and ET_B receptor subtypes (25). In this report, we describe the cloning and functional characterization of a novel ET_B receptor splice variant from human placenta.

EXPERIMENTAL PROCEDURES

The porcine cerebellum cDNA library (26) in pcDNA vector was screened by hybridization to nitrocellulose replicates using ³²P-labeled porcine ET_B-R cDNA coding sequence as a probe in 20% formamide, 5 × SSC (SSC is 150 mM NaCl, 15 mM sodium citrate), 5 × Denhardt's, 0.1% SDS, and 0.2 mg/ml Escherichia coli tRNA at 42 °C (27). Filters were washed with 2 × SSC, 0.1% SDS at 42 °C. Several positive recombinant clones were isolated from the porcine cerebellum library and characterized. Preliminary sequence analysis of these clones showed that 6 of these clones encode the ET_B-R clones except two clones which contain the same 5-coding region of ET_B-R and a diverge 3-coding sequences. In order to obtain the human homologue and eliminate the possibility that this variant is due to a recombination artifact, primers were synthesized corresponding to the amino termini of ET_B-R (5-TCTGGAGCAGGATCCAGCATGCAGCCGCCT-3) and the 3-untranslated region of the porcine ET_B-SVR (5-CCCGTGATATCTAAA TAGAATCCATATGGTGTG-3) and used to obtain the human full-length splice variant cDNA clone from human placental RNA using polymerase chain reaction (28).

The inserts of the porcine ET_B-SVR560 and the human ET_B-SVR cDNAs were sequenced on both strands using a modification of the dideoxy chain termination method (29) using the Sequenase II Kit (U. S. Biochemical Corp.). The Wisconsin Genetics Computer Group Software package (30) was used to assemble composite sequences from the various fragments and for future sequence analysis.

For Northern analysis, poly(A) RNA was isolated from various human tissues using the guanidinum thiocyanate acid-phenol method (31). One µg of each RNA was fractionated on 1% agarose-formaldehyde gels (32) and transferred to nitrocellulose membranes. Northern hybridizations were performed at 42 °C in 50% formamide, 5% SSPE, 5 × Denhardt's reagent, 0.1% SDS, and 100 µg/ml yeast tRNA (33). The blots were washed with 0.1 × SSC, 0.1% SDS at 50 °C and exposed to x-ray film for 4 days at 70 °C. Autoradiograms were analyzed by quantitative scanning densitometry.

Fragments containing the entire human ET_B-SVR cDNA and ET_B-R cDNA coding sequences was subcloned into the mammalian expression vector pRLDN (25). COS cells grown in 245 × 245-mm tissue culture plates were transfected with 75 µg of human ET_B-SVR or human ET_B-R cDNA and grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum for 2 days as described previously (26).

The COS cells were washed with Dulbecco's phosphate-buffered saline containing a protease inhibitor mixture (5 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, and 0.1 µg/ml aprotinin) and scraped in the same buffer. The membranes were prepared as described previously (25). Protein content was determined by the bicinchoninic acid method using bovine serum albumin as the standard.

¹²⁵I-ET-1 binding to membranes prepared from COS cells transfected with ET_B-R or ET_B-SVR was performed as explained (25) with the following modifications. The assay volumes were 50 µl, and the protein was 50-100 ng/tube. The procedure for ¹²⁵I-ET-3 and ¹²⁵I-IRL-1620 binding was exactly the same as that for ¹²⁵I-ET-1. Each experiment was done three times with separate transfections. The data presented are from one experiment which is representative of the others. Intra-experimental variation was <10%. ¹²⁵I-ET-1, ¹²⁵I-ET-3, and ¹²⁵I-IRL-1620 (specific activities 2200 Ci/mmol) were obtained from DuPont NEN.

COS cells transfected with human ET_B-R or ET_B-SVR receptor clones were treated with 1 µCi/ml myo-[³H]inositol for 24 h in serum-free medium. At the end of treatment, medium was removed, cells were washed with Dulbecco's phosphate-buffered saline, and then exposed to indicated concentrations of various agonists for 10 min at 37 °C. The inositol phosphates were separated using ion exchange chromatography following the procedure of Aiyar et al. (34).

The cytosensor microphysiometer was used to measure the intracellular pH change with a pH-sensitive silicon sensor which is part of a microvolume flow chamber (35). COS cells transfected with vector or human ET_BR or ET_B-SVR were allowed to attach in capsule cups (Molecular Devices, Sunnyvale, CA) for 24 h before transferring to the sensor chamber in the microphysiometer (Molecular Devices). After establishing a steady base line, acidification rate in response to ET was measured as a change in pH over time (35).

RESULTS AND DISCUSSION

The porcine ET_B-R cDNA, previously cloned in our laboratory (26) was used to probe porcine cerebellum cDNA library. Several positive clones were identified. Nucleotide sequence analysis revealed that several of these positive clones encoded for ET_B-R except for two novel ET_B-R clones which differed in the length and the amino acid sequence at the 3-coding region. In order to determine the presence of this novel ET_B-R form in human tissues, oligonucleotide primers corresponding to the 5-coding region of ET_B-R and the 3-noncoding region of the novel ET_B receptor form were used to polymerase chain reaction the human homologue using human placental cDNA as a template. Agarose gel electrophoresis identified a polymerase chain reaction product of 1400 base pairs. Sequence analysis of this product revealed a sequence that was identical to ET_B receptor from the 5-untranslated region through the putative seventh transmembrane domain, and then completely differed in the cytoplasmic domain and 3-untranslated region. The deduced polypeptide consisted of 436 amino acid residues with a calculated molecular mass of approximately 48 kDa (Fig. 1, A and B). This is different from the size of ET_B receptor which is 442 amino acids with a calculated molecular mass of 49 kDa. Several independent clones in our library were found to contain this sequence which appears to represent alternative splicing of the carboxyl-terminal tail of the ET_B receptor. The carboxyl-terminal region of the ET_B-SVR clone bore no significant homology with other known proteins. Several lines of evidence support the fact that the ET_B-SV and the ET_B receptors represent alternatively spliced variants of a single gene. First, the 3 terminal tail of the ET_B-SVR clone was used to search the data base for identical sequences. Interestingly this sequence was found to correspond to the extreme 3-untranslated region of ET_B-R. Further analysis of the ET_B-R genomic structure (36) revealed that exons 1 through 6 are identical in ET_B-SVR and the ET_B-R in terms of their nucleotide sequence composition and their splice site. However, the 3 terminal tail of the ET_B-SVR represents a putative splice site and an A(G/G)T sequence is found at the 5 donor junction which showed that this region is a splice site for the ET_B receptor (Fig. 2). Therefore, two putative splice sites were identified at exon 7 in the human ET_B receptor gene: the first splice site at nucleotide 1194 to produce a 2855-base pair exon which encodes the normal ET_B-R; and the second splice site at nucleotide 2970 to produce a 997-base pair exon which encodes the ET_B-SVR. Second, an mRNA transcript of 2.7 kb corresponds to the size of the ET_B-SV receptor was identified in various human tissues (Fig. 3).

Receptor subtypes can arise through divergent genes, e.g. ET_A and ET_B and in the case of intron-containing genes, additional variants within a subtype can arise by alternative RNA splicing. Recent studies have identified an ET_B receptor variant which contains an additional 10 amino acids in the second cytoplasmic domain of the ET_B receptor (37). This sequence was part of the ET_B receptor intron that separates the second and the third exons and therefore arises by alternative RNA splicing of a single gene. The identification of splice variants among the seven transmembrane receptors has been increasing at a phenomenal rate following the initial observation of two variant forms for dopamine D2 receptor (38). Recent splice variants have been identified for thyrotropin stimulating hormone, TRH (39), neurokinin receptors (40), prostaglandin EP3 receptor (41, 42), pituitary adenylyl cyclase-activating polypeptide, PACAP receptor (43), and monocyte chemoattractant protein (MCP-1) receptor (44). It is interesting to note that in all these examples the cytoplasmic domain of the receptor is altered indicating that alternative RNA splicing plays an important role in the generation of physiologically divergent receptor activity for the same ligand.

Northern hybridization analysis of RNA from various human tissues with the human ET_B-SV receptor cDNA clone indicated the presence of two major mRNA species at 4.4 and 1.7 kb and one minor band at 2.7 kb. The two major bands correspond to the full-length ET_B receptor clones resulted from two polyadenylation signals which are 32 and 29 base pairs upstream of the polyadenylation sites of the two mRNA species. The minor band of 2.7 kb corresponds exactly to the size of the splice variant ET_B receptor.

Northern blot was also used to examine the pattern of expression of the ET_B and the ET_B-SV receptor mRNA derived from different human tissues. As shown in Fig. 3, mRNA expressed by each ET_B-R species were specifically detected in all the RNA samples. The total amount of ET_B-R mRNA levels appeared to be higher in the lung, liver, kidney, and placenta. However, the relative ratio of expression of each individual ET_B-SVR mRNA does not represent more than 10% of the total ET_B-R expression, except the skeletal muscle where ET_B-SVR contributes to more than 39% of the total ET_B receptor expression (Fig. 3). In addition, the mRNA levels of normal as well as splice variant ET_B-R were examined by Northern blot as well as reverse transcriptase-polymerase chain reaction methods in smooth muscle and endothelial cells. While both displayed normal ET_B-R mRNA, the ET_B-SVR mRNA was not detectable in both cells (data not shown).

Addition of increasing concentrations of ¹²⁵I-ET-3 or ¹²⁵I-IRL-1620 to membranes prepared from COS cells transfected with ET_B-R or ET_B-SVR clone resulted in specific, saturable and high affinity binding as shown in Figs. 4 and 5. The nonspecific bindings were 5-25 and 5-35% for ¹²⁵I-ET-3 and ¹²⁵I-IRL-1620, respectively (Figs. 4, A and B, and 5, A and B, respectively). The Scatchard transformations of the specific binding from the saturation binding experiments are shown as Figs. 4C and 5C for ET_B-R and ET_B-SVR, respectively. The apparent dissociation constants (K_d values) for ¹²⁵I-ET-3 and ¹²⁵I-IRL-1620 were 53 and 145 pM for ET_B-R and 42 and 68 pM for ET_B-SVR. The maximum density of the receptors were comparable for both receptors with both radioligands (Figs. 4C and 5C), indicating that the expression of these two receptors in COS cells is comparable.

Competition binding data using ¹²⁵I-ET-1 and increasing concentrations of unlabeled ET-1, ET-3, S6c, and BQ123 (ET_A-selective antagonist) obtained with ET_B-R and ET_B-SVR are shown in Fig. 6, A and B. While ET-1, ET-3, and S6c gave superimposable monophasic competition curves with both receptors, BQ123 was totally inactive up to 10 µM in both receptors (Fig. 6), indicating that the binding properties of ET_B-SVR are very similar to those observed with ET_B-R. Similar binding profiles were obtained when the competition binding experiments were done with the ET_B-selective radioligand, ¹²⁵I-IRL-1620, and the ET_B-selective peptide antagonist, BQ788, and the nonselective nonpeptide antagonists, SB 209670 and SB 222802 (Fig. 6, C and D). Thus, the data presented so far clearly indicate that the modifications present at the COOH-terminal end of the receptor did not have any effect on the binding of the radioligands (¹²⁵I-ET-1, ¹²⁵I-ET-3, and ¹²⁵I-IRL-1620) or ET_B-selective peptide antagonist, ET_A-selective peptide antagonist, as well as nonselective-nonpeptide antagonists.

Since the intracellular regions of seven transmembrane G protein-coupled receptors have been implicated in the coupling of these receptors to the signal transduction pathway, it was of interest to test whether there were any differences in the coupling of these two variants to signal transduction pathways. ET receptors have been shown to activate phospholipase C, resulting in the generation of diacylglycerol and inositol triphosphate which, in turn, releases intracellular calcium. Exposure of myo-[³H]inositol-prelabeled COS cells expressing ET_B-R to increasing concentrations of ET-1, ET-3, or S6c resulted in a concentration-dependent increase in inositol phosphate accumulation as shown in Fig. 7A. The maximum stimulation (50-60% over basal) obtained with the three agonists as well as the EC₅₀ values (0.2-1.0 nM) of these three agonists for stimulation of inositol phosphate accumulation were very similar. In contrast, the COS cells transfected with ET_B-SVR and prelabeled with myo-[³H]inositol did not respond to any of the three agonists tested (Fig. 7B). Since ET peptides were ineffective in stimulating inositol phosphates accumulation in ET_BSVR-transfected COS cells, a more sensitive method, such as cytosensor microphysiometer, was used to test whether these receptors were capable of inducing a change in intracelluar pH in response to ET. As shown in Fig. 7C, COS cells transfected with human ET_B-R gave a nice increase in the acidification rate, whereas COS cells transfected with vector alone or ET_B-SVR gave very little response. These data agree well with the data obtained from the inositol phosphates accumulation and indicate that even though the splice variant displayed the same binding properties as the wild type, this receptor was incapable of stimulating a functional response. Similar results have been reported by Kuang et al. (45) for chemokine receptors MCP-1Ra and MCP-1Rb. These two receptors are alternately spliced receptors, and the differences between the two is the COOH-terminal intracellular domain. While MCP-1Rb coupled to both G16 and G14, MCP-1Ra did not couple to either of these G proteins. Thus, the data presented here and in the literature strongly suggest that the intracellular COOH-terminal domain of the 7TM receptors is critical for G protein coupling and functional responses. It is clear from the amino acid sequence comparison of the two receptors that the only difference between the ET_B-R and ET_B-SVR is the proximal 52 amino acids of the COOH-terminal end. The amino acid composition of the wild type has 5 cysteines, 9 serines, 2 tyrosines, and no threonine, whereas the splice variant has 1 cysteine, 2 serines, 2 threonines, and no tyrosines. The importance of these key amino acids (involved in palmitoylation as well as phosphorylation) in the coupling to signal transduction pathway is not clear. In an elegant study to identify the functional domains of human ET_A receptor, Adachi et al. (46) have reported that the regions involved in signal transduction for human ET_A receptor are amino acids 296-305 and 373-385. Comparison of human ET_A and ET_B in these regions indicate that these regions are >95% identical in both subtypes; suggesting the significance of this region in the signaling mechanism. Further analysis of the 52 amino acids located at the COOH-terminal tail of ET_B-R identify the sequence SCLC which is part of the 373-385 conserved sequence of ET_B-R and ET_A-R. Interestingly, this sequence in the ET_B-SVR was replaced by AGPH. These data suggest that these four amino acids are critical for the signal transduction of these receptors.

The main unanswered question is what is the physiological role of the ET_B-SVR receptor in the cell? It is tempting to hypothesize that this receptor might function as a clearance receptor for the ET peptides, since there was no observed difference in the binding characteristics between the two receptors. Another possibility is that ET exhibits many diverse physiological effects, and it is possible that the ET_B-SVR could mediate another, yet unidentified, response. It has been demonstrated recently that prostaglandin E₂ receptor has four alternatively spliced carboxyl-terminal tails (37, 38). Interestingly, the four isoforms couple to different G proteins to activate different second messengers.

Several laboratories have demonstrated that cells expressing seven transmembrane receptors could undergo homologous desensitization in response to repeated challenges with agonists (47, 48). The mechanism of homologous desensitization to ET-1 is not well understood because the binding of ET-1 to its receptor is irreversible which makes it rather difficult to study down-regulation after prolonged exposure to agonists. One commonly used mechanism for inactivation of seven transmembrane receptors is phosphorylation of the serines and/or threonines in the carboxyl-terminal tails by the -adrenergic receptor kinase, which belongs to a multigene family containing six known subtypes and are called G-protein-coupled receptor kinases (49, 50, 51). It is of interest in this regard that the ET_B-R has a total of 9 serines, whereas the ET_B-SVR has a total of 2 serines and 2 threonines. Differences in receptor phosphorylation by -adrenergic receptor kinase-like enzyme might lead to differences in the pattern of deactivation between the two forms of the ET_B-receptors, providing another mechanism for increasing the diversity of cellular responses to ETs. The availability of these clones will facilitate investigation of the potential roles of ET_B-SVR and its possible participation in physiological and pathophysiological processes.