RNA in oocytes from Xenopus laevis

Proc. Nati. Acad. Sci. USA Vol. 85, pp. 714-717, February 1988

Biochemistry

Receptors for neuropeptides are induced by exogenous poly(A)+ RNA in oocytes from Xenopus laevis (VI,

V2 receptors for vasopressin/thyrotropin-releasing hormone receptor/angiotensin H receptor)

WOLFGANG MEYERHOF*, STEVE MORLEY*, JURGEN SCHWARZt,

AND DIETMAR RICHTER* Eppendorf, Martinistrasse 52, 2000 Hamburg Universitats-Krankenhaus *Institut ffir Zellbiochemie und klinische Neurobiologie and tInstitut fur Physiologie,

20, Federal Republic of Genrany

Communicated by Tomas Hokfelt, October 1, 1987 (received for review August 11, 1987)

may, therefore, be suitable not only for studying biochemical aspects of peptide hormone receptors, but also as an assay system for molecular cloning experiments (3).

Receptors for the hormones vasopressin, ABSTRACT angiotensin II, and thyrotropin-releasing hormone have been studied electrophysiologically in Xenopus Laevis oocytes previously injected with poly(A)+ RNA from the respective receptor-containing tissues. The injected oocytes responded to the hormones by demonstrating oscillations in membrane currents as recorded by the voltage-clamp method. The response was dependent on the hormone concentrations and detectable between 5 and 1000 nM concentrations. Size fractionation of poly(A)+ RNA from the respective tissues showed that the mRNAs encoding the three hormone receptors were larger than 18S rRNA, suggesting a length of at least 2 kilobases. When vasopressin was added to the oocyte bath, an inward membrane current was generated in oocytes injected with rat poly(A)+ RNA from liver but not from kidney. This suggests that the V1-type (liver), not the V2-type (kidney), vasopressin receptor can be expressed and electrophysiologically identified in the oocyte. A Vl-specific, but not a V2-specific, antagonist suppressed the vasopressin-dependent effect. Application of angiotensin H to liver poly(A)+ RNA-injected oocytes elicited oscillations in membrane current, indicating that these oocytes also expressed receptors for angiotension II; the antagonist [Sarl, O-methionyl-Tyr4]angiotensin H blocked this effect. Poly(A)+ RNA from tumor-derived GH316 cells, known to contain receptors for thyrotropin-releasing hormone, injected into oocytes -induced receptors responding to thyrotropinreleasing hormone; the drug chlordiazepoxide suppressed the thyrotropin-releasing hormone response.

MATERIALS AND METHODS Antagonists and analogues were purchased from Bachem (Basel) and Peninsula (St. Helens, England), respectively. As antagonists for vasopressin receptors the following substances were used: V1, [1-(f-mercapto-f4p3-cyclopentamethylenepropionic acid),O-MeTyr2,Arg8]vasopressin; V2, [1-(J3mercapto-,3B,-cyclopentamethylenepropionic acid),DIle2,le4,Arg8]vasopressin; V1/V2, [1-(3-mercapto-3,4,-cyclopentamethyleneproprionic acid,O-EtTyr2,Arg ]vasopressin. [3-MeHis2]TRH, TRH free acid, and [3,4-dehydro-Pro3NH2]TRH were used as TRH analogues. The angiotensin II antagonist [Sar',O-methionyl-Tyr4]angiotensin II was provided by G. J. Moore, University of Calgary, Canada. The GH3B6 cells came from A. Tixier-Vidal and D. Gourdji, College de France, Paris, and the TRH antagonist chlordiazepoxide was from Hoffmann-La Roche, Basel. Preparation of Poly(A)+ RNA and Injection into Oocytes. Poly(A)+ RNA was prepared from rat liver and rat kidney and GH3B6 tumor-derived rat pituitary cells following procedures reported previously (4, 5). Briefly, RNA isolated from various receptor protein-containing tissues was selectively precipitated, with 0.025 vol 1 M acetic acid and 0.75 vol ethanol, from a 4 M guanidinium isothiocyanate tissue homogenate (10:1, vol/wt). The pellet was then redissolved in the same solution and layered onto a 5.7 M cesium chloride cushion. After centrifugation at 50,000 x g for 20 hr, the resulting RNA pellet was dissolved and passed twice over oligo(dT)-cellulose columns. Oocytes (stage V) from X. laevis were manually defolliculated before injection (5) of =50 nl of poly(A) + RNA (0.5 pug/ltd in 10 mM Tris-HCl, pH 7.5/100 mM NaCl). The injected oocytes were kept in Barth's medium at 200C for 2-5 days with daily changes of the bath. Although a few oocytes responded as early as 24 hr after injection, most were responsive only after 3-5 days of incubation. Electrophysiological Measurements. Whole-cell current measurements were done with a conventional two-microelectrode voltage clamp (6). Borosilicate glass capillaries were pulled and filled with a 3.0 M KCl solution. Voltage recording and current injection electrodes had dc resistances between 0.8 and 3.0 Mfl. The oocytes were placed in a small groove in an experimental chamber and continuously perfused with Ringer's solution (110 mM NaCl/2.5 mM KCl/1.8 mM CaCl2/5 mM Tris-HCI, pH 7.3). After insertion of the microelectrodes, the membrane potential initially dropped and then recovered to values between -30 and -60 mV. Membrane voltage was adjusted to -60 mV and voltage

Recent years have seen the elucidation of biosynthetic pathways at the molecular level for many neuropeptides expressed either in brain or peripheral organs (1). The least described part of the neuropeptide signaling pathway, however, remains the interaction of neuropeptides with their cognate receptor proteins. Major problems in studying the molecular structure-function relationship of neuropeptide receptors are the lack of sufficient material for microsequencing and their extreme lability when isolated from the environment of the lipid bilayer. To circumvent these problems we have adopted an approach based on earlier observations that poly(A)+ RNA from the electric organ of the electric ray, injected into oocytes of Xenopus laevis, induced synthesis of the nicotinic acetylcholine-activated ionchannel proteins, which could be detected by measurement of membrane currents using the voltage-clamp method (2). The data presented here show that certain neuropeptide receptors-namely, those for thyrotropin-releasing hormone (TRH), vasopressin, and angiotensin II, can be expressed and functionally identified by the voltage-clamp method in oocytes from X. laevis after injection of poly(A) + RNA from receptor-containing tissues. The oocyte expression system The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Abbreviations: TRH, thyrotropin-releasing hormone; [Arg']VP, ginine vasopressin.

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ar-

Biochemistry: Meyerhof et al. clamped. Membrane current signal was filtered with a corner frequency of 0.3 kHz. The assayed peptide hormones were perfused at the indicated concentrations. When not otherwise stated, 1 puM of the respective neuropeptide in frog Ringer's solution was used for measurements. The experiments were done at room temperature.

RESULTS Recent studies have revealed that channel and receptor proteins of the eukaryotic cell membrane fall into a few "superfamilies" based upon structural and functional similarities (7). Some of these proteins have in common that they can be functionally expressed and electrophysiologically recorded in the heterologous oocyte system (2, 8, 9). To see whether receptors for peptide hormones such as vasopressin can be expressed and functionally detected in oocytes, injections with poly(A) + RNA from liver, a tissue known to contain vasopressin receptors, were done. The vasopressin receptor system was chosen as a particularly suitable model because two types of receptors exist-namely, V1 and V2, each mediating their functions through different secondmessenger systems. The V1 receptor present in liver and responsible for vasopressor activity is thought to be coupled to inositol phosphate metabolism, causing the release of calcium from intracellular stores (10). The V2 receptor of the kidney is involved in the antidiuretic response to vasopressin and functions via cAMP as second messenger (11). Voltage-clamped oocytes, either not injected or mock injected with water, did not show any changes in membrane current when exposed to vasopressin, or to other neuropeptides, in the oocyte bathing medium (Fig. 1, control). In contrast, oocytes injected with poly(A) + RNA from rat liver (Fig. 1), but not from kidney (data not shown), were rendered responsive to vasopressin as demonstrated by inward membrane currents. Depending on exposure time of the hormone to the oocyte, different current responses were seen. Short 20-sec applications elicited a transient oscillating current response, whereas continued exposure resulted in significant oscillating currents lasting for >25 min. The amplitudes of the inward currents varied between preparations and ranged from 5-100 nA and were significantly TRH

Proc. Natl. Acad. Sci. USA 85 (1988)

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smaller than the current responses to serotonin, which could be as large as 1-2 AA (9). The response to vasopressin recorded in the oocyte was detected at concentrations between 150 and 1000 nM. A slight effect was seen with vasotocin but not with isotocin, oxytocin, or vasoactive intestinal peptide (data not shown), reflecting the known ligand specificity of the V1 vasopressin receptor type (12). The fact that V1-type vasopressin receptors were, indeed, acquired by membranes of oocytes injected with liver poly(A)+ RNA was further supported by experiments in which the response was measured in the presence of V1- or V2-receptor-specific antagonists (12). Only the V1 and the V1/V2, but not the V2, antagonist blocked the vasopressinspecific response of the oocyte (Fig. 2). Fig. 1 shows that after injection of liver poly(A)+ RNA, the oocyte membrane also apparently contained receptors for angiotensin II, representing a further example of a phosphatidyl inositol-linked receptor function (13). The voltage-clamped oocytes responded at concentrations between 0.1 and 1 uM of angiotensin II. The antagonist, [Sari, O-methionyl-Tyr4]angiotensin II (0.5 ,uM), suppressed this response when applied in 5-fold molar excess over the ligand

(data not shown). Receptors for TRH, also known to be coupled to ion channels through the inositol phospholipid pathway, have been studied in GH3B6 cells (14). Poly(A)+ RNA from GH3B6 cells injected into oocytes rendered them responsive to TRH (Figs. 1 and 3). Positive signals were obtained with TRH concentrations between 5 and 1000 nM (Fig. 3). TRH could be replaced by dehydro-TRH and the agonist methylTRH (15) but not by TRH free acid. The drug chlordiazepoxide antagonized the TRH-dependent membrane current change (Fig. 2B). Other peptides like vasoactive intestinal peptide, vasopressin, oxytocin, or substance P failed to elicit a significant response at the concentrations tested (1 ,uM) (data not shown). Size fractionation of poly(A)+ RNA preparations from various tissues on NaDodSO4 sucrose-density gradients (16) yielded mRNA fractions slightly larger than 18S rRNA evoking responses when injected into oocytes and exposed to the respective ligand. This suggests that the mRNAs encoding the receptor types reported here are at least 2 kilobases in size (Fig. 4). vasopressin

LI

angiotensin 11

control

FIG. 1. Whole-cell current record of voltage-clamped X. laevis oocytes. Oocytes were injected with 50 nl of poly(A) + RNA from rat liver (vasopressin and angiotension II receptors) or GH3B6 cells (TRH receptors). In the control experiment the oocyte was injected with 50 nl of buffer. The oocytes were exposed to 1 utM of the respective neuropeptide, as indicated by bars; otherwise the oocyte bath was perfused with frog Ringer's solution. Time and current scale is indicated by the right-angled bars: horizontal bars, 60 sec; vertical bars, 10 nA (except for the TRH experiment with 20 nA as scale).

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FIG. 2. Effects of arginine vasopressin, [Argq]VP, (AVP) and TRH antagonists on [Arg]VP or TRH-mediated membrane current responses of voltage-clamped oocytes injected with rat liver or GH3B6 poly(A) I RNA. (A) Vasopressin receptor. Bars, exposure time of the oocyte to [Arg8]VP or to the antagonists. Application of antagonists was followed by perfusion with Ringer's solution and finally by [Arg8]VP (1000 nM), each for a 2-min period. The following concentrations of the antagonists were used: 100 nM V1, 1000 nM V2, and 100 nM V1/V2. The angiotensin II receptor was not blocked by either the V1 or the V2 antagonist. (B) TRH receptor. Bars, exposure time of the oocyte to a solution of 200 nM chlordiazepoxide/100 nM TRH (1), followed by Ringer's perfusion and then by 100 nM TRH (2).

DISCUSSION The data presented here indicate that at least some receptors for neuropeptides can be identified in voltage-clamped oocytes from X. laevis when injected with poly(A)+ RNA from receptor-containing tissues. Peptide responsiveness of the oocyte is probably dependent upon the capability of the induced receptor to interact with an endogenous secondmessenger system. Presumably this occurs via G protein(s)

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FIG. 4. NaDodSO4 sucrose-density gradient centrifugation of poly(A)I RNA derived either from rat liver (1, 2) or GH3B6 cells (3). Individual fractions were precipitated with ethanol and analyzed

FIG. 3. TRH-mediated changes in membrane currents of voltage-clamped oocytes injected with poly(A)+ RNA extracted from GH3B6 cells. Various TRH concentrations were applied to the incubation bath for the time indicated by bars.

by injection of 50 nl into oocytes as reported earlier (16). After incubation for 3-5 days, the oocytes were voltage clamped and exposed to 1 A&M of either vasopressin (1), angiotensin 11 (2) or TRH (3). Bars, positions of the RNA fractions giving rise to positive responses by the voltage-clamped oocytes; the poly(A)I RNA encoding the receptors were found in a few fractions as rather distinct peaks, quite in contrast to the vasopressin fractions, which showed a broader distribution in the gradient.

Biochemistry: Meyerhof et al. of the frog oocyte that interact either directly or indirectly with endogenous Ca2"-activated ion channels, possibly of either the chloride or potassium type (17). It is remarkable that those peptide receptors responding positively in the oocyte are all thought to ultimately cause Ca2l release from intracellular stores via the inositol phosphate secondmessenger system. This is particularly clear in the case of liver V1 and kidney V2 receptors for vasopressin, where positive responses were recorded only with oocytes injected with liver poly(A) + RNA. On the other hand, the failure to identify the kidney vasopressin receptor in the voltageclamped oocyte may simply be due to a more complex assembly process of this receptor type, which apparently is composed of at least two subunits (11) and probably encoded by different mRNAs. It is well documented that the class of G proteins represent a coupling element in the transmembrane signaling pathway between extracellular membrane-localized receptors and intracellular effectors (18). G proteins may act either directly on ion channels or indirectly via enzymes that alter the concentrations of second messengers such as inositol phosphate, etc. It is tempting to speculate then that those peptide receptors, the presence of which has been functionally demonstrated in oocytes, recognize the same type of endogenous G protein(s); this would suggest that these receptor proteins are not only functionally, but also structurally, related by their interaction with common elements of the second-messenger signaling system of the oocyte. A structural grouping into protein families has been suggested for rhodopsin, the 3-adrenergic, and muscarinic acetylcholine receptors (19). Sequence comparisons reveal common features-e.g., the presence of several putative membranespanning domains, presumptive glycosylation sites at the hydrophilic, extracellularly localized NH2 terminus, and a long stretch of hydrophilic sequence at the COOH terminus that faces the cytoplasm. Close to the second hydrophobic membrane-embedded domain, a conserved amino acid sequence is found that is a candidate for interacting with G protein(s). Whether this domain is also present in the peptide receptors discussed here remains to be seen. The availability of the oocyte system offers a number of possibilities for studying the properties and structures of at least some receptor proteins. First, the system offers an opportunity to study the biochemistry of receptors in the context of a lipid bilayer environment, rather than as purified protein preparations. Second, the oocyte system may be useful for detecting expression of plasmid-encoded receptor cDNAs. This second aspect may prove particularly valuable in view of the difficulties encountered in obtaining purified


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preparations of certain receptor proteins as a starting point for classical cloning methods. We thank Dr. B. Sakmann, Gottingen, for his support in setting up the voltage-clamp method and thank W. Rust, H. Holtgreve-Grez, S. Engel-Haskiris, G. Ellinghausen, and G. Eikhof for skillful technical help. We are grateful to Dr. A. Tixier-Vidal for drawing our attention to the TRH antagonist and for providing the GH3B6 cell line. We also thank Deutsche Forschungsgemeinschaft for financial support to D.RL 1. Krieger, D. T. (1983) Science 222, 975-985. 2. Barnard, E. A., Beeson, D., Bilbe, G., Brown, D. A., Constanti, A., Conti-Tronconi, B. M., Dolly, J. O., Dunn, S. M. J., Mehraban, F., Richards, M. M. & Smart, T. G. (1983) Cold Spring Harbor Symp. Quant. Biol. 43, 109-124. 3. Lubbert, H., Hoffman, B. J., Snutch, T. P., van Dyke, T., Levine, A. J., Hartig, P. R., Lester, H. A. & Davidson, N. (1987) Proc. Nadl. Acad. Sci. USA 84, 4332-4336. 4. Chirgwin, T. M., Przybyla, A. E., MacDonald, R. J. & Rutter, W. J. (1979) Biochemistry 18, 5294-5299. 5. Kalthoff, H. & Richter, D. (1979) Biochemistry 18, 4144-4147. 6. Methfessel, C., Witzemann, V., Takahashi, T., Mishina, M., Numa, S. & Sakmann, B. (1986) Pflagers Arch. 407, 577-588. 7. Stevens, C. F. (1987) Nature (London) 328, 198-199. 8. Gundersen, C. B., Miledi, R. & Parker, I. (1983) Proc. R. Soc. London Ser. B 220, 131-140. 9. Takahashi, T., Neher, E. & Sakmann, B. (1987) Proc. Natl. Acad. Sci. USA 84, 5063-5067. 10. Creba, J. A., Downes, C. P., Hawkins, P. T., Brewster, G., Michell, R. H. & Kirk, C. J. (1983) Biochem. J. 212, 733-747. 11. Fahrenholz, F., Boer, F., Crause, P. & Toth, M. V. (1985) Eur. J. Biochem. 152, 589-595. 12. Jard, S., Gaillard, R. C., Guillon, G., Marie, J., Schoenenberg, P., Muller, A. F., Mannin, M. & Sawyer, W. H. (1986) Mol. Pharmacol. 30, 171-177. 13. Keppens, S., Vandenheede, J. R. & De Wulf, H. (1977) Biochim. Biophys. Acta 496, 448-457. 14. Gourdji, D., Tougard, C. & Tixier-Vidal, A. (1982) in Frontiers in Neuroendocrinology, eds. Ganong, W. F. & Martini, L. (Raven, New York), pp. 317-357. 15. Grousell, D., Gaivre-Bauman, A. & Tixier-Vidal, A. (1978) Neurosci. Lett. 7, 7-15. 16. Richter, D., Schmale, H., Ivell, R. & Schmidt, C. (1980) in Biosynthesis, Modiflcation, and Processing of Cellular and Viral Polyproteins, eds. Koch, G. & Richter, D. (Academic, New York), pp. 43-66. 17. Dascal, N., Ifune, C., Hopkins, R., Snutch, T. P., Lubbert, H., Davidson, N., Simon, M. I. & Lester, H. A. (1986) Mol. Brain Res. 1, 201-209. 18. Gilman, A. G. (1986) Trends Neurosci. 9, 460-463. 19. Kubo, T., Fukuda, K., Mikami, A., Maeda, A., Takahashi, H., Mishina, M., Haga, T., Haga, K., Ichiyama, A., Kangawa, K., Kojima, M., Matsuo, H., Hirose, T. & Numa, S. (1986) Nature (London) 323, 411-416.