expression of functional thrombin receptors in xenopus oocytes injected with human endothelial cell...

Vol. 171, No. 3,199O

September 28, 1990

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 913-919

Expression of functional thrombin receptors in Xenopus oocytes tn/ected with human endothefial cell mRNA

Eva Pipili-Synetos’ , Marvin C. Gershengorn, and Eric A. Jaffe*

From the Divisions of Hematology/Oncology and Endocrinology and Metabolism,

Department of Medicine and Specialized Center of Research in Thrombosis,

Cornell University Medical College, New York, NY 10021

Received August 7, 1990

Human endothelial cell thrombin receptors were functionally expressed in Xenopus laevis oocytes by injection of RNA extracted from human umbilical vein e

Yt!~$~~~~ EF$zSing injected with endothelial cell RNA responded to thrombin with a Ca current whose size depended on the amount of RNA injected. In oocytes expressing thrombin receptors, thrombin caused homologous but not heterologous desensitization. Both the catalytic and anion-binding exosites of thrombin were necessary to elicit depolarizing currer$$. Thus, Xenopus laevis oocytes injected with mRNA from human endothelial cells express Ca - dependent thrombin receptors which share many common features with thrombin receptors on intact endothelial cells. Xenopus oocytes may, therefore, be used as a screening system in the expression cloning of the endothelial cell thrombin receptor. 01990 Academic Press, Inc.

Thrombin, an important regulator of endothelial cell function, stimulates human umbilical

vein endothelial cells (HUVEq2 and causes rapid and concentration-dependent rises in

inositol-1,4,5-trisphosphate (IP3) and cytosolic calcium ([Ca2+]j) which precede rises in PG12

production (1,2). This process requires the catalytic site of thrombin suggesting that thrombin,

by proteolysis of an as yet uncharacterized EC extracellular surface protein, activates

phospholipase C via a G protein resulting in release of IP3, elevation of [Ca2+]i, and induction

of PG12 production (1-3). While some of the functional and biochemical aspects of the

interaction of thrombin with its putative receptor are known, the structure of this receptor

protein(s) is unknown.

As a first step in characterizing the thrombin receptor on HUVEC, we have attempted to

express functional thrombin receptors in the oocytes of female Xenopus laevis by injecting

HUVEC RNA. This system has been extensively used as a primary tool in the expression

cloning of cDNAs encoding for receptors for which there is neither sequence information nor

antibodies such as the phospholipase C-linked serotonin, glutamate, and TRH receptors (4-6).

* To whom correspondence should be addressed. ’ Dr. Pipili-Synetos is on sabbatical leave from the Department of Pharmacology, School of Medicine, University of Patras, Patras, Greece. 2 Abbreviations used: [Ca*+] cytosolic free calcium; HUVEC, human umbilical vein

endothelial cells; IP -1,4,5-trisphosphate; PGI , prostacyclin; PPACK, D-phenylalanyl- L-prolyl-L-arginine ketone hydrochloride, 3 RH, thyrotropin releasing hormone.

0006-291x/90 $1.50

913 Copyright 0 1990 by Academic Press, Inc.

All rights of reproduction in any form reserved.

Vol. 171, No. 3, 1990 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

In the present study, we demonstrate that injection of either total or poly (A)+ RNA derived

from HUVEC leads to the expression of thrombin receptors on oocytes which have character-

istics similar to those of the thrombin receptor on HUVEC.

MATERIALS AND METHODS

Materials Thrombin (a-thrombin and r-thrombin) were kindly supplied by Dr. J.W. Fenton II, NY State Dept. of Health, Albany, NY. D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone hydrochloride (PPACK) was obtained from Calbiochem. Fast Track mRNA isolation kits were obtained from Invitrogen. Culture of endothelial cells HUVEC were obtained from human umbilical cord veins and cul- tured as previously described (2,7,8). Preparation of RNA from human umbilical vein endothelial cells Passage 3 HUVEC grown to confluence in T75 flasks were washed once with HEPES buffered saline and RNA was extracted by a guanidinium isothiocyanate/CsCl procedure (9). The average yield of total RNA was 50 ,,g/T75 flask. Poly(A)+ RNA was isolated directly from lysed HUVEC by affinity chromatography on oligo-(dT)-cellulose microcolumns using a Fast Track mRNA isolation kit (Invitrogen). Poly(A)+ RNA recovery from HUVEC was about 250 - 500 ng/l75 flask (0.5 - 1% of the average yield of total RNA). Microiniection of total RNA and polv (A)+-RNA into Xenopus laevis oocvtes Oocyte - positive female Xenopus laevis frogs were purchased from Nasco (Fort Atkinson, WI). Oocytes were extracted and maintained as described previously (10). Stage V and VI oocytes were microinjected with total RNA, poly (A)+ RNA, or distilled water in a final volume of 50 nl. In one series of experiments, the oocytes were injected with total RNA and 48 hrs later they were reinjected with either sodium EGTA (50 nl of 10 mM solution) or distilled water (50 nl). experiments, poly (A)+ RNA or total RNA from HUVEC was co-inje

In other ted with positive control

RNA transcribed from cDNA clones encoding for the TRH receptor 5 (10,ll). B Electrophysiological experiments using a voltage clamp technique were performed as previously described (IO). Drugs were included in the perfusate and applied at a flow rate of 5 ml/min. The agonist applied in the perfusate took 5.5 set to reach the perfusion chamber. The time required to reach 50% of the maximal drug concentration in the chamber was 9.5 sec. Thrombin was applied for 45 set and TRH for 30 sec.

RESULTS

When either uninjected or water-injected, voltage-clamped X. laevis oocytes were

exposed to thrombin or TRH (thyrotropin releasing hormone), there was no change in electrical

activity (data not shown). These results suggest that there are no “intrinsic” receptors for

thrombin or TRH on the membrane of these oocytes. When, however, oocytes injected with

either HUVEC total RNA or poly(A)+ RNA were exposed to thrombin (Fig. l), thrombin elicited

an inward current whose magnitude varied with the amount of RNA injected.

The lag period between application of thrombin and initiation of the depolarizing current

was between 30-60 sec. The inward current consisted of 3 distinct components: a rapid

transient depolarizing current (D, component) was followed by a prolonged depolarizing

current (D2 component) with superimposed fluctuations (F component) of varying intensity.

These components are similar to those described for the endogenous acetylcholine receptor

(12) and the acquired TRH receptor (10) and are indicative of a [Ca2+]-dependent, inward Cl-

current. Expression of the receptor responding to thrombin was maximal between 2 to 3 days

after injecting the oocytes with HUVEC RNA.

‘R.E. Straub, G.C. Frech, R.H. Joho, and M.C. Gershengorn, submitted for publication,

914


bI 1 50nA

L lmln

L- lmln

T W

Figure l+ Thrombin causes an inward current in X laevis oocytes injected with either total or poly(A) RNA derived from human umbilical cord vein endothelial cells (HUVEC). X laevis oocytes were microinjected with (left panel) (a) 170 ng, (b) 250 ng, or (c) 350 ng HUVEC total RNA or with (right panel) (a) 35 ng, (b) 50 ng, or (c) 75 ng HUVEC poly(A)+ RNA. 45 hr later, the oocytes were tested for responsiveness to thrombin (T, 3 units/ml).

To determine whether the inward current elicited by thrombin is dependent on calcium,

oocytes were injected with total HUVEC RNA and incubated for 48 hr. The oocytes were then

injected with either EGTA or water and then exposed to thrombin (Fig. 2). Oocytes injected

with EGTA failed to respond to thrombin whereas water-injected oocytes responded to throm-

bin with an inward current.

To show that responses expressed after injecting oocytes with RNA extracted from HUVEC

were specific to thrombin, TRH was added to the perfusate and the electrical activity monitored.

TRH failed to induce an inward current in oocytes which responded to thrombin (data not

shown).

When a single oocyte which responded to thrombin was exposed to thrombin a second

time up to 15 min later, thrombin failed to cause any changes in electrical activity (Fig. 3a). To

test whether this desensitization was homologous or heterologous, oocytes were co-injected

with both total RNA from HUVEC and RNA transcribed from cDNA clones encoding for the

mouse TRH receptor and tested for responsiveness to thrombin and TRH (Fig. 3b). Prior

stimulation with thrombin failed to desensitize the oocyte to a subsequent stimulation with TRH.

915


NaEGTA C Injected

i 7 50nA

ii L lmin

Fiaure 2. Intracellular calcium is necessary for thrombin to elicit an inward current. X. laevis oocytes were microinjected with HUVEC total RNA (350 ng). 48 hr later the oocytes were microinjected with either sodium EGTA (EGTA, 50 nl of a 10 mM EGTA solution) or distilled water (C for control, 50 nl). 30 min later, the oocytes were exposed to thrombin (T, 3 units/ml).

Similarly, doubly injected oocytes stimulated initially with TRH responded to subsequent

thrombin stimulation (data not shown) suggesting that there is no cross desensitization

between the thrombin and TRH responses.

To demonstrate that the active site of thrombin is necessary to elicit an inward current,

PPACK-thrombin (a catalytically inactive thrombin derivative) (13) was added to oocytes

injected with poly (A)+ RNA (Fig. 4a). PPACK-thrombin failed to elicit an inward current.

However, when the same oocytes were subsequently exposed to catalytically active thrombin,

they responded with an inward current (Fig. 4a).

1 w TRH W

1 J I I b

T W

15min 1 +

50n*

L lmin

(a) X. laews Fiaure 3. Thrombin causes homologous but not heterologous desensitization. oocytes microinjected with 250 ng of HUVEC total RNA responded to an initial exposure to thrombin (T, 3 units/ml) but not to a second exposure to thrombin 15 min later. (b) X. laevis oocytes were microinjected with a mixture of HUVEC total RNA (250 ng) and RNA transcribed from cDNA clones encoding for the TRH receptor (200 pg). Oocytes were exposed to thrombin (T, 3 units/ml), washed, and when the electrical activity subsided, exposed to TRH (1 PM).

916


SAlA

L lhl

PPACK-T W T W %nA 111 1 8-T W T W

a 1 II 1

L lmln

b

Fiaure 4. The active catalytic and anion binding exosites of thrombin are necessary to elicit an inward current. (a) X. laevis oocytes were microinjected with HUVEC poly (A)+ RNA (75 ng). 48 hr later the oocytes were exposed to thrombin (20 nM or 3 units/ml) inactivated with PPACK (200 nM) (PPACK-thrombin), washed, and then exposed to intact thrombin (T, 3 units/ml). (b) X. laevis oocytes were microinjected with HUVEC total RNA (350 ng). 48 hr later the oocytes were exposed to +hrombin (r-T, equivalent to 3 units/ml), washed, and then exposed to intact thrombin (T, 3 units/ml).

Studies were performed to determine whether the anion-binding exosite of thrombin, a

site necessary for the recognition and cleavage of fibrinogen and disrupted in y-thrombin

(14,15), is necessary for the effect of thrombin on oocytes. When oocytes injected with total

HUVEC RNA were exposed to y-thrombin, no inward current was seen (Fig. 4b). In contrast,

when the same oocytes were then exposed to intact thrombin, these oocytes responded with

an inward current.

DISCUSSION

In the present study, we expressed a HUVEC receptor protein which responds to

thrombin in an heterologous system, Xenopus laevis oocytes, by injecting oocytes with total

and poly (A)+ RNA extracted from HUVEC. Injected oocytes responded to thrombin with a

Ca2+ dependent, inward Cl- current since oocytes injected with the Ca2+ chelator sodium

EGTA failed to respond to thrombin. Both the anion-binding exosite and the active site of

thrombin are required to evoke an electrical response in injected oocytes as indicated by the

inability of either r-thrombin or PPACK-thrombin to induce an inward current. These results are

consistent with observations made in intact HUVEC in which the catalytic site is necessary to

activate the cells (1,2). These results are also consistent with observations that r-thrombin or

thrombin inhibited with C-terminal fragments of hirudin, which interacts with the anion-binding

exosite and inhibits fibrin clotting activity but not the esterolytic activity of thrombin (1 S-19), fail

to activate HUVEC4.

4 JR Ngaiza, J. Grulich-Henn, G. Manley, 0. Cole, J. Krstenansky, and E.A. Jaffe, manu- script in preparation.

917


After the completion of this study, a report appeared showing that injection of RNA from

Chinese hamster lung fibroblasts can also lead to expression of thrombin receptors in Xenopus

oocytes (20). In fibroblasts, both the anion-binding exosite and the active site are required for

the mitogenic effect of thrombin (21,22). Near maximal fibroblast proliferation may be

stimulated by the simultaneous addition of 2 different molecules, one with binding activity but

no active site (DIP-thrombin) and one with an active site but no anion-binding exosite (r-

thrombin) even though these thrombin derivatives are mitogenically inactive when added

separately (22). Both the anion-binding exosite and the active site are also necessary for the

induction by thrombin of rises in [Ca2+]i in HUVEC (1 ,2)4. However, in contrast to fibroblasts,

these sites must reside on the same molecule for HUVEC activation to occur since

simultaneous addition of DIP-thrombin and r-thrombin failed to induce rises in [Ca2+]i in

HUVEC4. The above evidence suggests that the thrombin receptors may not be identical in

these two cell types.

An initial interaction of thrombin with intact HUVEC is known to result in receptor

desensitization as further additions of thrombin fail to increase [Ca2+]i or prostacyclin produc-

tion (1,2). Similarly, oocytes injected with HUVEC RNA and exposed to thrombin were

desensitized to further additions of this agonist. This desensitization appeared to be only ho-

mologous as oocytes co-injected with RNA from HUVEC and with transcript RNA from cDNA

clones encoding for the mouse TRH receptor3 responded to TRH after an initial exposure and

response to thrombin. Similarly, TRH, which causes homologous desensitization in TRH re-

sponsive oocytes (IO), did not cause heterologous desensitization of the thrombin responses.

The mechanism by which oocytes become desensitized to thrombin or TRH is not understood

and the site(s) of desensitization are unknown.

An interesting feature of the interaction between thrombin and the receptor expressed in

oocytes injected with HUVEC RNA is that the electrical activity persists for a period of 6-10 min

after thrombin is removed and may sometimes be initiated after thrombin has been removed

from the perfusion fluid. This observation is consistent with the idea that interaction of

thrombin with the receptor expressed in oocytes is not a simple equilibrium binding but may

involve proteolytic modification of the receptor and thus not require the continuous presence of

thrombin. It is possible that the lag period is due to the time required for proteolytic cleavage

of the receptor. However, it should be noted that electrical responses in oocytes by agonists

that are not enzymes, such as TRH, may also have prolonged delays (23).

In conclusion, the results of the present study suggest that the thrombin receptor expressed in oocytes injected with HUVEC RNA has many characteristics that are similar to

those of the native HUVEC thrombin receptor, In view of these data, Xenopus /aevis oocytes

appear to be a suitable screening system for the expression cloning of the human endothelial

cell thrombin receptor.

Acknowledaments This work was supported by National Institutes of Health Grants HL-18828 (Specialized

Center for Research in Thrombosis) and DK-43036. The authors thank Richard Straub and Elizabeth Raaka for their help and advice and George Lam for his technical assistance.

91R

Vol. 171, No. 3, 1990 BIOCHEMiCAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

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expression of functional thrombin receptors in xenopus oocytes injected with human endothelial cell...

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