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Molecular Characterization of a Dual Endothelin-1/Angiotensin II Receptor
Molecular Medicine volume 4, pages 96–108 (1998)
The molecular recognition theory (MRT) provides a conceptual framework that could explain the evolution of intermolecular and intramolecular interaction of peptides and proteins. As such, it predicts that binding sites of peptide hormones, and its receptor binding sites were originally encoded by and evolved from complementary strands of genomic DNA.
Materials and Methods
On the basis of principles underlying the MRT, we screened a rat brain complementary DNA library using an AngII followed by an endothelin-1 (ET-1) antisense oligonucleotide probe, expecting to isolate potential cognate receptors.
An identical cDNA clone was isolated independently from both the AngII and ET-1 oligonucleotide screenings. Structural analysis revealed a receptor polypeptide containing a single predicted transmembrane region with distinct ET-1 and AngII putative binding domains. Functional analysis demonstrated ET-1-and AngII-specific binding as well as ET-1- and AngII-induced coupling to a Ca2+ mobilizing transduction system. Amino acid substitutions within the predicted ET-1 binding domain obliterate ET-1 binding while preserving AngII binding, thus defining the structural determinants of ET-1 binding within the dual ET-1/AngII receptor, as well as corroborating the dual nature of the receptor.
Elucidation of the dual ET-1/AngII receptor provides further molecular genetic evidence in support of the molecular recognition theory and identifies for the first time a molecular link between the ET-1 and AngII hormonal systems that could underlie observed similar physiological responses elicited by ET-1 and AngII in different organ systems. The prominent expression of the ET-1 /AngII receptor mRNA in brain and heart tissues suggests an important role in cardiovascular function in normal and pathophysiological states.
The hydropathic character of an amino acid appears to be determined by the second base of a particular codon (1). A second base, U, generally specifies hydrophobic amino acids, while a second base. A, specifies hydrophilic amino acids (1). Since A and U are complementary, amino acid sequences derived from complementary DNA strands will generate peptides of inverted patterns of hydropathy. This fundamental observation provides the basis for the molecular recognition theory, which hypothesizes that complementary nucleotide sequences specify peptides that could interact through complementary structures as a result of their exactly inverted pattern of amino acid hydropathy (1). This theory further suggests that binding sites of interacting proteins, such as peptide hormone and receptors (2), and peptide substrate and enzyme (3), evolved from complementary strands of genomic DNA. This molecular paradigm further implies that the antisense peptide encoded by the antisense strand of peptide hormone mRNAs (spanning the hormone) represent the putative evolutionary precursor binding domain of corresponding peptide hormones. As a corollary, it can be envisioned that mutations in these precursor binding domains favoring an increased ligand-receptor affinity have occurred over time, leading to today’s respective binding domains.
Numerous biochemical studies have reported experimental evidence in support of this theory (4,5). However, the molecular recognition theory still remains controversial because of some negative biochemical and theoretical analysis (6–10). Our recent report identifying the AngII/AVP receptor represents the first molecular genetic evidence in support of this theory (11). Our current studies were designed to further test this theory by screening a rat brain complementary DNA library using an AngII followed by an endothelin-1 (ET-1) antisense oligonucleotide probe to potentially isolate additional AngII and ET-1 receptor isoforms. As with the AngII/AVP receptor (11), an identical cDNA clone was isolated independently from both the AngII and ET-1 oligonucleotide screenings. Functional and mutational studies of the predicted polypeptide elucidates a novel dual ET-1/AngII receptor, providing further molecular genetic evidence in support of the molecular recognition theory, as well as demonstrating for the first time a molecular link between the ET-1 and AngII hormonal systems. The identification of the structural determinants of hormone binding within the dual ET-1/AngII receptor provides new insight into the evolutionary origin of hormone-binding domains present in different iso-receptors.
Materials and Methods
cDNA Screening and Nucleotide Sequencing
An adult rat brain Agtl 1 cDNA library (Clontech) was screened twice independently with the ET-1 [5′ - CC AGATGATGTCCAGGTGGCAGAAGTAGA CACACTCTTTATCCATCAGGGAAGAGCAGGAG CA-3′] (12) and AngII [5′-AAAGGGGTGGATGT ATACGCGGTC-3′] (13) antisense oligonucleotide probes. Hybridizations were done as described (11). After the first round of hybridization, a significant number of potential positive signals were detected. Of these, we proceeded to isolate five cDNA clones hybridizing to the AngII probe and three cDNA clones hybridizing to the ET-1 probe. These clones showed the greatest signal to background ratio and they represented less than 5% of the potential positive signals. Among these clones we detected the 3.3-kb cDNA isolated in both AngII and ET-1 screenings. The 3.3-kb cDNAs were subcloned into Ml3 vectors and subsequently sequenced on both strands as described elsewhere (11). DNA sequence analysis was done using the GENEPRO computer DNA analysis program (Riverside Scientific Enterprises).
Expression Studies in Cos1pMAM-ET-1/AngII Transfectants
The 756-bp SspI-EcoRI fragment spanning nucleotides 2518–3274 of the full-length ET-1/AngII receptor cDNA was subcloned directionally (5′ – 3′) into the Nhel site of the pMAMneo expression vector (Clontech). The pMAM-ET-1/AngII expression vector was then transfected into Cosl cells and stable transfectants were selected and maintained in G418 for subsequent studies. Expression of mutant receptors was done similarly using the pMAM-ET-1/AngII R64G and pMAM-ET-1/AngII W65G expression vectors. 45Ca2+ efflux assays were done essentially as previously described (14). Cell cultures (P 35 dishes) were equilibrated with 45Ca2+ under standard growth conditions (DMEM + 10% FCS + 250 µg/ml G418 + 5 µCi/ml of 45CaCl2) for 18 to 20 hr. Assays were initiated by removal of the 45Ca2+ media and followed by rapid sequential washing and aspirations with three 1-ml aliquots of physiological buffer (PB composition in mmol/L: NaCl 140; KCl 5.4; CaCl2 1.8; MgCl2 1.6; D-glucose 5.5; Hepes 5, pH 7.4). One milliliter of assay solution containing appropriate ligands dissolved in PB were promptly added and efflux was allowed to proceed for the specific time intervals. Efflux was terminated by rapid washing and aspiration with three 1-ml aliquots of MgCl2-free PB containing 5 mmol/L LaCl3. Residual 45Ca2+ content was expressed relative to total isotope present in cultures that received the wash protocol without any intervening efflux interval (time 0 = 12,050 ± 941 cpm). All assays were done at 37°C. 125I-ET-1 binding experiments were performed on intact cells in Dulbecco’s modified Eagle medium (DMEM) supplemented with 20 mmol/L Hepes, pH 7.3 and 0.1 % bovine serum albumin (BSA). Incubations were performed at 37°C for 60 min. 125I-AngII binding experiments were performed as described elsewhere (11). Specific binding was determined as the difference between the total radioactivity bound to cells and the radioactivity bound to blanks containing 1 µmol/L ET-1 or 1 µmol/L AngII. Affinity constants were determined by Scatchard analysis (RADLIG, Version 4 Program, McPherson).
A polyclonal rabbit antipeptide antibody (custom-made by Multiple Peptide Systems, San Diego, CA) was raised against the synthetic peptide P51LLTSLGSKE60 spanning a portion of the predicted extracellular domain of the ET-1/AngII receptor. Control CoslpMAMneo and test Cos1pMAM-ET-1/AngII cells were reacted with this antibody (1:500) and immunostained as described previously (11).
Mutants were constructed by oligonucleotide-directed mutagenesis using the Transformer Site-Directed Mutagenesis Kit (Clontech) and the pMAM-ET-l/AngII756 as template. Mutagenic oligonucleotides were as follows: 5′-CCA-GTT-CCA-GCC-AGA-CTT-CAT-CTC-3′ (antisense strand) replacing the codon CGC (R64) by GGC (G64) and 5′-CCA-GTT-CCC-GCG-AGA-CTT-CAT-CTC-3′ (antisense strand) replacing the codon TGG (W65) by GGG (G65). Both mutants were verified by nucleotide sequencing of the entire amino acid coding region of the ET-1/AngII receptor to ensure the absence of unwanted mutations. Mutant recombinants were called pMAM-ET-1 /AngII R64G and pMAM-ET-1/AngII W65G.
Isolation of the Identical cDNA Clone from Independent AngII and ET-1 Screenings
The isolation of the ET-1/AngII receptor cDNA was accomplished by using the same strategy employed for the identification of the dual AngII/ AVP receptor (11). In this particular case, we screened 0.5 × 106 recombinants from an adult rat brain cDNA library using a 24-base AngII antisense oligonucleotide probe followed by a 6 3-base ET-1 antisense oligonucleotide probe (see Materials and Methods). Unexpectedly, an identical cDNA clone, approximately 3.3 kb in length, was isolated independently from the AngII and ET-1 oligonucleotide screening. With the precedence of a dual AngII/AVP receptor (11), the logical prediction was that this 3.3-kb brain cDNA might encode a dual ET-1 /AngII receptor.
Structural Analysis of the ET-1/AngII Receptor cDNA
Nucleotide sequence analysis of the 3.3-kb ET-1/AngII receptor cDNA revealed a single open reading frame (ORF) encoding a protein of 127 amino acids with a predicted molecular mass of 13,698 daltons (Fig. 1A). A single region with significant homology was found for each antisense peptide sequence in the 127 amino acid (aa) ORF. Comparison of the ET-1 and AngII cRNA (complementary RNA) sequences with the nucleotide and amino acid sequences of the ET-1/AngII receptor identified E60MKSRWNW67 as a potential ET-1 binding domain (Fig. 1C), and G41AASMQV47 as a potential AngII binding domain (Fig. 1C) within the ET-1/AngII receptor. This analysis also delineates the antisense peptide from frame 2 as the putative evolutionary ET-1 precursor binding domain (Fig. 1C), whereas the antisense peptide from frame 3 is the putative evolutionary AngII precursor binding domain (Fig. 1C). Consistently, the single region with the highest nucleotide sequence homology to the AngII oligonucleotide probe (63% identity spanning nucleotides [nt] 2802–2820) and the single region with the highest nucleotide sequence homology to the ET-1 oligonucleotide probe (65% identity spanning nt 2859–2881) are distinct and correspond to the AngII and ET-1 antisense peptide homology regions (Fig. 1). According to the molecular recognition theory, these antisense homology regions should identify putative AngII and ET-1 binding domains, respectively.
Hydropathy analysis using the Kyte-Doolittle scale (15) and GES hydropathy plot (16) predicts a single transmembrane domain (H-l; Fig. 1A). H-l (n = 20 amino acids) is predicted to cross the plasma membrane considering that a transmembrane region of 12–14 amino acids have been experimentally proven to be sufficient to cross the plasma membrane (17). The existence of a single transmembrane-spanning region in conjunction with the identification of the putative AngII and ET-1 binding domains within the amino terminal end predict the localization of the ET-1/AngII receptor amino terminal end to the extracellular side (Fig. 1B). Two potential phosphorylation sites for cAMP-dependent protein kinase, S91 and T108 (18,19), can be noted in the predicted cytoplasmic carboxyl-end (Fig. 1). A potential internalization recognition sequence (IRS) is also found in the cytoplasmic carboxyl-end: Y111RRP114 (Fig. 1), resembling the IRS present in human lysosomal acid phosphatase (20,21). The structural features of the ET-1/AngII polypeptide are consistent with a single-transmembrane dual hormone receptor containing distinct ET-1 and AngII binding domains.
Functional Analysis of the ET-1/AngII Receptor
Although no other significant ORFs were detected in the cDNA, because of the unusual structure of the ET-1/AngII receptor and the localization of the ET-1/AngII receptor ORF to the 3′ end (Fig. 1A), we tested the possibility that this mRNA could be polycistronic in nature, thus encoding additional polypeptides within the long 5′ UT (approximately 2.7 kb) necessary for receptor function. For this purpose, two different expression vectors were constructed. The 3274-bp ET-1/AngII receptor cDNA was subcloned directionally (5′ to 3′) into the NheI site of the pMAMneo expression vector (Clontech) to generate pMAM-ET-l/AngII3274. Similarly, a 756-bp SspI-EcoRI fragment spanning nucleotides 2518–3274 of the full-length cDNA (including the 127 aa ORF, Fig. 1A) was subcloned into the pMAMneo expression vector to generate pMAM-ET-l/AngII756. Both expression vectors were stably transfected into Cos 1 cells and assayed for 125I-AngII and 125I-ET-1 binding. These binding experiments showed equivalent expression of ET-1/AngII receptors in CoslpMAM-ET-1/AngII3274 and Cos1pMAM-ET-1/AngII756 transfectants (data not shown). These results demonstrate that the predicted 127 aa open reading frame is necessary and sufficient for receptor function and that the 127 aa polypeptide can interact specifically with ET-1 and AngII as predicted by the structural analysis (Fig. 1). Control nontransfected and mock-transfected cell did not reveal any binding or activation of second messenger systems upon addition of ET-1 and AngII. All subsequent experiments presented below were conducted with CoslpMAM-ET-1/AngII756 transfectants (abbreviated as Cos1pMAM-ET-1/AngII).
Because ET-1 receptors are functionally coupled to a Ca2+ mobilizing transduction system involving phospholipase C (22), the Cos1pMAM-ET-1/AngII transfectants were tested for their ability to support peptide hormone-induced Ca2+ mobilization. Analysis of a panel of peptide hormones revealed that Cos1pMAM-ET-1/AngII transfectants responded to ET-1 and AngII (Fig. 2A, B). Measured as the % 45Ca2+ efflux activity indicated by % 45Ca2+ retained (14), 100 nmol/L ET-1 and 100 nmol/L AngII elicited equivalent amounts of Ca2+ mobilization (Fig. 2B). No responses were elicited by either arginine-vasopressin (AVP) or bradykinin. In comparison to ET-1, ET-2 induced 60% 45Ca2+ efflux activity, whereas ET-3 induced none at all. Similarly, compared with AngII, AngIIl induced 25% 45Ca2+ efflux activity, whereas Angl induced none at all (Fig. 2B). The effects of antagonists on the ET-1/AngII receptor were also analyzed. An AngII-specific antagonist (Sar1, Ala8-AngII) efficiently blocked both AngIIand ET-1-induced 45Ca2+ efflux (Fig. 2B). This is consistent with the notion that AngII- and ET-1-induced responses were mediated by the same receptor.
The specificities of the ET-1 and AngII responses of the dual ET-1/AngII receptor were then assessed in concentration dependence assays measuring ET-1- and AngII-induced 45Ca2+ efflux. The response to both ET-1 and AngII were similar with equivalent effector concentration for half-maximal response (EC50) values of 7 ± 1.4 pmol/L (Fig. 2C) and 6 ± 0.9 pmol/L (Fig. 2D), respectively. Pharmacologic specificity was further ascertained by ligand dissociation studies. As shown in Fig. 2E and F, high (KH) and low (KL) affinity sites were detected for both ET-1 and AngII with KH of 0.05 ± 0.01 nmol/L for ET-1 (Bmax = 6.0 ± 1.2 fmol 10−6 cells) and 0.27 ± 0.046 nmol/L for AngII (Bmax = 5.4 ± 0.8 fmol 10−6 cells). The KL for ET-1 was 50 ± 13.5 nmol/L (Bmax = 72 ± 11.0 fmol 10−6 cells); KL for AngII was 16 ± 2.4 nmol/L (Bmax = 78 ± 9.4 fmol 10−6 cells). Interestingly, 125I-ET-1 was not displaced by AngII, nor was 125I-AngII displaced by ET-1 (Fig. 2E, F). Displacement of ET-1 by ET-2 was also detected with high- and low-affinity sites with a KiH of 1 ± 0.13 nmol/L and KiL of 300 ± 33 nmol/L (Fig. 2E). In contrast, ET-3 displayed a single low-affinity site with a KiL of 20 ± 1.6 nmol/L (Fig. 2E). Displacement of AngII by the AT1 receptor-specific antagonist, Losartan, was detected with a KiH of 0.7 ± 0.14 nmol/L and KiL of 160 ± 19 nmol/L (Fig. 2F). The AT2-specific antagonist, PD123319, did not displace AngII binding up to concentrations of 10−5 mol/L (Fig. 2F). These results define the ET-1/AngII receptor as a novel AT1 receptor isoform coupled to Ca2+-mobilizing pathways.
Immunocytochemical Localization of the ET-1/AngII Receptor
In order to ascertain the cytolocalization and predicted topography of the ET-1/AngII receptor polypeptide, immunocytochemical studies were done on intact (nonpermeabilized) CoslpMAM-ET-1/AngII transfectants utilizing a polyclonal antipeptide antibody raised against amino acids 51–60 of the predicted extracellular domain (Fig. 1). As shown in Fig. 3, prominent staining was observed in pMAM-ET-1/AngII transfectants (Fig. 3B). In contrast, the cells transfected with the parental expression vector, CoslpMAMneo, were not stained (Fig. 3A). These results corroborate the predicted topography and cell membrane localization of the ET-1/AngII receptor.
Tissue Distribution of the ET-1/AngII Receptor mRNA
Analysis of the ET-1 / AngII receptor mRNA tissue distribution by Northern blot analysis (Fig. 4) revealed an approximately 4-kb mRNA prominently expressed in brain and heart tissues. Much lower but detectable levels can be observed in aorta, adrenal gland, and lung tissues. Northern analysis did not detect ET-1/AngII receptor sequences in kidney and liver. However, reverse transcriptase-polymerase chain amplification of 5′ untranslated RNA sequences could detect the ET-1/AngII mRNA in all tissues tested, including kidney and liver (data not shown).
Site-Directed Mutagenesis of the ET-1 Binding Domain
In order to determine whether the putative ET-1 binding domain, as delineated by the homology to antisense peptide sequences (Fig. 1), indeed represents the structural determinants for ET-1 binding within the ET-1/AngII receptor, we performed mutational studies on the putative ET-1 binding domain while preserving the structure of the putative AngII binding domain. More specifically, our strategy involved modifying the ET-1 binding domain without affecting, or only minimally so, the AngII binding site which would serve as an ideal internal control for potential “unwanted conformation effects” of the mutagenesis directed towards the ET-1 binding domain. We therefore chose to replace R64 and W65, amino acids that contain the largest side chains among the naturally occurring amino acids, for glycine (G), an amino acid that contains the smallest side chain (hydrogen). Such drastic changes in size of the side chain at amino acid positions 64 and 65 were expected to have a prominent effect on the putative conformation of this peptide region with concomitant detrimental effect on ET-1 binding.
The results of the mutational studies (Fig. 5, Table 1) support the hypothesis that motif E60MKSRWNW67 is a structural determinant of ET-1 binding in the ET-1/AngII receptor. Substitution of R64 by glycine (R64G; Fig. 5C, D, Table 1) and substitution of W65 by glycine (W65G; Fig. 5E, F, Table 1) completely abrogate ET-1 binding without or minimally affecting AngII binding. It is noted that the R64G substitution did not affect AngII binding (Table 1), whereas W65G substitution decreased the AngII affinity by 5.7-fold (from wild-type KH of AngII = 1.30 ± 0.30 nmol/L to W65G KH AngII = 7.36 ± 1.50 nmol/L; Table 1).
The elucidation of the ET-1/AngII receptor as a single transmembrane domain receptor is unique among AngII (11,23–27) and ET (28–30) seven-transmembrane domain G-protein-coupled receptors. The dual ET-1/AngII receptor is functionally coupled to a Ca2+-mobilizing transduction system, responding equivalently to both ET-1 and AngII in a highly specific manner. The ET-1 and AngII EC50 values of 7 pmol/L and 6 pmol/L, respectively, strongly suggest that this receptor is fully functional under normal physiological conditions as their respective EC50 values fall well within the range of normal circulating levels of respective peptide hormones (31,32). Interestingly, Sar1-Ala8-AngII (AngII-specific antagonist) blocked both the AngII- and ET-l-induced Ca2+ response (Fig. 2B). The molecular mechanism involved in the inhibition of the ET-1 response by the AngII-specific inhibitor, Sar1-Ala8-AngII, most likely involves inhibition of ET-l-induced receptor conformational transition necessary to activate the downstream transducing system effected by ET-1 since neither AngII (Fig. 2E) nor Sar1-Ala8-AngII (data not shown) were able to displace 125I-ET-1-specific binding.
ET-1 and AngII can evoke a number of similar responses in different organ systems (33). The existence of the dual ET-1/AngII receptor could partially explain these observations. Recently, it has been reported that both ET-1 and AngII stimulated fibronectin and type IV collagen mRNA expression and mitogenesis in rat mesangial cells (34). Unexpectedly, Losartan (a specific AT1 receptor antagonist) inhibited the ET-1-mediated effects whereas BQ-123 (a specific ETA receptor antagonist) inhibited the AngII-induced fibronectin synthesis and mesangial cell proliferation (34). These data could well be explained by the presence and mediation of these physiologic responses by the ET-1/AngII receptor in rat mesangial cells. The inhibition of ET-1-mediated responses by Losartan (34) resembles the inhibition of ET-l-induced Ca2+ mobilization by Sar1-Ala8-AngII noted above (Fig. 2B). We have detected ET-1/AngII receptor mRNA in kidney; however, its existence in mesangial cells remains to be determined.
Our results demonstrate that the motif E60MKSRWNW67 is necessary for ET-1 binding to the ET-1/AngII receptor. This is in contrast with previous structure-functional studies in which extensive regions within endothelin receptors spanning extracellular, transmembrane, and intracellular portions of the receptor have been invoked to be involved in endothelin binding (35–37). These studies, however, did not exclude the possibility of conformational effects induced by the mutagenic changes because of the lack of “internal control” that could account for induced conformational/allosteric effects. It is interesting to note that the antisense peptide delineating the ET-1 binding domain encompasses the carboxyl-terminal 8 amino acids of the ET-1 peptide, thus indicating that these 8 residues most likely represent the motif within ET-1 that interact with the ET-1/AngII receptor. This is consistent with previous observations implicating the carboxyl-terminal domain of endothelin peptides in their binding to their corresponding receptors (38–40), respectively.
The isolation and characterization of the ET-1/AngII receptor elucidates a new paradigm involved in the evolution of hormone-binding domains in different receptors, thus refining a principle of the molecular recognition theory. The data presented above identified the antisense peptides from frames 2 and 3 as the related evolutionary precursor binding domains for ET-1 and AngII, respectively, in the ET-1/AngII receptor (Fig. 1). On the other hand, the antisense peptides in frame 1 for both AngII and AVP were delineated as the corresponding precursor binding domains within the AngII/AVP receptor (11). These findings suggest that antisense peptides originating from the three possible frames can be used as potential precursor binding domains in the evolutionary pathway of different isoreceptors. This intrinsic property of derived antisense peptides probably had a significant impact in the evolution of peptide hormone-cognate receptor systems by effectively increasing the possibility of producing putative cognate receptors. We anticipate that as more molecular genetic, physicochemical, and crystallographic evidence accumulates in the near future, a more complete understanding of the evolution and principles underlying peptide and protein-protein interaction will emerge, thus opening a window into a fundamental organizational principle that governs structure and function in living cells.
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We thank Dr. Joan Keiser (Parke-Davis) for providing us with PD123319, Dr. Ronald D. Smith (Dupont Merck) for providing us with Losartan potassium, and A. Tsikoudakis for preparation of the graphs and manuscript.
Communicated by S. H. Orkin.
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Ruiz-Opazo, N., Hirayama, K., Akimoto, K. et al. Molecular Characterization of a Dual Endothelin-1/Angiotensin II Receptor. Mol Med 4, 96–108 (1998). https://doi.org/10.1007/BF03401733
- AngII Receptor
- Molecular Recognition Theory (MRT)
- Identical cDNA Clones
- Antisense Oligonucleotide Probes
- Molecular Genetic Evidence