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Molecular Endocrinology, doi:10.1210/me.2006-0343
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Molecular Endocrinology 21 (5): 1192-1204
Copyright © 2007 by The Endocrine Society

Cloning and Functional Identification of Novel Endothelin Receptor Type A Isoforms in Pituitary

Noriyuki Hatae, Nadia Aksentijevich, Hana W. Zemkova, Karla Kretschmannova, Melanija Tomic and Stanko S. Stojilkovic

Section on Cellular Signaling, Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), Bethesda, Maryland 20892-4510

Address all correspondence and requests for reprints to: Stanko S. Stojilkovic, Section on Cellular Signaling, National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), Building 49, Room 6A-36, 49 Convent Drive, Bethesda, Maryland 20892-4510. E-mail: stankos{at}helix.nih.gov.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mammalian endothelin (ET) receptors, termed ETAR and ETBR, are derived from two intron-containing genes and the functional splice variants of ETBR but not ETAR have been identified. Here, we report about the isolation of cDNAs of ETAR transcripts from rat anterior pituitary, which are generated by alternative RNA splicing. Deletion of exon 2 and insertion of fragments from intron 1 and 2 accounted for formation of three misplaced proteins, whereas the insertion of a fragment from intron 6 resulted in generation of a functional plasma membrane receptor, termed ETAR-C13. In this splice variant, the C-terminal 382S-426N sequence of ETAR was substituted with a shorter 382A-399L sequence, resulting in alteration of the putative domains responsible for coupling to Gq/11 and Gs proteins and the endocytotic recycling, as well as in deletion of the predicted protein kinase C/casein kinase 2 phosphorylation sites. The mRNA transcripts for ETAR-C13 were identified in normal and immortalized pituitary cells and several other tissues. The pharmacological profiles of recombinant ETAR and ETAR-C13 were highly comparable, but the coupling of ETAR-C13 to the calcium-mobilizing signaling pathway was attenuated, causing a rightward shift in the potency for agonist. Furthermore, the efficacy of ETAR-C13 to stimulate adenylyl cyclase signaling pathway and to internalize was significantly reduced. These results indicate for the first time the presence of a novel ETA splice receptor, which could contribute to the functional heterogeneity among secretory pituitary cell types.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE ENDOTHELIN (ET) family of peptides is composed of three endogenous isoforms, ET-1, ET-2, and ET-3, which are each encoded by different genes (1, 2). The peptides are differentially expressed in tissues of the periphery and central nervous system and have profound effects on cardiovascular, neuroregulatory, and endocrine functions (3, 4). The actions of ETs are mediated by two plasma membrane ET receptor (ETR) subtypes, ETAR (5) and ETBR (6). These receptors are members of the superfamily of G protein-coupled receptors (GPCRs) that signals through variable G proteins, depending on the cell type in which they are expressed (7). The ETAR is selective for ET-1 and ET-2 compared with ET-3, whereas the ETBR is unable to distinguish among these peptides (8). The ETC receptor cloned from Xenopus leavis dermal melanophores is ET-3 specific (9); however, the mammalian homolog for the ETC receptor does not exist. Numerous antagonists of these receptors have been developed, including the ETAR-specific BQ-123 and ETBR-specific BQ-788 (8).

ETs are also produced by pituitary cells (10) and functional ETRs are expressed in all five major secretory cell types (11, 12). In gonadotrophs, stimulation of these receptors leads to activation of the Gq/11 signaling pathway accompanied with the oscillatory Ca2+ release from intracellular pools and gonadotropin secretion (13). The stimulatory action of these receptors on Ca2+ signaling and secretion in gonadotrophs is transient due to their rapid desensitization and internalization (14). In lactotrophs and somatotrophs, ETs also activate the Ca2+-mobilization pathway and transiently stimulate hormone release (15, 16). In contrast to gonadotrophs, the stimulatory effect of ET is followed by inhibition of prolactin (PRL) and GH release below the basal levels (15, 17). The inhibitory phase lasts for several hours (12, 18), arguing against rapid desensitization of these receptors. Although there is parallelism in the actions of ETs on PRL and GH secretion and voltage-gated Ca2+ influx (19, 20), the ET-1-induced inhibition of PRL release is mediated by the Gz ß/{gamma} dimer (20), whereas inhibition of voltage-gated Ca2+ influx is mediated by the Gi/o signaling pathway (16, 19). Such a difference in the actions of ETs in gonadotrophs vs. lactotrophs/somatotrophs would be consistent with the expression of both subtypes of these receptors, but experiments demonstrated that the BQ-123-sensitive receptor subtype is responsible for ETs effects on signaling and secretion in three cell types (14, 21, 22).

Based on these experimental observations, an elegant theoretical model was developed to describe the complex signaling pathway in lactotrophs, with the main idea that one receptor is coupled to many G proteins (23). We may also speculate that multiple receptor subtypes are generated by alternative RNA splicing of the pituitary ETAR and exhibit comparable binding characteristics but are coupled to different G proteins and intracellular signaling. In support of the second hypothesis, ETAR and ETBR arise through divergent intron-containing genes and the alternative splice forms of mRNAs have been reported (8). Some splice isoforms of ETBRs are functional and different from the wild-type receptor with respect to the coupling to intracellular signaling pathways (24, 25, 26). The human ETAR gene has also been proposed to give rise to at least three alternative splice isoforms, corresponding to the deletion of exon 3, exon 4, and exon 3 plus exon 4, which produced truncated proteins containing two, three, and five membrane-spanning domains, respectively. However, the truncated receptors were nonfunctional when expressed in COS cell lines (27, 28). Here we report on the novel splice forms of ETAR, with the focus on the functional ETAR-C13 receptor subtype. Our results indicate that the ligand-binding properties of wild-type and C-13 splice receptors are similar and that the variable C-terminal structures of the two proteins may determine the receptor-specific coupling to G proteins.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cloning and Sequence Analysis of the Rat ETAR Splice Isoforms
Four splice variants of ETAR, termed ETAR-L, ETAR-U3, ETAR-U8 and ETAR-C13, were isolated from cDNAs of rat anterior pituitary gland by RT-PCR using specific primers that covered the coding region of the rat ETAR cDNA (Table 1Go). The nucleotide sequence of the largest splice form, termed ETAR-C13 (supplemental Fig. 1 published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org), did not correspond to any previously described ETAR splice variant sequence. A nucleotide fragment of approximately 480 bp was inserted in the region between exon 6 and exon 7 of the wild-type ETAR sequence (Fig. 1Go, A and B). The putative splicing cassette, 5'-splicing site, 3'-splicing site, and branch point in the intron sequence were predicted by the UCSC Genome Bioinformatics Site Program (http://genome.ucsc.edu/index.html). This resulted in substitution of the C-terminal 382S-426N sequence of the wild-type ETAR with 382A-399L sequence of C13 isoform without affecting the other regions (Fig. 2Go; GenBank accession no. DQ832324). The C-terminal region of this isoform bore no significant homology with other known proteins, as predicted by Protein-Protein BLAST Program of NCBI (http://www.ncbi.nlm.nih.gov/BLAST).


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  		Table 1. Oligonucleotide Sequences of Primers

 

Figure 1
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  		Fig. 1. Structural Organization of the Rat ETAR Gene

A, Schematic representation of cDNA for ETAR splice variants. Gray boxes indicate putative translational regions, and solid lines indicate 5'- and 3'-untranslated regions. Exons in the ETAR-wt cDNA and their corresponding regions of the splice variant cDNAs are connected by broken lines. Inserted and deleted fragments lead to shifts in the open reading frame and to premature termination of polypeptides. B, Detailed characterization of ETAR-C13 cDNA. The ETAR genome structure is shown as solid bars (introns) and gray boxes (exons). Transmembrane (TM-I to TM-VII) and extracellular/intracellular domains are indicated as black and open boxes, respectively. Exons in the genomic DNA and their corresponding regions of the cDNAs for wild-type and splice receptors are connected by broken lines.

 

Figure 2
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  		Fig. 2. Amino Acid Sequence of the Rat ETARs

The deduced amino acid sequences of wild-type and ETAR-C13 receptors are shown, starting with the initial methionine residue. Positions of the putative transmembrane regions (TM-I to TM-VII) are indicated by gray areas. Asterisks indicate identical amino acids for both receptors.

 
The nucleotide sequence structures of ETAR-L, ETAR-U3, and ETAR-U8 are shown in supplemental Fig. 2, published as supplemental data on The Endocrine Society’s Journals Online web site, and the predicted amino acid sequences in supplemental Fig. 3 (GenBank accession nos: DQ839232, DQ839233, and DQ839234). Schematic representation of cDNA for these splice variants is shown in Fig. 1AGo. Comparison of the sequence for ETAR-L with that of the ETAR gene revealed that the missing sequence of this transcript corresponds to exon 2. This rat ETAR splice transcript is equivalent to the ETAR-{Delta}3 transcript previously described in the human placenta (28). Two other transcripts are novel, and inserted fragments of intron 1 and 2 accounted for their formation (Fig. 1AGo). Inserted and deleted nucleotide fragments lead to shifts in the open reading frame and to premature termination of the polypeptides, from 426 residues present in the wild-type receptor, to 259 residues for ETAR-U3, 161 residues for ETAR-L, and 144 residues for ETAR-U8. Furthermore, the last four, 10, and 21 predicted residues of ETAR-U8, ETAR-U3, and ETAR-L, respectively, differed from the corresponding sequences of ETAR-wt (supplemental Fig. 3).

Expression of ETAR Splice Variants
To characterize the presence of mRNA transcripts for ETARs in pituitary and other tissues, we designed three different pairs of primers, termed B, C, and E/F (Fig. 3Go, A and D, and Table 1Go). Using primers B, the RT-PCR analysis of the total mRNAs from anterior pituitary cells revealed the presence of several bands of different sizes (Fig. 3BGo). The bands were identified by PCR using cDNAs from wild-type and ETAR-L, ETAR-U3, and ETAR-U8 as templates. The presence of wild-type and ETAR-C13 mRNA transcripts in total pituitary mRNAs was done using primers C. As shown in Fig. 3CGo, two bands were isolated from total mRNAs from pituitary and identified by PCR using cDNAs from the wild-type and C13 splice variant as templates. Primers E/F were designed to identify only the presence of C13 splice variant mRNA, and single band was found in both pituitary and GH3 cells (Fig. 3EGo). In other tissues, the expression of mRNA transcripts for this splice form was lower or not present (Fig. 3FGo).


Figure 3
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  		Fig. 3. Expression of mRNA Transcripts for ETARs in Rat Tissues

A–C, RT-PCR analysis of the total mRNA from primary cultures of mixed anterior pituitary cells. A, Schematic representation of PCR primer pairs used for amplification of ETARs. The oligonucleotide sequences of primers are shown in Table 1Go. B and C, The bands were identified by PCR using cDNAs from wild-type and splice variants as templates. The sizes of PCR products were: 503 bp for ETAR-wt, 375 bp for ETAR-L, 577 bp for ETAR-U3, and 635 bp for ETAR-U8 (B), and 399 bp for ETAR-wt and 803 bp for ETAR-C13 (C). The PCR products were sequenced. D, Schematic representation of PCR primer pairs used for amplification of ETAR-C13 (for details see Table 1Go). E, RT-PCR analysis of total mRNAs from primary cultures of mixed anterior pituitary cells and immortalized GH3 cells. No PCR products were detected in controls containing all components except the reverse transcriptase. F, PCR analysis was performed using RT products of variable rat tissues. In both panels, the PCR products for ETAR-C13 are shown as 474-bp bands and for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as 469-bp bands.

 
Subcellular Localization and Binding Characteristics of ETAR Splice Variants
To evaluate the subcellular distribution of receptors, we constructed the AcGFP1 fusion receptors and analyzed the green fluorescent protein (GFP) fluorescence pattern by confocal microscopy. In Chinese hamster ovary (CHO)-K1 cells 24 h after transfection, the majority of ETAR-wt and ETAR-C13 were inserted into the plasma membrane (Fig. 4AGo, left panels). Similar localization of GFP-tagged receptors was also observed in stably transfected human embryonic kidney (HEK)293 cells (Fig. 4AGo, right panels). In contrast, ETAR-L and ETAR-U8 proteins were not inserted into the plasma membrane, but were associated with intracellular membranes, presumably the endoplasmic reticulum membranes (Fig. 4BGo, left panels), whereas the GFP vector expressed alone and the ETAR-U3 protein were identified in cytosol (Fig. 4BGo, right panels). In agreement with these findings, the [125I]ET-1-specific binding was observed in intact HEK293 cells transiently expressing ETAR-wt and ETAR-C13 receptors, but not in cells expressing ETAR-L, ETAR-U3, and ETAR-U8 receptors (Fig. 4CGo).


Figure 4
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  		Fig. 4. Expression of Wild-Type and Splice ETARs

A, The plasma membrane insertion of wild-type and C13 splice receptors 24 h after transfection in CHO-K1 cells (left panels) and in stably transfected HEK293 cells (right panels). B, The subcellular distribution of ETAR-L, ETAR-U3, ETAR-U8, and vector in CHO-K1 cells 24 h after transfection. C, Total [125I]ET-1 binding in HEK293 cells transiently expressing variable ETARs. Binding was determined in intact cells after 60-min incubation time at 4 C. Vec, Vector; WT, wild type.

 
Using the membrane fractions from HEK293 cells stably expressing wild-type and C13 splice nonmodified receptors, we further characterized the binding properties of two receptors at 37 C. As shown in Fig. 5Go, A and B, the ligand binding was displaced by ET-1, BQ-123, and ET-3 in a concentration-dependent manner; in both preparations ET-1 was most potent, followed by BQ-123, and ET-3. Such a potency order is consistent with the presence of typical ETAR binding sites in membrane fractions from cells expressing two receptors (29). No significant differences in the IC50 values for ET-1 or BQ-123 were observed in two preparations (Fig. 5CGo), further indicating that changes in the C-terminal sequence at ETAR-C13 do not affect the expression and ligand specificity of receptor.


Figure 5
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  		Fig. 5. Characterization of [125I]ET-1 Binding to Wild-Type and C13 Splice Receptors in Membranes from Stably Transfected HEK293 Cells

A and B, Displacement of [125I]ET-1 binding for wild-type (A) and C13 (B) receptors. Membranes were incubated with [125I]ET-1 in the absence or presence of increasing concentrations of unlabeled ET-1, BQ-123 (the ETAR-specific antagonist), or ET-3 (the ETBR-selective agonist) for 1 h at 37 C. ET-1, open circle; BQ-123, solid circles; ET-3, open squares. Data shown are mean ± SEM values (n = 3). C, The calculated EC50 values for ETAR ligands.

 
Coupling of ETAR-C13 to Intracellular Signaling
The ETAR signals through the coupling with Gq/11 heterotrimeric proteins, the concomitant activation of phospholipase C signaling pathway, and calcium mobilization from the intracellular inositol triphosphate-sensitive pools (29). When stimulated with 100 nM ET-1, both wild-type and C13 receptors responded with comparable peak amplitude of calcium transients in the presence of extracellular calcium (Fig. 6AGo) as well as in calcium-deficient medium (data not shown). However, the EC50 value for ET-1 was rightward shifted in cells expressing the C13 isoform (Fig. 6BGo), indicating that the splicing-induced changes in the C-terminal structure affect the sensitivity of receptor-Gq/11 coupling.


Figure 6
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  		Fig. 6. ET-1-Induced [Ca2+]i Response in Stably Transfected HEK293 Cells Expressing Wild-Type and ETAR-C13 Clones

A, ET-1-induced calcium signals in single HEK293 cells transfected with wild-type and ETAR-C13 receptor isoforms. The traces shown are mean values derived from a single experiment. B, Concentration-dependent effects of ET-1 on the peak calcium response. Shown are percentage changes in basal calcium levels. ETAR-wt, open circles; ETAR-C13 isoform, solid circles. Data shown are means ± SEM values (n > 10).

 
When expressed in CHO-K1 cells, the wild-type and splice receptors also responded to ET-1 application with increase in cAMP accumulation in a time- (Fig. 7AGo) and concentration-dependent (Fig. 7BGo) manner. Similar results were observed in stably transfected HEK293 cells and transiently transfected GH3 immortalized pituitary cells (data not shown). In contrast to calcium signals, the amplitude of cAMP response in cells expressing splice receptor was significantly lower than in cells expressing ETAR-wt in all time and concentration points (Fig. 7Go), although the expression efficiency (measured as specific [125I]ET-1 binding and percentage of GFP-positive cells) for two receptors was comparable. To test the hypothesis that the decreased potency of ETAR-C13 to elevate cAMP reflects its dual coupling to Gs and Gi/o/Gz signaling pathway, CHO-K1 cells and HEK293 cells were pretreated with pertussis toxin overnight and stimulated with 100 nM ET-1. As shown in Fig. 7CGo for CHO-K1 cells, there was no effect of this treatment on cAMP accumulation in cells expressing wild-type and splice receptors. Shutting off the Gz signaling pathway by phorbol 12-myristate 13-acetate (30) in these cells also did not alter the efficacy of two receptors to elevated cAMP levels (data not shown).


Figure 7
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  		Fig. 7. Stimulation of Adenylyl Cyclase Activity by ETAR and ETAR-C13

A, Time course of cAMP production in cells expressing ETAR-wt (open circles) and ETAR-C13 (closed circles). B, Concentration-dependent effects of ET-1 on cAMP accumulation. C, Pertussis toxin (PTX) insensitivity of ET-1-induced cAMP accumulation. Cells with inhibited endogenous activity of phosphodiesterases by 1 mM IBMX were incubated at 37 C for indicated times (A) or 30 min (B). For experiments shown in panel C, cells were pretreated with or without 250 ng/ml of PTX for 16 h at 37 C, and after washing, cells were incubated for 30 min at 37 C in the presence or absence of 100 nM ET-1. In all experiments, cAMP was measured in cell content and medium and the combined values (means ± SEM values; n = 4) are shown.

 
The stimulatory effect of ET-1 on cAMP accumulation does not necessarily support the splice receptor coupling with G{alpha}s, but could reflect its signaling through the ß-arrestin/Akt pathway, as documented for the wild-type dopamine receptor (31). To test this hypothesis, we blocked the Akt signaling by 3 mM lithium and examined effects of 100 nM ET-1 on cAMP accumulation in cells expressing wild-type and splice receptors. However, this treatment was completely ineffective in preventing cAMP accumulation in both cultures (data not shown). We also tested a hypothesis that two receptors stimulate cAMP accumulation through Gq/11 coupling and calcium and/or protein kinase C-dependent modulation of adenylyl cyclase/phosphodiesterase activity. To do this, we blocked the Gq/11 coupling to phospholipase C by U73122 and analyzed effects of such treatment on ET-1-induced cAMP accumulation in CHO-K1 cells expressing two receptors. As shown in Table 2Go, 100 nM ET-1-stimulated cAMP production was significantly reduced in cells expressing ETAR-wt and was practically abolished in cells expressing ETAR-C13.


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  		Table 2. Effects of Inhibition of Phospholipase C on ET-1-Induced cAMP (pmol/well) Accumulation in CHO-K1 Cells Expressing Wild-Type and C13 Splice ETARs

 
Desensitization and Internalization of ETARs
To examine the desensitization pattern of wild-type and splice ETARs, we stimulated CHO-K1 cells expressing two receptors with 100 nM ET for 5 min, followed by 30-min washing period. The ET-1-induced rise in intracellular calcium concentration ([Ca2+]i) declined to plateau levels during the 5-min application. Consistent with findings in pituitary cells (14), there was no recovery in calcium response 30 min after the initial stimulation (supplemental Fig. 4 published as supplemental data on the Endocrine Society’s Journals Online web site). The lack of response for both receptors was also observed 1, 2, and 3 h after removal of agonist (data not shown).

In intact HEK293 cells incubated at 37 C, the binding of [125I]ET-1 was dependent on the time of exposure, and there was no difference in the specific binding between the two receptors (Fig. 8AGo), a finding consistent with the comparable levels of their insertion into the plasma membrane. To separate the plasma membrane bound from internalized ligands, the surface-bound ligand was washed out by incubating cells in an acid-containing medium as described in Material and Methods. These experiments revealed significant differences in the surface-bound ligand (Fig. 8BGo) and the levels of internalized receptors (Fig. 8CGo) between cultures expressing wild-type and splice receptors in all time points examined.


Figure 8
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  		Fig. 8. ET-1 Mediated ETAR Endocytosis in Cells Expressing Wild-Type and C13 Splice Receptors

A–C, Stably transfected HEK293 cells were incubated with [125I]ET-1 for the indicated times at 37 C and the intracellular and cell surface-attached radioactivity was determined. The values for internalized receptors were expressed as intracellular radioactivity divided by total radioactivity and multiplied by 100. Data shown are means ± SEM values (n = 4). D and E, Transiently transfected CHO-K1 cells were preincubated with [125I]ET-1 for 1 h at 4 C, washed, and incubated for indicated times at 37 C, and total, cell surface, and intracellularly trapped radioactivity was determined. D, Time course of internalization of ligand in cells expressing wild-type (left) and splice (right) receptors. E, Comparison of internalization level of wild-type and splice receptors. Values are the means ± SEM of tetraplicate experiments.

 
To test the cell specificity in the internalization of two receptors, additional experiments were done in CHO-K1 cells. Cells expressing two types of receptors were incubated at 4 C for 1 h in the presence of ligand, the unbound tracer was then washed, cells were incubated at 37 C for variable times, and the surface-bound and internalized tracers were measured. Elevation in the incubation temperature was followed by internalization of ligand in both cultures (Fig. 8DGo), but the level of internalization was more obvious in cultures expressing ETAR-wt (Fig. 8EGo). These results indicate that splicing-induced changes in the C-terminal structure affect the agonist-induced trafficking of receptor.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
ETRs are members of GPCRs, a superfamily of proteins encoded by more than 800 genes that share a common structure of seven {alpha}-helix transmembrane segments, signal via interactions with the intracellular G proteins, and exhibit low overall sequence similarity to each other. However, ETRs differ from other GPCRs in two respects. First, the majority of GPCR genes (>90%) are intronless in their open reading frame (32), whereas ETRs are encoded by two intron-containing genes (25, 27). At the present time, it is unknown whether most GPCR genes are derived relatively recently in evolutionary history from a single intronless common progenitor, or whether mammalian GPCR genes might originally have had introns that have subsequently been lost (32). Second, although GPCRs frequently signal through more than one G protein type, the ability of ETRs to couple with different G proteins is unusually wide. For example, ETAR signals through Gq/11 (33), Gs (34), Gi/o (19), Gz (20), and G12/13 (35) pathways. It is also unknown to which extent the presence of introns in GPCR genes, which permits generation of receptor splice variants, contributes to the ability of receptors for multi G protein coupling. Interestingly, the receptor domains involved in interactions with G proteins and other intracellular proteins are the predominant sites of variation arising through splicing (36).

The rat dopamine D2 receptor was reported in 1989 as the first GPCR for which multiple variants were produced by alternative splicing (37). Since then, several other GPCR genes containing introns in their open reading frames were also identified, including serotonin 5HT4, corticotropin-releasing factor, {gamma}-aminobutyric acidB, and mGluR6 receptors (reviewed in Ref. 36). The structures of three regions of GPCRs, the N- and C-terminal tails and the third intracellular (i3) loop, are most frequently changed by alternative splicing. Splice variants of the N-terminal were reported for {gamma}-aminobutyric acid B and corticotropin-releasing factor receptors. The splice variants of the intracellular (i) 3 loop were reported for dopamine D2, histamine H3, pituitary adenylate cyclase-activating polypeptide, and cholecystokinin B receptors (36). It is interesting that in the majority of the verified GPCR splice isoforms, including TRH (38), prostaglandin EP3 (39, 40), and pituitary adenylate cyclase-activating polypeptide (41) receptors, the C-terminal domain of receptor is altered.

The ETBR C-terminal splice receptors were also identified in human (25) and porcine (26) cerebellum, as well as the splice variant of i2 loop in the human brain (24). The last two splice receptors were functional, whereas the splice variant identified by Elshourbagy and colleagues (25) did not respond to agonist stimulation with inositol phosphate accumulation. The human ETAR gene has also been reported to generate at least three alternative splice transcripts corresponding to deletion of exon 3, exon 4 (27), and exon 3 plus exon 4 (28). This resulted in transcripts, which would be translated into proteins containing two, three, and five transmembrane domains, respectively. The mRNA transcripts for these receptor variants were observed in various tissues, including the lung, aorta, atrium, kidney, and placenta. The lack of specific agonist binding in cells expressing these proteins is consistent with the finding that all seven transmembrane domains were important in ligand binding to ETR (42). The rat pituitary ETAR-L splice transcript described here corresponds to the ETAR-{Delta}3 transcript described in the human placenta (28). The ETAR-U3 and ETAR-U8 transcripts are novel, and inserted fragments of intron 1 and 2 account for their formation. All three receptors are misplaced, as shown by confocal studies and by the lack of specific ligand binding in cells expressing nontagged proteins. Here we also show the presence of a novel ETAR C-terminal splice variant in the pituitary cells and several other tissues.

As expected, the wild-type and ETAR-C13 showed no changes in the binding characteristics. However, the coupling of this splice receptor to heterotrimeric G proteins and its internalization were affected. This is consistent with numerous studies showing that the i3 loops and C-terminal tails of GPCRs are important in G protein coupling (43). It has also been suggested that 10 amino acid residues in the i3 loop of human ETAR together with the 13 amino acid residues at the proximal C-tail play a role in the receptor coupling to the calcium-mobilizing pathway (44). Nine of these 13 residues in the C-terminal tail are preserved in ETAR-C13, whereas the SCLC amino acid sequence at the position from 382–385 in wild-type receptor was substituted with the ALLW sequence. This substitution did not abolish the coupling of receptors to the calcium mobilization pathway, but it affected the sensitivity of coupling, as indicated by a rightward shift in the EC50 value for ET-1.

In some cell types, the ETAR subtype is coupled to Gs signaling pathway, leading to the elevation in cAMP production (45, 46). In contrast, activation of the native ETARs in pituitary cells inhibited cAMP production through Gz signaling pathway (20), and in other cell types the receptor is negatively coupled to the adenylyl cyclase signaling pathway through Gi/o (47). Experiments with chimeric ETAR and ETBR suggested that i2 and i3 loops are major determinants of the selective coupling of ETAR and ETBR with Gs and Gi, respectively (48). Here we show that the wild-type and C-13 splice ETARs stimulated cAMP production when expressed in HEK293 and CHO-K1 cells. However, the maximum in cAMP accumulation was ranging between 10–20% of that observed in controls.

In general, such reduced efficacy of the splice C-13 receptor to stimulate adenylyl cyclase could mirror the lower level of receptor expression. That was probably not the case in our experiments, because we could not observe any difference in the specific ligand binding in cells in population, or a decrease in the percentage of the GFP-positive cells and the plasma membrane insertion of two receptors. The differential cross-coupling of two receptors to Gi/o and Gz signaling pathway was also excluded. Furthermore, the lack of effect of lithium, a blocker of ß-arrestin/Akt signaling pathway, on ET-1-induced cAMP accumulation argues against the hypothesis that the residual activity of ETAR-C13 on cAMP production reflects the G protein-independent action, as documented for other cellular functions controlled by dopamine receptors (49). We also addressed the hypothesis that such activity reflects the Gq/11-mediated action, through activation of phospholipase C, elevation in calcium, and increase in basal activity of calcium-sensitive adenylyl cyclase (50) and/or phospodiesterase isoforms (51). Consistent with this hypothesis, in ETAR-C13-expressing cells with blocked phosphodiesterases, inhibition of phospholipase C signaling pathway practically abolished ET-1-induced cAMP production, whereas in cells expressing ETAR-wt it was only partially inhibited.

The C terminal region of GPCRs contributes not only to the G protein coupling, but also to receptor desensitization and internalization initiated by C-terminal phosphorylation by kinases (43), as well as to receptor dimerization and interaction with intracellular adaptor proteins (36). The human ETAR and ETBR each have C-terminal tail domains containing numerous serine and threonine residues (52, 53). Receptors desensitize during prolonged stimulation (14, 54), and this process corresponds temporarily with agonist-induced phosphorylation of ETRs (55, 56). The C-terminus of rat ETAR has nine serine and three threonine residues, including the protein kinase C (TER) and protein kinase C/casein kinase 2 (SHK) phosphorylation sites, predicted by MOTIF Search Cite Program (http://www.genome.jp). Although all thee threonine and eight serine residues are missing in ETAR-C13, we did not observe an obvious difference in the rates of wild-type and splice receptor desensitization. This is in agreement with findings that C-terminal splice variants of other GPCRs frequently show a reduced number of phosphorylation sites, accompanied with a relatively modest effect on the rates of receptor desensitization (36).

On the other hand, two receptors differed in the time courses of accumulation of the tracer in the cytosol under different experimental conditions, which indicated the subtype-specific internalization of receptors. Based on the level of accumulation of [125I]-ET-1 in cells, it is reasonable to conclude that the increase in the ETAR-C13 density at the cell surface, when compared with the wild-type receptor, might be predominantly a function of slower internalization rather than the faster recovery of receptors to the membrane. This is in agreement with the finding that internalization of GPCRs is highly dependent on the presence of serine-threonine-rich regions in the C-terminal tail of receptors (57). As discussed above, most of these residues are missing in the C-13 splice receptor. Changes in the rates of trafficking of receptors are not unique for the ETA C-terminal splice receptors, but were also observed for rat µ-opioid (58) and human mGluR1 (59) receptors. The recycling of receptors is specific for ETAR, as the ETBRs subtype is sorted to the late endosomal/lysosomal pathway (60, 61). Consistent with our observations, the truncated mutants of human ETAR revealed the relevance of the C-terminal 390–406 amino acid sequence for receptor recycling (62). An additional study indicated that the ETAR C-terminal mutants were not able to undergo recycling (63).

In conclusion, here we show for the first time the existence of a functional ETAR-C13 splice receptor, which belongs to the largest group of the C-terminal tail splice variants. The receptor is expressed in anterior pituitary cells and several other tissues. Neither the ligand binding characteristics and localization of receptors nor the rate of receptor desensitization was obviously affected. However, the coupling potency of the splice receptor to Gq/11 signaling pathway was reduced, and the coupling to the Gs signaling pathway was practically abolished. The internalization of the splice receptor was also changed, which could influence the rates of trafficking of receptors. ETRs make functional homo- and heterodimers (64), suggesting that native pituitary ETAR receptors could represent the heterodimers of wild-type and spliced receptors. Further studies should also clarify whether the protein-protein interactions involving intracellular domains of splice receptors are also affected, and which pituitary cell types express the splice form.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Chemicals
ET-1 and ET-3 were obtained from Peninsula Laboratories, Inc. (San Carlos, CA), BQ-123 was from TOCRIS Bioscience (Ellisville, MO), and 3-isobutyl-1-methylxanthine (IBMX) and BSA were from Sigma Chemical Co. (St. Louis, MO). TRIZOL reagent, Dnase I, Superscript II, pCR2.1 TOPO TA cloning kit, LipofectAMINE 2000, fura-2/AM, cell culture medium, and serum were from Invitrogen (Carlsbad, CA). EcoRI, SacI, SalI were from New England Biolabs (Ipswich, MA), and Ex Taq polymerase was from PanVera Corp. (Madison, WI). Total RNAs from rat tissue panel were purchased from BD Biosciences (Mountain View, CA) and (3-[125I]iodotyrosyl)ET-1 (2000 Ci/mmol) was from Amersham Biosciences Corp. (Piscataway, NJ).

Cell Culture
Anterior pituitaries were isolated from normal female Sprague Dawley rats (Taconic Farms, Hudson, NY). Cells were cultured in medium 199 with Earl’s salts, 10% horse serum, 100 U/ml penicillin G, and 100 µg/ml streptomycin sulfate under humidified air containing 5% CO2 at 37 C. Immortalized pituitary GH3 cells were maintained in F12K nutrient mixture containing 2.5% heat-inactivated fetal bovine serum, 15% horse serum, and 100 µg/ml gentamicin sulfate under humidified air containing 5% CO2 at 37 C. HEK293 and CHO-K1 cells were cultured in DMEM-F12 medium with 2.4 g/liter NaHCO3, 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin G, and 100 µg/ml streptomycin sulfate under humidified air containing 5% CO2 at 37 C.

RT-PCR
Total RNAs from pituitary primary cells and GH3 cells were isolated using TRIZOL reagent and treated Dnase I for degradation of contaminated genomic DNA. First-strand cDNA was synthesized from 5 µg total RNA using Superscript II reverse transcriptase and random hexamers in a reaction volume of 20 µl. The PCR was performed with Ex Taq polymerase using 1 µl of aliquot of the resulting single-strand cDNA in a reaction volume of 30 µl. For cloning of rat ETAR cDNAs and detection of mRNAS for wild-type and splice receptors, we used the primers described in Table 1Go. The thermal cycle programs for RT-PCR were: 94 C for 2 min, followed by 30 cycles at 94 C for 0.5 min, 60 C for 0.5 min, 72 C for 1 min (for detection of ETARs) or 2 min (for cloning of ETAR cDNAs), followed by 72 C for 7 min. Samples were visualized on 1.2% agarose gel. The same volume of samples used for ETAR-C13 mRNA analysis was also subjected to PCR using glyceraldehyde phosphate dehydrogenase-specific primers base pairs (469 bp); sequences for sense and antisense primers were 5'-GATGGTGAAGGTCGGTGTG-3' and 5'-GGGCTAAGCAGTTGGTGGT-3', respectively. No PCR products were detected from controls containing all components except reverse transcriptase, ruling out the possibility of genomic DNA contamination.

Isolation of cDNA Encoding Rat ETAR Transcripts
The cloning of the rat ETAR cDNA revealed the presence of the expected 1.4-kb product corresponding to the wild-type ETAR, as well as the presence of the 1.9-kb product. The portions of agarose gels corresponding to the longer and shorter transcripts were recovered, purified, and subcloned into the pCR2.1 vector. The sequences were determined by the dideoxy chain termination method (Veritas, Inc., Rockville, MD). Both strands on denatured plasmid templates were used for sequence analysis.

cDNA Expression in HEK293 and CHO-K1 Cells
The subcloned ETAR-wt, ETAR-L, ETAR-U3, and ETAR-U8 inserts were digested with EcoRI and ligated into EcoRI sites of the pIRES2-EGFP expression vector. The sequence of ETAR-C13 was digested with SacI and XhoI and ligated into SacI and SalI sites of the pIRES2-EGFP expression vector. For construction of AcGFP1 fusion receptors, the stop codons of wild-type and C13 splicing isoform ETAR cDNAs were deleted by PCR mutagenesis. The PCR fragments were digested with EcoRI and KpnI sites and inserted into the corresponding sites of pAcGFP1-N3 expression vector. The cDNA expression was done in HEK293 and CHO-K1 cells. For cAMP measurements, cells were seeded in 60-mm dishes (1 x 106 cells) overnight, washed, and treated by a mixture of 1 µg of plasmid DNAs, 10 µl of LipofectAMINE 2000, and 2 ml Opti-MEM. After incubation for 3 h at 37 C, cells were dispersed, reseeded in 24-well plates, and cultured in DMEM-F12 medium for 24 h at 37 C. For confocal analysis and calcium measurements, cells were seeded on glass coverslips placed in 35-mm dishes (50,000 cells per dish) and cultured for 48 h in DMEM-F12 medium. Transfection was done using 2 µg plasmid DNA with 10 µl of LipofectAMINE 2000 in 2 ml Opti-MEM. After incubation for 3 h at 37 C, cells were washed and cultured in DMEM-F12 medium for 24–48 h at 37 C.

Stable Expression of the Wild-Type and C13 ETARs in HEK293 Cells
Both pIRES2-EGFP and pAcGFP1-N3 expression vectors containing cDNA of wild-type and splice receptors were used for generation of stably transfected cells as previously described (65). The cDNA-transfected cells were selected by 1 mg/ml of G418, and cell lines were established by single-cell cloning. The control HEK293 cells were transfected only with the vector and isolated. The expression of wild-type and splice ETARs was assessed by [125I]ET-1 ligand binding assay.

[125I]ET-1 Ligand Binding
Ligand binding assay was done in intact cells and in cell membrane fractions. For intact cell binding assay, ETAR-expressing and nonexpressing cells (2.5 x 105 cells per well) were seeded in 12-well plates and incubated for 48 h under humidified air containing 5% CO2 at 37 C. Then the cells were washed twice with DMEM-F12-HEPES containing 0.1% BSA, pH 7.4, followed by the addition of 0.9 ml incubation medium and 0.1 ml radioligand. Cells were incubated for variable times at 37 C. In some experiments, cells with radioligand were preincubated at 4 C for 1 h. Subsequently, the cells were washed and incubated in 1 ml ligand-free medium for variable times at 37 C. The cell-bound radioactivity was measured after removing the incubation mixture and washing the cells with ice-cold DMEM-F12. To determine the surface-bound and internalized [125I]ET-1, the cells monolayers were washed at 4 C with 0.2 M acetic acid (pH 2.5) twice for 10 min each time. The remaining cell-associated radioactivity was quantized after solubilization of cells in 1 M NaOH containing 0.1% sodium dodecyl sulfate. For ligand binding assay using membrane fraction, the ETARs-expressing cells were washed in ice-cold PBS and harvested in 1 ml of 25 mM Tris-HCl (pH 7.5) containing 2 mM EDTA, 0.25 M sucrose, and protease inhibitors. The cells were homogenized on ice using Dounce homogenizer, followed by centrifugation at 50,000 x g for 15 min. The membrane pellets were resuspended in 25 mM Tris-HCl (pH 7.5) containing 2 mM EDTA, 100 mM NaCl, 0.1% BSA, and protease inhibitors. The membrane fraction (100 ng) was incubated with radioligand in the presence of increasing doses of unlabeled ligands for 1 h at 37 C. The samples were filtrated through Brandel GF/C filters presoaked with 0.1% BSA to remove unbound ligand. After three washes with 0.5 M NaCl, the filters were removed and subjected to {gamma}-spectrometric analysis.

Confocal Microscopy
Stably ETAR-expressing HEK293 cells and transiently ETAR-expressing CHO-K1 cells (6 x 105 cells per sample) were seeded onto 25-mm poly-L-lysine-coated coverslips and incubated for 24 h under humidified air containing 5% CO2 at 37 C. The distribution of GFP-tagged receptors within live cells was examined by laser scanning confocal microscopy. The culture medium was replaced with phenol red-free Krebs-Ringer buffer, and coverslips with cells were mounted on the stage of Zeiss LSM 510 Inverted Meta System (Carl Zeiss, Oberkochen, Germany). Images were collected under x63 objective lens, and further zoom (x2) was also applied. The same detector gain and settings were used for all images.

cAMP Measurements
For cAMP accumulation assay, the ETARs stably expressing HEK293 cells and transiently transfected CHO-K1 cells were seeded in 24-well plates and incubated overnight under humidified air containing 5% CO2 at 37 C. Then the plates were washed and preincubated in 1 mM IBMX-containing DMEM-F12-HEPES medium at 37 C for 10 min, after which medium was replaced with fresh 1 mM IBMX-containing DMEM-F12-HEPES medium containing variable doses of ET-1. After incubation for 30 min at 37 C, medium and cell pellets were collected and analyzed for cAMP content by RIA using specific antiserum provided by Albert Baukal (National Institute of Child Health and Human Development (NICHD), National Institutes of Health, Bethesda, MD).

Measurement of Calcium Ion Concentration
For [Ca2+]i measurements, cells were incubated in Krebs-Ringer buffer supplemented with 5 µM fura-2/AM. Coverslips with cells were washed with this buffer and mounted on the stage of an Axiovert 135 microscope (Carl Zeiss) attached to the Attofluor Digital Fluorescence Microscopy System (Atto Instruments, Rockville, MD). Cells were examined under a x40 oil immersion objective during exposure to alternating 340- and 380-nm light beams, and the intensity of light emission at 520 nm was measured. The ratio of light intensities, F340/F380, which reflects changes in Ca2+ concentration, was followed in several single cells simultaneously.

Calculations
Concentration-response relationships were fitted to a four-parameter logistic equation using a nonlinear curve-fitting program, which derives the EC50 and IC50 values (Kaleidagraph; Synergy Software, Reading, PA). Where appropriate, the results were expressed as means ± SEM, with n = 4 or higher in one of at least three similar experiments, and significant differences, with P < 0.01, were determined by Student’s t test.


   ACKNOWLEDGMENTS
 
Microscopy imaging was performed at the Microscopy and Imaging Core of the National Institute of Child Health and Human Development (NICHD) with the assistance of Dr. Vincent Schram. We thank Dr. Takayo Murano for help in cAMP measurements and Dr. Laszlo Hunyady for helpful discussion.


   FOOTNOTES
 
This work was supported by the Intramural Research Program of the National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH).

Disclosure Statement: The authors have nothing to declare.

First Published Online February 20, 2007

Abbreviations: [Ca2+]i, Intracellular calcium concentration; CHO, Chinese hamster ovary; ET, endothelin; ETR, ET receptor; GFP, green fluorescent protein; GPCR, G protein-coupled receptor; HEK, human embryonic kidney; i3 loop, third intracellular loop; IBMX, 3-isobutyl-1-methylxanthine; PRL, prolactin.

Received for publication August 18, 2006. Accepted for publication February 15, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Yanagisawa M, Inoue A, Ishikawa T, Kasuya Y, Kimura S, Kumagaye S, Nakajima K, Watanabe TX, Sakakibara S, Goto K, Masaki T 1988 Primary structure, synthesis, and biological activity of rat endothelin, an endothelium-derived vasoconstrictor peptide. Proc Natl Acad Sci USA 85:6964–6967[Abstract/Free Full Text]
  2. Inoue A, Yanagisawa M, Takuwa Y, Mitsui Y, Kobayashi M, Masaki T 1989 The human preproendothelin-1 gene. Complete nucleotide sequence and regulation of expression. J Biol Chem 264:14954–14959[Abstract/Free Full Text]
  3. Simonson MS, Dunn MJ 1990 Cellular signaling by peptides of the endothelin gene family. FASEB J 4:2989–3000[Abstract]
  4. Stojilkovic SS, Catt KJ 1996 Expression and signal transduction pathways of endothelin receptors in neuroendocrine cells. Front Neuroendocrinol 17:327–369[CrossRef][Medline]
  5. Arai H, Hori S, Aramori I, Ohkubo H, Nakanishi S 1990 Cloning and expression of a cDNA encoding an endothelin receptor. Nature 348:730–732[CrossRef][Medline]
  6. Sakurai T, Yanagisawa M, Takuwa Y, Miyazaki H, Kimura S, Goto K, Masaki T 1990 Cloning of a cDNA encoding a non-isopeptide-selective subtype of the endothelin receptor. Nature 348:732–735[CrossRef][Medline]
  7. Milligan G, Kostenis E 2006 Heterotrimeric G-proteins: a short history. Br J Pharmacol 147(Suppl 1):S46–S55
  8. Davenport AP 2002 International Union of Pharmacology. XXIX. Update on endothelin receptor nomenclature. Pharmacol Rev 54:219–226[Abstract/Free Full Text]
  9. Karne S, Jayawickreme CK, Lerner MR 1993 Cloning and characterization of an endothelin-3 specific receptor (ETC receptor) from Xenopus laevis dermal melanophores. J Biol Chem 268:19126–19133[Abstract/Free Full Text]
  10. Kanyicska B, Lerant A, Freeman ME 2001 Endothelin-like immunoreactivity in lactotrophs, gonadotrophs, and somatotrophs of rat anterior pituitary gland are affected differentially by ovarian steroid hormones. Endocrine 14:263–268[CrossRef][Medline]
  11. Stojilkovic SS, Iida T, Merelli F, Catt KJ 1991 Calcium signaling and secretory responses in endothelin-stimulated anterior pituitary cells. Mol Pharmacol 39:762–770[Abstract]
  12. Kanyicska B, Burris TP, Freeman ME 1991 Endothelin-3 inhibits prolactin and stimulates LH, FSH and TSH secretion from pituitary cell culture. Biochem Biophys Res Commun 174:338–343[CrossRef][Medline]
  13. Stojilkovic SS, Merelli F, Iida T, Krsmanovic LZ, Catt KJ 1990 Endothelin stimulation of cytosolic calcium and gonadotropin secretion in anterior pituitary cells. Science 248:1663–1666[Abstract/Free Full Text]
  14. Stojilkovic SS, Balla T, Fukuda S, Cesnjaj M, Merelli F, Krsmanovic LZ, Catt KJ 1992 Endothelin ETA receptors mediate the signaling and secretory actions of endothelins in pituitary gonadotrophs. Endocrinology 130:465–474[Abstract]
  15. Lachowicz A, Van Goor F, Katzur AC, Bonhomme G, Stojilkovic SS 1997 Uncoupling of calcium mobilization and entry pathways in endothelin-stimulated pituitary lactotrophs. J Biol Chem 272:28308–28314[Abstract/Free Full Text]
  16. Tomic M, Zivadinovic D, Van Goor F, Yuan D, Koshimizu T, Stojilkovic SS 1999 Expression of Ca(2+)-mobilizing endothelin(A) receptors and their role in the control of Ca(2+) influx and growth hormone secretion in pituitary somatotrophs. J Neurosci 19:7721–7731[Abstract/Free Full Text]
  17. Kanyicska B, Livingstone JD, Freeman ME 1995 Long term exposure to dopamine reverses the inhibitory effect of endothelin-1 on prolactin secretion. Endocrinology 136:990–994[Abstract]
  18. Samson WK, Skala KD, Alexander BD, Huang FL 1990 Pituitary site of action of endothelin: selective inhibition of prolactin release in vitro. Biochem Biophys Res Commun 169:737–743[CrossRef][Medline]
  19. Tomic M, Van Goor F, He ML, Zivadinovic D, Stojilkovic SS 2002 Ca(2+)-mobilizing endothelin-A receptors inhibit voltage-gated Ca(2+) influx through G(i/o) signaling pathway in pituitary lactotrophs. Mol Pharmacol 61:1329–1339[Abstract/Free Full Text]
  20. Andric SA, Zivadinovic D, Gonzalez-Iglesias AE, Lachowicz A, Tomic M, Stojilkovic SS 2005 Endothelin-induced, long lasting, and Ca2+ influx-independent blockade of intrinsic secretion in pituitary cells by Gz subunits. J Biol Chem 280:26896–26903[Abstract/Free Full Text]
  21. Samson WK 1992 The endothelin-A receptor subtype transduces the effects of the endothelins in the anterior pituitary gland. Biochem Biophys Res Commun 187:590–595[CrossRef][Medline]
  22. Kanyicska B, Freeman ME 1993 Characterization of endothelin receptors in the anterior pituitary gland. Am J Physiol 265:E601–E608
  23. Bertram R, Tabak J, Toporikova N, Freeman ME 2006 Endothelin action on pituitary lactotrophs: one receptor, many GTP-binding proteins. Sci STKE 2006:pe4
  24. Shyamala V, Moulthrop TH, Stratton-Thomas J, Tekamp-Olson P 1994 Two distinct human endothelin B receptors generated by alternative splicing from a single gene. Cell Mol Biol Res 40:285–296[Medline]
  25. Elshourbagy NA, Adamou JE, Gagnon AW, Wu HL, Pullen M, Nambi P 1996 Molecular characterization of a novel human endothelin receptor splice variant. J Biol Chem 271:25300–25307[Abstract/Free Full Text]
  26. Nambi P, Wu HL, Ye D, Gagnon A, Elshourbagy N 2000 Characterization of a novel porcine endothelin(B) receptor splice variant. J Pharmacol Exp Ther 292:247–253[Abstract/Free Full Text]
  27. Miyamoto Y, Yoshimasa T, Arai H, Takaya K, Ogawa Y, Itoh H, Nakao K 1996 Alternative RNA splicing of the human endothelin-A receptor generates multiple transcripts. Biochem J 313:795–801[Medline]
  28. Bourgeois C, Robert B, Rebourcet R, Mondon F, Mignot TM, Duc-Goiran P, Ferre F 1997 Endothelin-1 and ETA receptor expression in vascular smooth muscle cells from human placenta: a new ETA receptor messenger ribonucleic acid is generated by alternative splicing of exon 3. J Clin Endocrinol Metab 82:3116–3123[Abstract/Free Full Text]
  29. Rubanyi GM, Polokoff MA 1994 Endothelins: molecular biology, biochemistry, pharmacology, physiology, and pathophysiology. Pharmacol Rev 46:325–415[Medline]
  30. Fields TA, Casey PJ 1995 Phosphorylation of Gz {alpha} by protein kinase C blocks interaction with the ß {gamma} complex. J Biol Chem 270:23119–23125[Abstract/Free Full Text]
  31. Beaulieu JM, Sotnikova TD, Marion S, Lefkowitz RJ, Gainetdinov RR, Caron MG 2005 An Akt/ß-arrestin 2/PP2A signaling complex mediates dopaminergic neurotransmission and behavior. Cell 122:261–273[CrossRef][Medline]
  32. Gentles AJ, Karlin S 1999 Why are human G-protein-coupled receptors predominantly intronless? Trends Genet 15:47–49[CrossRef][Medline]
  33. Shraga-Levine Z, Sokolovsky M 2000 Functional coupling of G proteins to endothelin receptors is ligand and receptor subtype specific. Cell Mol Neurobiol 20:305–317[CrossRef][Medline]
  34. Rebsamen MC, Church DJ, Morabito D, Vallotton MB, Lang U 1997 Role of cAMP and calcium influx in endothelin-1-induced ANP release in rat cardiomyocytes. Am J Physiol 273:E922–E931
  35. Gohla A, Schultz G, Offermanns S 2000 Role for G(12)/G(13) in agonist-induced vascular smooth muscle cell contraction. Circ Res 87:221–227[Abstract/Free Full Text]
  36. Minneman KP 2001 Splice variants of G protein-coupled receptors. Mol Interv 1:108–116[Abstract/Free Full Text]
  37. Monsma Jr FJ, McVittie LD, Gerfen CR, Mahan LC, Sibley DR 1989 Multiple D2 dopamine receptors produced by alternative RNA splicing. Nature 342:926–929[CrossRef][Medline]
  38. de la Pena P, Delgado LM, del Camino D, Barros F 1992 Two isoforms of the thyrotropin-releasing hormone receptor generated by alternative splicing have indistinguishable functional properties. J Biol Chem 267:25703–25708[Abstract/Free Full Text]
  39. Namba T, Sugimoto Y, Negishi M, Irie A, Ushikubi F, Kakizuka A, Ito S, Ichikawa A, Narumiya S 1993 Alternative splicing of C-terminal tail of prostaglandin E receptor subtype EP3 determines G-protein specificity. Nature 365:166–170[CrossRef][Medline]
  40. Sugimoto Y, Negishi M, Hayashi Y, Namba T, Honda A, Watabe A, Hirata M, Narumiya S, Ichikawa A 1993 Two isoforms of the EP3 receptor with different carboxyl-terminal domains. Identical ligand binding properties and different coupling properties with Gi proteins. J Biol Chem 268:2712–2718[Abstract/Free Full Text]
  41. Charo IF, Myers SJ, Herman A, Franci C, Connolly AJ, Coughlin SR 1994 Molecular cloning and functional expression of two monocyte chemoattractant protein 1 receptors reveals alternative splicing of the carboxyl-terminal tails. Proc Natl Acad Sci USA 91:2752–2756[Abstract/Free Full Text]
  42. Sakamoto A, Yanagisawa M, Sawamura T, Enoki T, Ohtani T, Sakurai T, Nakao K, Toyo-oka T, Masaki T 1993 Distinct subdomains of human endothelin receptors determine their selectivity to endothelinA-selective antagonist and endothelinB-selective agonists. J Biol Chem 268:8547–8553[Abstract/Free Full Text]
  43. Savarese TM, Fraser CM 1992 In vitro mutagenesis and the search for structure-function relationships among G protein-coupled receptors. Biochem J 283:1–19[Medline]
  44. Hashido K, Adachi M, Gamou T, Watanabe T, Furuichi Y, Miyamoto C 1993 Identification of specific intracellular domains of the human ETA receptor required for ligand binding and signal transduction. Cell Mol Biol Res 39:3–12[Medline]
  45. Aramori I, Nakanishi S 1992 Coupling of two endothelin receptor subtypes to differing signal transduction in transfected Chinese hamster ovary cells. J Biol Chem 267:12468–12474[Abstract/Free Full Text]
  46. Eguchi S, Hirata Y, Imai T, Marumo F 1993 Endothelin receptor subtypes are coupled to adenylate cyclase via different guanyl nucleotide-binding proteins in vasculature. Endocrinology 132:524–529[Abstract]
  47. Khac LD, Naze S, Harbon S 1994 Endothelin receptor type A signals both the accumulation of inositol phosphates and the inhibition of cyclic AMP generation in rat myometrium: stimulation and desensitization. Mol Pharmacol 46:485–494[Abstract]
  48. Takagi Y, Ninomiya H, Sakamoto A, Miwa S, Masaki T 1995 Structural basis of G protein specificity of human endothelin receptors. A study with endothelinA/B chimeras. J Biol Chem 270:10072–10078[Abstract/Free Full Text]
  49. Bibb JA 2005 Decoding dopamine signaling. Cell 122:153–155[CrossRef][Medline]
  50. Birnbaumer L 2000 Adenylyl cyclase. In: Conn PM, Means AR, eds. Principles of molecular regulation. Totowa, NJ: Humana Press; 249–260
  51. Conti M 2000 Phosphodiesterases and cyclic nucleotide signaling in endocrine cells. Mol Endocrinol 14:1317–1327[Free Full Text]
  52. Sakamoto A, Yanagisawa M, Sakurai T, Takuwa Y, Yanagisawa H, Masaki T 1991 Cloning and functional expression of human cDNA for the ETB endothelin receptor. Biochem Biophys Res Commun 178:656–663[CrossRef][Medline]
  53. Hosoda K, Nakao K, Hiroshi A, Suga S, Ogawa Y, Mukoyama M, Shirakami G, Saito Y, Nakanishi S, Imura H 1991 Cloning and expression of human endothelin-1 receptor cDNA. FEBS Lett 287:23–26[CrossRef][Medline]
  54. Cyr CR, Rudy B, Kris RM 1993 Prolonged desensitization of the human endothelin A receptor in Xenopus oocytes. Comparative studies with the human neurokinin A receptor. J Biol Chem 268:26071–26074[Abstract/Free Full Text]
  55. Freedman NJ, Ament AS, Oppermann M, Stoffel RH, Exum ST, Lefkowitz RJ 1997 Phosphorylation and desensitization of human endothelin A and B receptors. Evidence for G protein-coupled receptor kinase specificity. J Biol Chem 272:17734–17743[Abstract/Free Full Text]
  56. Cramer H, Muller-Esterl W, Schroeder C 1997 Subtype-specific desensitization of human endothelin ETA and ETB receptors reflects differential receptor phosphorylation. Biochemistry 36:13325–13332[CrossRef][Medline]
  57. Hunyady L, Catt KJ, Clark AJ, Gaborik Z 2000 Mechanisms and functions of AT(1) angiotensin receptor internalization. Regul Pept 91:29–44[CrossRef][Medline]
  58. Koch T, Schulz S, Schroder H, Wolf R, Raulf E, Hollt V 1998 Carboxyl-terminal splicing of the rat µ opioid receptor modulates agonist-mediated internalization and receptor resensitization. J Biol Chem 273:13652–13657[Abstract/Free Full Text]
  59. Ciruela F, McIlhinney RA 1997 Differential internalisation of mGluR1 splice variants in response to agonist and phorbol esters in permanently transfected BHK cells. FEBS Lett 418:83–86[CrossRef][Medline]
  60. Bremnes T, Paasche JD, Mehlum A, Sandberg C, Bremnes B, Attramadal H 2000 Regulation and intracellular trafficking pathways of the endothelin receptors. J Biol Chem 275:17596–17604[Abstract/Free Full Text]
  61. Oksche A, Boese G, Horstmeyer A, Furkert J, Beyermann M, Bienert M, Rosenthal W 2000 Late endosomal/lysosomal targeting and lack of recycling of the ligand-occupied endothelin B receptor. Mol Pharmacol 57:1104–1113[Abstract/Free Full Text]
  62. Paasche JD, Attramadal T, Kristiansen K, Oksvold MP, Johansen HK, Huitfeldt HS, Dahl SG, Attramadal H 2005 Subtype-specific sorting of the ETA endothelin receptor by a novel endocytic recycling signal for G protein-coupled receptors. Mol Pharmacol 67:1581–1590[Abstract/Free Full Text]
  63. Paasche JD, Attramadal T, Sandberg C, Johansen HK, Attramadal H 2001 Mechanisms of endothelin receptor subtype-specific targeting to distinct intracellular trafficking pathways. J Biol Chem 276:34041–34050[Abstract/Free Full Text]
  64. Gregan B, Jurgensen J, Papsdorf G, Furkert J, Schaefer M, Beyermann M, Rosenthal W, Oksche A 2004 Ligand-dependent differences in the internalization of endothelin A and endothelin B receptor heterodimers. J Biol Chem 279:27679–27687[Abstract/Free Full Text]
  65. Maeda S, Wu S, Juppner H, Green J, Aragay AM, Fagin JA, Clemens TL 1996 Cell-specific signal transduction of parathyroid hormone (PTH)-related protein through stably expressed recombinant PTH/PTHrP receptors in vascular smooth muscle cells. Endocrinology 137:3154–3162[Abstract]




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