AbstractKinetics of ligand/receptor interactions using ET-1, ET-3 and SRTX-b were studied and cross-linking experiments carried out in guinea pig ileum and rat cerebellar preparations. Dissociation studies indicate that the two regions are characterized by different receptor subtypes and different modes of ligand binding. Autoradiographic patterns obtained following cross-linking of ET-1 and ET-3 to the different issues support these conclusions

Bdolah, Avner

Galron, Ronit

Kloog, Yoel

Kochva, Elazar

Sokolovsky, Mordechai

Wollberg, Zvi

Elsevier - Publisher Connector 

Volume 2iI), number I, 11~,14 FEllS Ot~T~4 Ma~ 19ql 
Kinetic and cross-linking studies indicate different receptors for 
endothelins and sarafotoxins in the ileum and cerebellum 
Ronit Galron =, Avner Bdolah a. Ela~ar Kochva", Zvi Woilberg=, Yo¢l KlOog ~ and Mordechai Sokolovsky = 
' Lt#bnramrr tV'Neurnbtoehemt, ar, l', Dep~mment t~fBInehemlstr.v trod ~Department ofZt~ol#,~y, Genrge S. Wise b'tte,dO' t¢Life 
Sciences. Tel A viv Unlrrr,~lO', 7"¢1 aviv dP~78, laraei 
Received 7 M arch 199 ! 
Kinetics of lil!and/,~'ce~ptor inl¢rctelio~s usintl ~'r. h ET.~ etnd SRTX.b were studied and ert~=t-linkintl expcriment~ carried out in lluine=t pi~ ileum 
and rat ¢eret~llar preparations. Dissociation ~tudies indicate th;tl the two re~ions are ¢haraeteri~ed b~ different receptor subtypes and difl'erent 
modes of lilland bindintl. At~toradiotlraphie pnttert~ obluined Ibllt~win~ ero~s.llt~kin~ of ET. i and ET-3 to the difl'erent tis~ue~ support hese ¢o~t¢lu. 
~itons, 
Rweptor subtype: Endolhdin. !: Endolhdin-~; Rtue of dissochflion: Affinity labelinlj; Brain: Peripheral: S=trafotoxin 
1. INTRODUCTION 
Data obtained from binding studies and affinity 
cross-linking experiments suggest the possibility of 
heterogeneity among the endothelin/sarafotoxin 
(ET/SRTX) receptors in certain tissues [1-3]. On the 
other hand, a recent study of the contractile effects of 
the ET/SRTX family of peptides in the ileum and the 
binding of these peptides to guinea pig ileum suggests 
the presence of only one cell surface receptor subtype 
in this preparation [4]. These findings raise the 
possibility that the receptors present in the guinea pig 
ileum are different from those found in brain prepara- 
tions, In order to investigate this possibility we con, 
ducted kinetic studies aimed at determining the rate 
constants that characterize the binding of three ligands, 
ET-i, ET-3 and SRTX-b, to their receptors in two dif- 
ferent tissue preparations and examined their affinity 
cross-linking to the membrar:al target. One of the tissue 
preparations used, namely membranes prepareci from 
guinea pig ileum, presumably contains homogeneous 
receptor binding sites [4], while the other, from rat 
cerebellum, is thought to contain multiple receptor sub- 
types [5]. 
2. MATERIALS AND METHODS 
2.1, Materials" 
[I:'~I-Tyrt'IET-3 (-2000 Ci/mmol) was purchased from Amershmn 
International (UK). Iodinated [~2Sl]STRX-b and ET-I were prepared 
as described previously [6]. ET-I and ET-3 were from American Pep. 
Cotv'espondence address': M. Sokolovsky, Laboratory of 
Neurobiochemistry, Dept, of Biochemistry, Tel Aviv University, Tel 
Aviv 69978, Israel, Fax: (972) (31 415053 
Ode Co, (Santa Clara, CA). DSS and DSP were rrom Pierce 
(Rockford, ILL 
2,2 Tissue preparatitm 
Cerebellar tissue (fronl adult male Charles River derived rats) and 
exclsed guinea pig ileum were washed anti homogenized in 50 mM 
Tris-HCl buffer, pH 7.4. ¢ontaininit protein inhibitors (5 units/,nl 
aprolinin, Ss~g/ml pcpslatin, 0,1 mM phenylmetl~anesulfonyl 
fluoride, 3 mM EDTA and 1 mM ELII'A), 
The cerebellar ho~nogenates were centrifuged '-it 40000 X //and the 
pellets resuspended in about SO vols of 10 rt~M Tris buffer containing 
10/~M MgCIz and the above protease inhibitors The I~uinea pi~ 
homogenatcs were filtered throu~dh three layers of cheesecloth and 
centrifuged twice at 30000 × g for 20 rain. Tile final pellet was 
rcsuspt, nded in Tris buffer contamin~ the same proteasc inhibitors, 
2,3, Bindinl~ experiments 
Binding of [~Z'~llpeptides (ET.I, ET-3 and SRTX.b) to tlae mctrJ. 
branes was carried out at 25°C for 1 h as previously described [4,61, 
2,3.1. Dissociation of [~:'~l]peptidcs 
Receptor/ligand complexes were formed by preincubation of naem- 
brane preparations for 1 h at 25°C with 10 taM [s~'Sl]peptides (ET.I, 
ET.3 or STRX-b). Tile membranes were pelleted out by centrifuga. 
finn (15000 x g, 20rain), washed with ice-co'd buffer, and 
resuspended in the original volume of buffer (5 nd), Dissociation 
reactions were initiated by the addition of unlabeled peptide (1 tiM 
final concen|ration). Reactions were terminated either immediately 
upon addition of the unlabeled peptide (zero time) or at the indicated 
times, 
Specific cross-linking of [J2'~i]derivatives of ET-1 and ET-3 was 
performed as described in detail previously [5], employing DSP 
(1 raM) and DSS (1.5 raM), respectively, as thecross.linklng reagent. 
3. RESULTS AND DISCUSSIGN 
Data obtained previously from binding isotherms in 
guinea pig ileum mebranes indicate that a single recep- 
tor binds ET-1, ET-3 and SRTX-b with similar af- 
finities (Kd values of 0.7, 0.85 and 0.73aM, 
Published by Elsevier Science Publishers B. V. 1 1 
Volum~ 28;~, number I FEBS LETTERS M.), 1991 
respectively) [41, In cerebellar preparations the cot. 
responding K,~ values were 10, 12, and 3 aM. Hilly ct 
al. {71 also found in rat cerebellum rather similar af- 
finities for these ligands, although the affinities they 
report "are somewhat higher, probably due to the u~e of 
different experimental protocols 12,6]. Similar h',t 
values do not, however, necessarily reflect site identity, 
and a more accurate indication is provided by kinetic 
analysis of binding. One such parameter, which is 
relatively simple to measure, is dissociation rate. 
Dissociation of bound ==Sl.labeled peptides from 
their preformed receptor/ligand complexes was in- 
itiated and the rates measured as described in section 2. 
As shown in Fig. 1, the dissociation rates for all three 
I!~ands were significantly lower in the cerebellar than in 
the ileum preparations. In addition, in both prepara. 
tions the rates of dissociation of ET-3 from the receptor 
were significantly lower than those of ET-I or SRTX.b. 
The it/= values in the ileum are about 18 rain for ET.I 
and 7 rain for SRTX-b, in contrast o more than 2 h for 
ET-3; in the cerebellum the t=/2 values are more than 
2-3 h f('r SRTX.b and ET-I, while for ET-3 the rate of 
dissociation from the receptor is negligible (t~nder these 
experimental conditions). The fact that for each of the 
three ligands the dissociafon rate in tire cerebellar 
preparation was significantly different from that in the 
ileum suppo,rts the suggested existence of different 
receptor subtypes in these two regions. Alternatively, 
these findings might be explained in terms of interac- 
tions of the same receptor vAth differen~ membrane 
components in the different issues. Also worth noting 
are the differences in dixsociative behavior between 
ET'3 vs ET-I and SRTX-b in both the cerebellum and 
the ileum. These differences could stem from 
dissimilarlti~:s in the nature of the reeeptor/lilland com- 
plexes (i.e. different modes of bindtn$} [81. Recent 
kinetic studies of (he binding of ET- 1 and ET-2 to Swiss 
3T3 fibroblasts [9] disclosed ifferent rates and extents 
of dissociation rot the two i~oforms. It shotdd be noted 
that the dissociation curves (Pig. tj were not 
monophasic, Such ~ behavior is indicative of either site 
heterogeneity or Ill, and induced conformational 
changes in the receptor= or both. These possibilities are 
currently under investigation i  our laboratory. 
Specific cross-linking of the [la~llderivatives of ET-1 
and ET-3 in membranes was achieved by binding of" the 
ligand to the receptor (1 aM, I h, room temperature), 
wa.~hing of" the comple× to remove free or loosely 
bound ligand, and incubation with the bifunctional 
reagent DSS (1.5 raM) or DSP (1 raM) (1 rain, room 
temperature, 2rag protein/ml), followed by SDS- 
PAGE and atttoradiotlraphy [5]. Autoradiographs 
from guinea pig ileum membranes in which [t=Sl]ET, l 
and [==Sl]ET-3 were cross-linked are shown in Fig. 2; In 
both cases, densitometry disclosed one major labeled 
protein (60-7007o), with an apparent M, of 74-75 kDa. 
Also detected were two additional bands, which 
demonstrated weaker labeling at 53 i(t)a (10-15070) and 
at 38 kDa (10-15%). The labeling was specific, as in- 
dicated by the fact that it did not occur when unlabeled 
ligand (5 x 10 -~ M) was present during binding. The 
labeled adducts were not seen in the absence of cross- 
G8 
0.7] 
o.81 
0,. ~ 
{).4 
O. ~ . 
• "CEREBELLUM 
a -ILEUM 
\ 
\ 
\ 
\ 
\ 
\ 
\ 
\ 
"'!-st-3__ [.N.. '"X-S~TX'bh 
\ 
\ 
\ 
\ 
\ 
' \  
\ 
TIME (hours)  
Fig, !. First-order plots of  dissociation of iodinated ET-1, ET-3 and SRTX-b receptor complexes. Dissociation of  the preformed corn plexes was 
initiated by the addition of  1 .uM unlabeled peptide (see section 2), Bo = amount  of radioligand bound at zero time; Bt = amount  of radiol igand 
bound at time t. Data are the mean values from four experiments, in which individual data points were obtained in triplicate; Standard deviation 
was 5-10070 of the measured values. 
12 
VOIm1~ 2~1, number ! FEBS LETT~R,S M;ty 1991 
tt4 
67--  
43- -  
30 - -  
20--  
1 2 3 4 5 6 
- ÷ - ÷ - ÷ 
Fill, 2, Autoradiol~raphie pattern~ derived From el<'~lrophorede 
amtly~ts or I~"~llpeplitles ero~,~.linked to lluinea pil~ ileum 
nlembrmoex. Mernbrane~ (2(t0-300/,~ or protein) incubi|led with 
I nb,'l II='~IIET.I (hme~ I and 21, II"'~IIET.3 (lanet 3 and 4), and 
lJ;~l ISRTX.b (lane.~ $ and 61 were cros~.lhlked a~, described in ~¢¢1io1'1 
2 anti then ~ubjecled to SDS.PAGEon 1(1% ~1~, (~) Tolal binding; 
(+) non,~pe¢iflc binding, (in the i~re~ence of 5 × 10 "7 M unlabeled 
lilland). The positiom, t~r molt'¢ular ma~s slandard~ are shown (At, 
I0=~). 
linking. Cross.linking of iodinated peptides alone did 
not produce bands corresponding to those observed 
upon cross-linking in the presence of the membranes 
(not shown). Two recent reports have described the ex- 
istence of art endothelin receptor with an apparent Mr 
of about 70000 [3,10]. The former report describes the 
existence of two receptor subtypes having different 
molecular weights, i.e. 73000 and 60000. 
In our previous tudy [5] conducted in rat cerebellar 
preparations labeled with [~zSI]ET.I we observed one 
major labeled band with an apparent Mr of 53 kDa 
(80-90°70). Cross-linking of [~2"~I]ET-3 with cerebellar 
tissue showed the presence of two bands of M, 53000 
and 38000; densitometry disclosed that the former 
polypeptide accounts for 60-70°70 of the binding and 
the latter for 30-40°70. 
Cross-linking experiments performed at 4°C (not 
shown) yielded results identical with those obtained at 
25°C. In view of this, and because the two types of 
tissue preparations were labeled simultaneously and 
under identical conditions, it seems unlikely that the M~ 
38 000 polypeptide represents a product of proteolysis. 
A number of possible explanations for the different 
labeling patterns obtained by various laboratories have 
been presented elsewhere ([5] and references therein). 
These results clearly indicate that in the ileum the 
majority of endothelin-binding sites are located on a 
polypeptide of apparent M, 70000, while in the 
cerebellum the receptor polypeptides are smaller, hav- 
ing apparent M~ of 50000 and 35000. They therefore 
support the assumption derived from tile kinetic date, 
namely that bindin~ in the two tissues is ~ssociated with 
different receptor(s). This could imply, a~ we have suB- 
gested elsewhere [2,5,81, that the molecular structure of 
the receptor/ET complex in brain lissu¢ differs from 
that in peripheral tissues, One cannot, however, dis- 
count the alternatiw or additional possibility of inter- 
species differences in the receptor system (e.g. [I !]). 
The fact that in the ileum ET-I and ET-3 yielded 
sin-tilar labeling patterns but different dissociation data 
strongly supports the existence of different recep- 
torlligand complexes resuhin8 from structural dtf. 
ferences between the two ligands (as discussed in detail 
by Kloog, and Sokolovsky tll). 
Despite the evidence that the majority of receptor 
subtypes in the ileal preparation have a molecular 
weight of around 70 kDa, one cannot rule out the 
possibility that the physiological response is also 
mediated by one or both of the minor bands (50 or 
38 kDa). 
The cloning of two endothelin receptors ~as reported 
recently. Arai et al. [12] cloned the receptor from a 
bovine lung eDNA library, and reported a sequence of 
42"1 anaino acids, corresponding toa molecular mass of 
48 kDa. The other receptor, cloned from a rat lung 
eDNA library [13], corresponds toa polypeptide of 415 
amino acids (47 kDa). It is not yet known whether these 
two receptors represent the same subtype; comparison 
of the reported sequences indicates differences. The 
differences between the predicted molecular masses and 
tl~ose reported earlier by affinity labeling ([3,4,10] and 
references cited therein) could reflect different degrees 
of glycosylation and/or other factors affecting the 
estin'mtion of molecular mass, e.g. by SDS-PAGE, as 
suggested earlier [13]. In view o f  the above, the on. 
dothelin receptor subtype identified in the cerebellum 
might be equivalent to one of these cloned receptors. 
As for the receptor identified in the ileum, the 
molecular weight and different pharmacological pro- 
perties indicate that it is a different subtype of the 
ET/SRTX receptor. However, at present one cannot 
discount he possibility that it is basically similar to the 
cerebellar receptor but undergoes a heavier glycosyla- 
tion. For example, in the case of muscarinic 
acetylcholine r ceptors, glycosylation was shown to be 
responsible for 25-28% of the estimated molecular 
mass [14,15]. 
The physiological findings [4] demonstrate hat com. 
pared with ET-3, ET-I is significantly more potent and 
its removal by washing from the ileum segment occurs 
more slowly. Since the rate of removal by washing is 
usually related to the dissociation rate, one might ex- 
pect ET-3 to dissociate more rapidly than ET-I. This, 
however, is clearly not the case (Fig. 11. There are 
several possible xplanations for this phenomenon, for 
example: (i) differences between the preparations 
employed in the two types of studies(an i tact ileal seg- 
13 
Volume 283, number i FEBS LETTERS May 1091 
mont vs. homogena|=); ( i i )  involvement or n dcsen- 
s i t izat ion process with di f ferent kinetics for  ET-!  and 
ET.3.  These an~| other possibil it ies are currently under 
invest igat ion in our laboratory, 
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14 


Kinetic and cross-linking studies indicate different receptors for endothelins and sarafotoxins in the ileum and cerebellum 

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Kinetic and cross-linking studies indicate different receptors for endothelins and sarafotoxins in the ileum and cerebellum

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