Expression of Receptors for Cholecystokinin and Other Ca2+-Mobilizing Hormones in Xenopus Oocytes (Biosynthesis) JOHN A
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Proc. Nati. Acad. Sci. USA Vol. 85, pp. 4939-4943, July 1988 Physiological Sciences Expression of receptors for cholecystokinin and other Ca2+-mobilizing hormones in Xenopus oocytes (biosynthesis) JOHN A. WILLIAMS*, DENNIS J. MCCHESNEY, M. CLARA CALAYAG, VISHWANATH R. LINGAPPA, AND CRAIG D. LOGSDONt Departments of Physiology and Medicine, University of California, San Francisco, CA 94143; and Cell Biology Laboratory, Mount Zion Hospital and Medical Center, San Francisco, CA 94120 Communicated by Viktor Mutt, March 14, 1988 ABSTRACT The expression of receptors for cholecystoki- by the electrophysiological effects of receptor occupancy. nin (CCK) and other similar acting Ca2 -mobilizing hormones We made use of the fact that the receptors for CCK mobilize was studied in Xenopus laevis oocytes. Poly(A)+ RNA was intracellular Ca2+ to develop a simple functional assay based prepared from pancreatic AR42J cells, which normally express on the increased efflux of 45Ca2. Oocytes injected with receptors for CCK and bombesin and the RNA injected into mRNA from a CCK-sensitive pancreas cell line (AR42J cells) oocytes. The presence of these pancreatic receptors on the were shown to express CCK receptors with normal CCK oocytes was then demonstrated by hormone-induced mobiliza- analog and antagonist sensitivity. We also present data tion of &5Ca2 CCK receptors were present 1 day (maxnum, showing that the CCK receptor is encoded by a 3-kilobase 2 days) after injection ofRNA and were generally proportional (kb) mRNA and that the assay developed can also document to the amount of poly(A)+ RNA injected (1-50 ng). Oocyte the expression of receptors for bombesin, angiotensin, and CCK receptors retained selectivity for CCK analogs (CCK8 > vasopressin when RNA from appropriate cells is injected. unsulfated CCK8 > CCK4) and were blocked by the specific CCK receptor antagonist CR 1409. When poly(A)+ RNA was METHODS subjected to size fractionation on sucrose gradients, activity- inducing CCK receptors showed a single peak centered at 3 Cell Culture. AR42J cells were maintained as subconfluent kilobases. The generality of this oocyte system for expressing cultures in Dulbecco's modified Eagle's medium containing Ca2+-mobilizing hormone receptors was further shown by penicillin, streptomycin, amphotericin B, and 10o fetal bovine expression of a response to bombesin after injection of AR42J serum (from the Cell Culture Facility, University ofCalifornia, cell RNA and a response to vasopressin and angiotensin 11 when San Francisco, CA). In some cases, cells were treated with poly(A)+ RNA from rat liver was injected. No response to CCK dexamethasone (100 nM) for 48 hr prior to harvesting. was demonstrable after injection of liver RNA, demonstrating PreparationofmRNA. RNA was isolated from AR42J cells, the specificity of this assay. rat pancreas, and rat liver by the method of Chirgwin et al. (21). AR42J cells (2 x 107) were suspended by trypsinization, Cholecystokinin (CCK) is a gastrointestinal hormone that washed in phosphate-buffered saline, lysed in 10 ml of a acts to regulate secretion of the exocrine and endocrine buffer containing 4 M guanidine thiocyanate, 50 mM Tris HCl pancreas, gallbladder contraction, and gastric emptying (1, (pH 7.5), 10 mM EDTA, 0.5% sodium lauroylsarcosine, and 2). Moreover, it is also present in brain and other neural tissue 0.1 M mercaptoethanol. RNA was separated from DNA and where it appears to function as a neurotransmitter or neuro- protein by centrifugation through cesium chloride (22). modulator (3-6). The actions of CCK on peripheral target Poly(A) + RNA was obtained by oligo(dT)-cellulose chroma- cells are mediated by enhanced inositol phospholipid turn- tography (23) and was quantified spectrophotometrically over, diacylglycerol formation, and mobilization of intracel- (A26 unit = 40 jig of RNA per ml). lular Ca2" (7). These actions are initiated by specific mem- Size fractionation of RNA was carried out by sucrose brane receptors, which have been characterized as to num- gradient centrifugation as follows: Sucrose gradients (10- ber, specificity, size, and subunit composition (8-12). 50%) were prepared by a modification ofthe method ofLuthe Although the CCK receptor has been solubilized with reten- (24). Briefly, five solutions of 10%o, 20%o,'30%o, 40%o, and 50% tion of binding, complete purification has proven difficult. sucrose (wt/vol) were prepared in a buffer consisting of 50 In the present work, we have concentrated instead on the mM Tris'HCl (pH 7.6), 50 mM KCl, and 5 mM EDTA, and mRNA encoding the receptor. While most cell-free systems 2.5 ml ofeach was sequentially added to quick-seal centrifuge will not correctly synthesize and process membrane recep- tubes (16 x 76 mm) (Beckman Instruments) and quick frozen tors to a functional state, cellular systems such as the on dry ice. Gradients were thawed at 40C for at least 12 hr Xenopus oocyte are able to do so (13). Oocytes, by nature of prior to use. Poly(A)+ RNA (100 tug) was dissolved in H20, their size, can be readily injected and have been shown, after heated to 70'C for 30 min, and fractionated by centrifugation injection ofmRNA or cDNA, to express foreign receptors for at 55,000 rpm for 16 hr in a Beckman Ti7O rotor. Fractions epidermal growth factor, insulin, nicotinic cholinergic neu- (=0.5 ml) were collected from the bottoms of the tubes and rotransmitters, serotonin, substance P, and other neurotrans- precipitated in ethanol. mitters (14-20). While epidermal growth factor and insulin For qualitative analysis, fractions were usually pooled and receptors have generally been recognized by immunoprecip- either injected into oocytes or translated in a wheat germ itation with specific antibodies, the presence of foreign neurotransmitter receptors has generally been documented Abbreviation: CCK, cholecystokinin. *To whom reprint requests should be sent at present address: Department ofPhysiology, 7744 Medical Sciences II, University of The publication costs of this article were defrayed in part by page charge Michigan, Ann Arbor, MI 48109. payment. This article must therefore be hereby marked "advertisement" tPresent address: Department ofPhysiology, University ofMichigan in accordance with 18 U.S.C. §1734 solely to indicate this fact. Medical School, Ann Arbor, MI 48109. 4939 Downloaded by guest on September 28, 2021 4940 Physiological Sciences: Williams et al. Proc. Natl. Acad. Sci. USA 85 (1988) cell-free system (25). in the latter case, the products were When oocytes were injected with mRNA from AR42J cells, then separated by polyacrylamide gel electrophoresis (26). they responded with increased45Ca2+ efflux to CCK and to For determination of the average size of mRNA molecules in a lesser extent to bombesin (Fig. 1). This increased efflux was each fraction, total rat pancreas RNA was fractionated in the maximal in the first 5min and subsided over the next 30min. same manner and the fractions were then electrophoresed on Uninjected oocytes or those injected with H20 responded to a 1.2% agarose gel and stained with ethidium bromide, and neither hormone, although they still responded to carbachol. the bands visualized by UV light were compared to an RNA To determine the time course of responsiveness to CCK sizing ladder (Bethesda Research Laboratories). after mRNA injection, oocytes were studied immediately after Oocyte Injection and Culture. Mature oocyte-positive fe- injection and at daily intervals. Although a significant increase male Xenopus laevis were obtained from Xenopus I (Ann in45Ca2` efflux was always observed after 1 day, the maximal Arbor, MI), maintained in dechlorinated tap water, and fed response was observed after 2 or 3 days (Fig. 2). Most Purina Trout Chow twice weekly. Animals were anesthetized experiments were therefore carried out 2 days after injection, in 0.3% Tricaine solution and ovarian tissue was removed via since at longer times some oocytes appeared to deteriorate. an abdominal incision.Oocytes were teased free under a The magnitude of the response varied between batches of dissecting microscope and large oocytes (Dumont stage V- oocytes from a 4- to 60-fold increase in 45Ca2+ efflux, but VI) were selected for use and stored in modified Barth's within each assay results were quite consistent between wells. saline solution (MBSH) with Hepes buffer (pH 7.4), genta- When oocytes were injected with various amounts of AR42J mycin, and penicillin/streptomycin (27). mRNA, there was a dose-dependent response in CCK- After 24 hr at 18'C, oocytes were examined under a induced45Ca2+ efflux over the range of 1 to 50 ng of injected dissecting microscope, damaged oocytes were removed, and mRNA not To ensure a maximum response, healthy appearing ones were prepared for microinjection (data shown). essentially as described (27).Oocyteswere injected in the oocytes were injected in most cases with 50 ng of mRNA, vegetal pole with 50 nl of ultrapure water or water containing although when it was desired to assess relative mRNA activity poly(A)+ RNA, usually at 1ug/jul. After injection, oocytes in size fractions, lesser amounts were used. were stored at 18'C in MBSH containing 10%6 fetal calf serum Further Characterization ofOocyte CCK Receptors. The that had been dialyzed against MBSH; this medium was properties of CCK receptors on AR42J cells have been changed daily. At the indicated time, usually 48 hr after characterized and are generally similar to those on normal injection, oocytes were washed three times with fresh MBSH pancreas (30). We next tested whether CCK receptors on without serum and then used for the45Ca2+ efflux assay. oocytes preserved the agonist and antagonist selectivity of 4'Ca2" Efflux Assay.Oocytes were incubated in 1 ml of their parent cell. The sulfated CCK octapeptide increased MBSH containing45Ca2 + (50, Ci/ml; 1 Ci = 37 GBq) for 2.5 45Ca2 + efflux over the concentration range of 10-10 to10-7 hr at18°C. They were then washed three times with MBSH M.