©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structural Basis of G Protein Specificity of Human Endothelin Receptors
A STUDY WITH ENDOTHELINCHIMERAS (*)

Yasutaka Takagi , Haruaki Ninomiya , Aiji Sakamoto (§) , Soichi Miwa , Tomoh Masaki (¶)

From the (1) Department of Pharmacology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The endothelin (ET) family of peptides acts via two subtypes of guanine nucleotide-binding regulatory protein (G protein)-coupled receptors termed ETand ET. ET-1 stimulated cAMP formation in Chinese hamster ovary (CHO) cells stably expressing human wild-type ET(CHO/hETcells) while it inhibited cAMP formation in CHO cells expressing human wild-type ET(CHO/hETcells), and pharmacological evidence indicated that the opposite effects were due to the selective coupling of each receptor subtype with Gs/Gi. To find out a receptor domain(s) that determined the selective coupling, a series of chimeric receptors between hETand hETwas expressed on CHO cells, and the effect of ET-1 on cAMP formation in each cell line was tested. hETwith the replacement of second and/or third intracellular loop (ICLII and/or -III) to the corresponding region(s) of hETfailed to transmit the stimulatory effect of ET-1. hETwith the replacement of ICLIII to the corresponding region of hETfailed to transmit the inhibitory effect of ET-1. A chimeric receptor with ICLII of hETand with ICLIII of hETfailed to transmit both effects. In cells expressing chimeric receptors with ICLII of hETand with ICLIII of hET, ET-1 inhibited cAMP formation while it stimulated cAMP formation when cells were pretreated with pertussis toxin. These results indicated the roles of ICLII and -III of hETR as a major determinant of the selective coupling of hETand hETwith Gs/Gi, respectively. We also demonstrated that each receptor subtype expressed on the same cell could work independently, i.e. for hETto activate Gs and for hETto activate Gi, resulting in dose-dependent dual effects of ET-1 on cAMP formation.


INTRODUCTION

Guanine nucleotide-binding regulatory protein (G protein)() -coupled receptors can be thought of as having two binding domains, a ligand-binding domain on the extracellular side and a G protein-binding domain on the intracellular side. As for the G protein-binding domain, early studies on adrenergic and muscarinic receptors using receptor chimeras, deletions, or point mutations indicated the importance of the third intracellular loop (ICLIII) (1, 2, 3, 4, 5, 6, 7) . Subsequent studies including those using peptides that mimic or inhibit receptor-G protein interaction, however, suggested the presence of multiple points of interaction between receptors and G proteins (8, 9) , and now it is generally assumed that the binding surface comprises, at least, regions from ICLII, ICLIII, and carboxyl-terminal cytoplasmic tail; each receptor region contributes to G protein specificity in a combinatorial fashion, enhancing or restricting interaction with particular G proteins (10, 11) . Recent identifications of splice variants with differential G protein specificities of prostaglandin E receptor (12) and of pituitary adenylyl cyclase-activating polypeptide receptor (13) illustrated the examples of in vivo tuning of G protein selection by receptor sequences in carboxyl terminus and ICLIII, respectively.

The endothelins (ETs) are a family of potent vasoactive peptides termed endothelin-1, -2, and -3 (ET-1, -2, and -3) (14, 15) . cDNA cloning of cell-surface receptors for ET-1 identified two subtypes of ET receptors (ETRs), denoted ETand ET, both of which belong to a family of G protein-coupling receptors (16, 17) . The two subtypes can be pharmacologically distinguished by different rank orders of affinity toward the three ET isopeptides; ETis ET-1 selective, showing an affinity rank order of ET-1 ET-2 ET-3, whereas ETexhibits similar affinities to all three isopeptides (16, 17) . In other words, ET-1 is a nonselective agonist, and ET-3 is an ET-selective agonist. Human ETand ET(hETand hET) exhibit a high polypeptide sequence identity to each other (55% overall, 74% within the putative transmembrane helices) (18, 19) . Of the receptor subdomains, the extracellular amino terminus and the intracellular carboxyl terminus display the least sequence similarities, whereas the sequences both in ICLII and -III are relatively well conserved (16 out of 21 residues of ICLII and 21 out of 29 residues of ICLIII are identical). The significance of the heterogeneity of either amino or carboxyl terminus in the subtype-specific receptor functions has not yet been elucidated.

Initial studies indicated that both subtypes of hETR coupled with pertussis toxin (PTX)-insensitive G protein, possibly a member(s) of Gq family, to activate phospholipase C (20) . It is, however, now apparent that both of them belong to a subfamily of G protein-coupled receptors with a promiscuous nature, which can activate multiple types of G proteins. Interaction of ETR with Gs has been suggested by studies demonstrating ET-1-induced increase in cAMP formation in rat vascular smooth muscle cells (21) , bovine tracheal cells (22) , or Chinese hamster ovary (CHO) cells expressing rat ET(23) . Interaction of ETR with Gi has been suggested by studies demonstrating ET-1-induced decrease in cAMP formation in bovine endothelial cells (21) , C6 glioma cells (24) , or CHO cells expressing bovine ET(23) .

In a preliminary study, we found that hETand hET, when expressed on CHOK1 cells, transmitted opposite effects on cAMP formation in the cells. Because pharmacological evidence indicated that the opposite effects were due to the selective coupling of each receptor subtype with Gs/Gi, we set out to reveal the structural basis for it using hETchimeras. To circumvent the potential influence of variable receptor densities on cell responses, we generated stable transfectants of each receptor construct and subjected clones with similar receptor densities to the study.

Expression of ETand ETare not exclusive to each other in native cells; some types of cells express both mRNAs for the receptor subtypes (25) . In an attempt to obtain an experimental model that mimicked the situation, we also generated a clonal cell line that co-expressed hETand hETand studied the signaling events activated by ET-1.


MATERIALS AND METHODS

Reagents

Materials were obtained from the following sources: HAM F12 medium, fetal calf serum, lipofectamine, G418, and blasticidin from Life Technologies, Inc. (Tokyo); synthetic human ET-1 and ET-3 from Peptide Institute (Osaka, Japan); I-ET-1 (74 TBq/mmol) and cAMP radioimmunoassay assay kit from Amersham (Buckinghamshire, United Kingdom); PTX and U-73122 (1-(6-[(17-3-methoxyestra-1,3,5 (10) -trien-17-yl)amino]hexyl)-1H-pyrrole-2,5-dione) from Funakoshi Co. Ltd. (Tokyo, Japan); Fura-2 acetoxymethyl ester from Dojin Chemicals (Tokyo); BCA microprotein assay kit from Pierce. The ETantagonist 97-139 (27- O-3-[2-(3-carboxy-acryloylamino)-5-hydroxyphenyl]-acryloyloxy myricerone) was kindly provided by Shionogi & Co. (Osaka, Japan), and the ETantagonist BQ788 ( N- cis-2,6-dimethylpiperidinocarbonyl - L - - methylleucyl - D 1 - methoxycarbonyltryptophanyl-D-norleucine) was kindly provided by Ciba-Geigy Japan Ltd. (Hyogo, Japan). All other chemicals were of reagent grade and were obtained commercially.

Stable Expression of Wild-type and Chimeric hETRs in CHO Cells

To obtain a cell line stably expressing hETR, we used SR promoter-based mammalian expression vector pME18Sf, which carried cDNA construct encoding wild-type or chimeric hET. Procedures for construction and subcloning of wild-type and chimeric receptor cDNA were exactly the same as those previously described (26) . Each expression plasmid was cotransfected with pSVneoplasmid into CHOK1 cells by lipofection, using lipofectamine according to the manufacturer's instructions. Cell populations expressing neogene product were selected in HAM F12, 10% fetal calf serum containing G418 (0.5 mg/ml). From these selected cell populations, clonal cell lines were isolated by colony lifting and maintained in the same selection medium. The density of receptors expressed in each clone was determined by saturation isotherms of I-ET-1 binding to the crude membrane preparations as described below. We have isolated several clones for each expression plasmid with various receptor densities. Cell clones showing similar levels of receptor densities were used for the present study (see ``Results'' for the actual binding capacities). To obtain a cell line that expressed both hETand hET, wild-type hETcDNA was cotransfected with pSVbsrplasmid into CHOK1 cells stably expressing wild-type hET, and the cells were selected for resistance against blasticidin (10 µg/ml). The ratio of the densities of hET/hETexpressed on each clone was determined by displacement study of I-ET-1 binding as described below.

I-ET-1 Binding Assay

Receptor densities were determined by I-ET-1 binding assay according to the protocols previously described (17) with slight modifications. In brief, crude membrane preparations were prepared by lysis of the cells suspended in PBS by sonication followed by centrifugation at 100,000 g for 30 min. The pellets were suspended in PBS, and protein concentrations were determined by BCA microprotein assay kit. The membrane preparations (0.5-1.0 µg of protein/assay) were incubated with various concentrations (10-1000 pM) of I-ET-1 in 0.2 ml of PBS, 0.2% bovine serum albumin. After incubation for 60 min at 25 °C, bound ligand was separated from free ligand by filtration through Whatman GF/C glass fiber filters pre-immersed in the ice-cold binding medium. The filters were washed with ice-cold PBS and counted for radioactivity in a -counter. Nonspecific binding was defined as the binding in the presence of 100 nM unlabeled ET-1 and was always less than 10% of the total binding activity. For displacement study of I-ET-1 binding, the membrane preparations were incubated with 100 pM of I-ET-1 and various concentrations (1 pM-1 µM) of either ET-1 or ET-3.

PTX Pretreatment of the Cells and Measurement of Cyclic AMPFormation

Cells in 48-well plates at 50% confluency were incubated for 16 h with or without PTX (50 ng/ml). The cells were washed twice with PBS and then incubated at 37 °C for 10 min with 0.25 ml of PBS containing a phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) (1 mM). IBMX alone caused no significant change in basal cAMP formations (data not shown). ETR antagonists, when used, were included in the preincubation medium. The cells were then stimulated with ET-1 at 37 °C for 10 min at the concentrations indicated. The reaction was stopped by addition of 10% (w/v) trichloroacetic acid, and the cAMP content in the trichloroacetic acid-soluble cell extract was measured using a radioimmunoassay kit (Amersham).

Data Analysis

All of the experiments were done in duplicate unless otherwise indicated. When necessary, statistical analysis was done by analysis of variance.


RESULTS

Stable Expression of Wild-type and Chimeric hETRs

By cotransfecting CHOK1 cells with pMEsf/hETR and pSVneoconstructs and then selecting for resistance against G418, we obtained more than three individual clonal cell lines that stably expressed hETRs at various levels for each receptor construct. Scatchard analysis of the data obtained from saturation isotherms with I-ET-1 detected a single class of binding sites on each cell clone. Cell clones showing similar levels of receptor densities were used in the subsequent study. The Kand Bvalues for the receptors expressed on each clone adopted are listed in . Functional expression of each receptor construct was verified by the [Ca]response evoked by ET-1 according to the procedures previously described (26) . When loaded with Ca-sensitive fluoroprobe Fura-2 and stimulated by ET-1, every clone showed essentially the same pattern of [Ca]transient, i.e. 1) the response was biphasic, consisting of an initial robust peak followed by a sustained increase, 2) maximal initial peak increments (1 µM) were obtained by 10 nM ET-1, and 3) ECvalues for ET-1 to give half-maximum increments of the initial peak were 0.5 nM. Pretreatment of the cells with PTX did not affect the [Ca]response evoked by ET-1 in any of the clones (data not shown). A phospholipase C inhibitor, U-73122, at the concentration of 10 µM, completely suppressed the [Ca]response evoked by ET-1 (10 nM) (data not shown).

Effects of ET-1 on cAMP Formation in CHO Cells Expressing Wild-type hETR

In contrast to the apparently identical [Ca]responses, there were distinct differences in the effects of ET-1 on cAMP formation between CHO/hETand CHO/hETcells. First, ET-1 stimulated cAMP formation in CHO/hETcells but not in CHO/hETcells (Fig. 1, left). The ECvalue for the stimulatory effect of ET-1 was 6.5 ± 0.3 nM (mean ± S.E., n = 3), and the maximum effect was an 8-fold increase above basal formation. Second, ET-1 inhibited forskolin-induced cAMP formation in CHO/hETcells but not in CHO/hETcells (Fig. 1, right). The ECvalue for the inhibitory effect of ET-1 was 68 ± 5 pM (mean ± S.E., n = 3), and the maximum effect was 50% inhibition. These differential effects of ET-1 were attributed to a direct and selective coupling of hETand hETwith Gs and Gi, respectively, based on the following observations. First, PTX pretreatment of the cells caused no change in the stimulatory effect of ET-1 on cAMP formation in CHO/hETcells while it completely abolished the inhibitory effect of ET-1 in CHO/hETcells (Figs. 2 and 3). Second, ET-1, when applied together with GTP, stimulated cAMP formation in membrane preparations from CHO/hETcells but not in those from CHO/hETcells (data not shown). Third, effects of arachidonic acid metabolites such as thromboxane Aor prostacyclin, whose production could be stimulated by the activation of phospholipase C/Ca/protein kinase C signaling pathway in some cell types, were excluded because 1) a phospholipase C inhibitor, U-73122, at the concentration of 10 µM failed to affect both the stimulatory effect of ET-1 in CHO/hETcells and the inhibitory effect of ET-1 in CHO/hETcells and 2) a cyclooxygenase inhibitor indomethacin (100 µM) also failed to affect both effects (data not shown).


Figure 1: Effects of ET-1 on cAMP formation in CHO cells expressing wild-type hET () or hET (). Cells were stimulated with increasing concentrations of ET-1 for 10 min with ( right) or without ( left) 100 µM forskolin in the presence of 1 mM IBMX. The contents of cAMP in the trichloroacetic acid-soluble cell extracts were determined by radioimmunoassay. Values are the means ± S.E. of three determinations each done in duplicate.



Stimulatory Effects of ET-1 on cAMP Formation in CHO Cells Expressing Chimeric hETR

To find out a receptor domain(s) that determined the selective coupling of hETwith Gs, the effects of ET-1 on cAMP formation in stable transfectants of chimeric receptors were tested (Fig. 2). In the series of B/A chimeras, substitutions of hETdomains from amino terminus up to third transmembrane helices (TMIII) to the corresponding regions of hETdid not affect the coupling, whereas further substitutions including ICLII resulted in a complete loss of coupling. In the series of A/B chimeras, substitutions of hETdomains from carboxyl terminus up to TMVII to the corresponding regions of hETdid not affect the coupling, whereas further substitutions including ICLIII resulted in an apparent loss of coupling. The effect of ET-1 transmitted through a chimeric receptor, A(N-IV)B(V-C), was peculiar in that, although lesser in degree compared with the effect of ET-1 on cells expressing wild-type hET, ET-1 did cause a significant stimulation of cAMP formation when cells were pretreated with PTX. The same was the case for a chimeric receptor A(N-IV)B(V-VI)A(VII-C) in which ICLIII of hETwas replaced with that of hET.


Figure 2: Stimulatory effects of ET-1 on cAMP formation in CHO cells expressing wild-type ( wt) or chimeric hETR and effects of PTX pretreatment. Cells, either untreated ( open bars) or pretreated ( hatched bars) with PTX (50 ng/ml for 16 h), were stimulated with 10 nM ET-1 for 10 min in the presence of 1 mM IBMX. The contents of cAMP in the trichloroacetic acid-soluble cell extracts were determined by radioimmunoassay. Each bar represents the mean ± S.E. of three determinations each done in duplicate. *, p < 0.01 (significantly different from the values of basal formation).



Inhibitory Effects of ET-1 on cAMP Formation in CHO Cells Expressing Chimeric hETR

To find out a receptor domain(s) that determined the selective coupling of hETwith Gi, the effects of ET-1 on forskolin-stimulated cAMP formation in cells expressing chimeric receptors were tested (Fig. 3). In the series of A/B chimeras, substitutions of hETdomains from amino-terminal head up to TMV to the corresponding regions of hETdid not affect the coupling, whereas further substitutions including ICLIII resulted in a complete loss of coupling. In the series of B/A chimeras, substitutions of hETdomains from carboxyl-terminal tail up to TMVII to the corresponding regions of hETdid not affect the coupling, whereas further substitutions including ICLIII resulted in a complete loss of coupling. ET-1 inhibited forskolin-stimulated cAMP formation in cells expressing A(N-IV)B(V-VI)A(VII-C), which carried ICLIII of hETand ICLI, -II, and -C termini of hET. In every case where the inhibitory effect of ET-1 was observed, it was completely blocked by PTX pretreatment of the cells.


Figure 3: Inhibitory effects of ET-1 on cAMP formation in CHO cells expressing wild-type ( wt) or chimeric hETR and effects of PTX pretreatment. Cells, either untreated ( open bars) or pretreated ( hatched bars) with PTX (50 ng/ml for 16 h), were stimulated either with forskolin (100 µM) alone or with forskolin (100 µM) and ET-1 (10 nM) for 10 min in the presence of IBMX (1 mM). The contents of cAMP in the trichloroacetic acid-soluble cell extracts were determined by radioimmunoassay. The values were expressed as relative to the forskolin-stimulated formation (100%). There were no significant differences in the absolute values of forskolin-stimulated formations in the cell clones examined. Each bar represents the mean ± S.E. of three determinations each done in duplicate. *, p < 0.01 (significantly different from the values from of cAMP formation stimulated with forskolin alone).



Effects of ET-1 on cAMP Formation in CHO Cells Expressing a Chimeric Receptor A(N-IV)B(V-VI)A(VII-C)

A detailed dose-response analysis of the effects of ET-1 on cAMP formation in cells expressing A(N-IV)B(V-VI)A(VII-C) confirmed the findings described above (Fig. 4). When cells were untreated with PTX, ET-1 alone did not stimulate cAMP formation while it inhibited forskolin-stimulated cAMP formation. Both the ECvalue for the inhibitory effect of ET-1 (19 ± 2 pM, mean ± S.E., n = 3) and the maximum effect (50% inhibition) were compatible with those observed for the effect of ET-1 in CHO/hETcells. When cells were pretreated with PTX, ET-1 alone stimulated cAMP formation while it failed to inhibit forskolin-stimulated cAMP formation. The ECvalue for the stimulatory effect of ET-1 was 5.7 ± 0.8 nM (mean ± S.E., n = 3), which was similar to that obtained in CHO/hETcells, and the maximum effect was a 4-fold increase, which was about half of the maximum effect of ET-1 in CHO/hETcells.


Figure 4: Effects of ET-1 on cAMP formation in CHO cells expressing a chimeric receptor A(N-IV)B(V-VI)A(VII-C). Cells were either untreated () or pretreated () with PTX (50 ng/ml for 16 h). They were then stimulated with increasing concentrations of ET-1 for 10 min with ( right) or without ( left) 100 µM forskolin in the presence of 1 mM IBMX. The contents of cAMP in the trichloroacetic acid-soluble cell extracts were determined by radioimmunoassay. Values are the means ± S.E. of three determinations each done in duplicate.



Effects of ET-1 on cAMP Formation in CHO Cells Expressing Both hET and hET

Clonal cell lines co-expressing both hETand hETwere isolated as described under ``Materials and Methods.'' Displacement study using I-ET-1 and ET-3 revealed the expression of two classes of binding sites with different affinities for ET-3. One with the higher affinity was supposed to be hET, and the other with the lower affinity was supposed to be hET. Of the several clones obtained, one with the approximately equal densities of each receptor subtype was adopted for the subsequent study (Fig. 5). When the cells were not pretreated with PTX, ET-1 caused dose-dependent dual effects on cAMP formation (Fig. 6). One was the inhibitory effect observed at the concentrations higher than 100 pM, and the other was the stimulatory effect observed at the concentrations higher than 3 nM. PTX pretreatment oppositely modulated the two effects of ET-1 (Fig. 6); the pretreatment completely abolished the inhibitory effect of ET-1 while it enhanced the stimulatory effect. These apparently complex effects of ET-1 reflected independent and simultaneous activations of both receptor subtypes as revealed by applications of selective antagonists. The ET-specific antagonist 97-139 (27) completely suppressed the stimulatory actions of ET-1 on cAMP formation both in PTX-untreated and pretreated cells (Fig. 7) while it caused no change in the inhibitory actions of ET-1 (Fig. 8). The ET-specific antagonist BQ788 (28) completely suppressed the inhibitory actions of ET-1 on cAMP formation both in PTX-untreated and pretreated cells (Fig. 8) while it enhanced the stimulatory actions of ET-1 in PTX-untreated cells but not in PTX-pretreated cells (Fig. 7).


Figure 5: Displacement by either ET-1 () or ET-3 () of I-ET-1 binding to membrane preparations from CHO cells expressing both hET and hET. Isolation of cells co-expressing both receptor subtypes and displacement study on the membrane preparations with I-ET-1 and unlabeled ET-1/ET-3 were conducted as described under ``Materials and Methods.'' The concentration of I-ET-1 was 100 pM. Shown are means of triplicate determinations obtained in a single experiment. Similar results were obtained in two other separate experiments.




Figure 6: Effects of ET-1 on cAMP formation in CHO cells expressing both hET and hET. Cells were either untreated () or pretreated () with PTX (50 ng/ml for 16 h). They were then stimulated with increasing concentrations of ET-1 for 10 min with ( right) or without ( left) 100 µM forskolin in the presence of 1 mM IBMX. The contents of cAMP in the trichloroacetic acid-soluble cell extracts were determined by radioimmunoassay. Values are the means ± S.E. of three determinations each done in duplicate. *, p < 0.01 (significantly different from the values of basal formation).




Figure 7: Effects of ETR antagonists on the stimulatory effects of ET-1 on cAMP formation in CHO cells expressing both hET and hET. Cells, either untreated ( left) or pretreated ( right) with PTX (50 ng/ml for 16 h), were incubated for 10 min with or without the ETR antagonist indicated and then stimulated with 10 nM ET-1 for 10 min. The concentrations of both 97-139 and BQ788 were 1 µM. IBMX (1 mM) was included throughout the preincubation and stimulation periods. The contents of cAMP in the trichloroacetic acid-soluble cell extracts were determined by radioimmunoassay. Each bar represents the mean ± S.E. of three determinations each done in duplicate.




Figure 8: Effects of ETR antagonists on the inhibitory effects of ET-1 on cAMP formation in CHO cells expressing both hET and hET. Cells, either untreated ( left) or pretreated ( right) with PTX (50 ng/ml for 16 h), were incubated for 10 min with or without the ETR antagonist indicated and then stimulated with 10 nM ET-1 and 100 µM forskolin for 10 min. The concentrations of both 97-139 and BQ788 were 1 µM. IBMX (1 mM) was included throughout the preincubation and stimulation periods. The contents of cAMP in the trichloroacetic acid-soluble cell extracts were determined by radioimmunoassay. The values were expressed as relative to the forskolin-stimulated formation (100%). There were no significant differences in the absolute values of forskolin-stimulated formations under the indicated conditions. Each bar represents the mean ± S.E. of three determinations each done in duplicate.




DISCUSSION

Both hETand hET(and also all of the chimeric receptors examined) could couple with PTX-insensitive G protein (possibly a member(s) of Gq family) to induce [Ca]response of the cells. The [Ca]responses of the various clones were indistinguishable from each other, suggesting that any combinations of ICLII, ICLIII, and carboxyl terminus of either hETor hETcould confer the full ability both to select and activate Gq. These results are consistent with the observation on deletion mutants of hET(29) that both 10-amino acid residues in the carboxyl-terminal stalk region of ICLIII and 13-amino acid residues in the proximal stalk region of carboxyl terminus were required to induce ET-1-dependent increase in [Ca]. hETand hETshare the common 10-amino acid residues in the carboxyl-terminal region of ICLIII and a quite similar sequence in the proximity of carboxyl terminus; 11 out of the 13 amino acid residues are identical.

Aramori and Nakanishi (23) demonstrated the selective coupling of bovine ETand rat ETwith Gs and Gi, respectively, when expressed on CHO dhfr cells. The same was the case for hETand hETexpressed on CHOK1 cells (Fig. 1), suggesting that the G protein specificity was a characteristic of ETR conserved over species differences. The present study demonstrated the roles of ICLII and -III but not that of carboxyl terminus or of hETR in the selective activation of Gs/Gi.

The data on the stimulatory effect of ET-1 on cAMP formation indicated the roles of both ICLII and -III in the selective activation of Gs (Fig. 2). hETwith the replacement of ICLII and/or -III to the corresponding region(s) of hETfailed to transmit the stimulatory effect of ET-1. In cells expressing chimeric receptors with ICLII of hETand with ICLIII of hET, ET-1 stimulated cAMP formation only when cells were pretreated with PTX. These results can be interpreted as follows. 1) Both ICLII and III of hETwere required for full activation of Gs; 2) of the two ICLs, ICLII was an absolute requirement for the coupling with Gs, and ICLIII played an ancillary role to confer the higher efficacy of coupling; and 3) in chimeric receptors with ICLII of hETand ICLIII of hET(A(N-IV)B(V-C) and A(N-IV)B(V-VI)A(VII-C)), concomitant activation of Gi counteracted the activation of Gs. A study on chimeric muscarinic/-adrenergic receptors suggested a similar requirement for the original set of ICLII and -III of -adrenergic receptors (AR) to confer the full ability to activate Gs (7) .

The data on the inhibitory effects of ET-1 indicated the role of ICLIII of hETR as a determinant for the selective activation of Gi (Fig. 3). All of the chimeric receptors with ICLIII of hET, regardless of the various combinations with ICLII/carboxyl terminus derived either from hETor hET, could transmit the effects that were compatible with that transmitted by wild-type hET. All the chimeric receptors with ICLIII of hETfailed to transmit the effect. These results suggested that ICLIII of hETwas necessary and also sufficient to confer the full activation of Gi. Studies on chimeras between muscarinic receptor subtypes (4, 5, 6) also indicated the role of ICLIII of m2/m4 as a major determinant for coupling with Gi.

That the characteristics of structural basis for the selective coupling are shared by hETand AR to activate Gs and by hETand muscarinic receptors to activate Gi (as described here) points to the notion that both the three-dimensional architecture of the surface that binds G proteins and the mechanism that activates G proteins are highly conserved in all G protein-coupled receptors (11) . The carboxyl-terminal stalk portion of ICLIII has been proposed to be the most likely region involved in receptor/G protein coupling by mutagenesis studies of the receptor-G protein pairs, including both AR-Gs and m2/m4-Gi (30) . Because hETand hETshare the common sequence in the carboxyl-terminal stalk region of ICLIII, the region cannot determine the G protein specificity but is quite likely to be involved in the activation mechanism common to Gs and Gi (and also to Gq).

There was a large difference in the ECvalues for the stimulation and inhibition of cAMP formation by hETand hET, respectively (Fig. 1), and similar differences have been observed for the efficacies of AR to stimulate cAMP formation and of muscarinic receptors to inhibit it (3) . These results suggested that, besides the structural basis for the selective coupling, the differential efficacies to stimulate/inhibit cAMP formation may be a common feature to all the Gs/Gi-coupled receptors. The changes in cAMP formation, however, are the net results of receptor-G protein-adenylate cyclase interactions, and detailed analyses will be required to clarify the molecular basis for the differential efficacies.

We also demonstrated in the present study that each receptor subtype expressed on the same cell could work independently, i.e. for hETto activate Gs and for hETto activate Gi, resulting in dose-dependent dual effects of ET-1 on cAMP formation (Fig. 6). Some cell types including cardiomyocytes express mRNAs for both receptor subtypes (25) and possibly both receptor proteins. An intriguing observation is that, in isolated cardiomyocytes from guinea pig, ET-1 exerted inhibitory effect on cAMP formation to counteract AR stimulation, and this effect was apparently transmitted via ETcoupled with Gi (31, 32) . Co-expression of the two receptor subtypes, as evidenced by the data presented, cannot be the reason for the coupling of ETwith Gi. Further study is necessary to verify alternative explanations such as a tissue-specific expression of receptors with structural differences or a selective sequestration of G proteins by the cells.

  
Table: Densities and affinities of wild-type and chimeric hETexpressed on CHO cells

Clonal cell lines expressing each receptor construct were isolated as described under ``Materials and Methods.'' Binding parameters were determined by saturation isotherms of I-ET-1 binding assay using membrane preparations. Shown are the values obtained from a single experiment on each clone. These clones were selected for use in the present study because of the similar receptor densities as listed. N, I-VII, and C designate the extracellular amino terminus, transmembrane helices (TM) I-VII, and cytoplasmic carboxyl terminus, respectively, plus the adjacent loop regions when applicable. For example, the chimeric receptor A(N-VII)B(C) has hETsequences of amino terminus, TMI-VI, and a part of VII plus adjacent loop regions (Metto Asn) and hETsequences of a part of TMVII and cytoplasmic carboxyl terminus (Serto Ser). See Ref. 26 for detailed description of each receptor construct. Progressive substitutions from the amino-terminal side of the subdomains of hETto the corresponding domains of hETgave B/A chimeras, while those from the carboxyl-terminal side gave A/B chimeras.



FOOTNOTES

*
This work was supported in part by research grants from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Molecular Cell Pharmacology, National Children's Medical Research Ctr., Tokyo 154, Japan.

To whom correspondence should be addressed. Tel.: 81-75-753-4477; Fax: 81-75-753-4402.

The abbreviations used are: G protein, guanyl nucleotide-binding regulatory protein; ET, endothelin; hETR, human endothelin receptor; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; ICL, intracellular loop; TM, transmembrane helices; IBMX, 3-isobutyl-1-methylxanthine; PTX, pertussis toxin; AR, -adrenergic receptor.


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