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. Similar 45Ca mobilization could be produced by the at room temperature and aliquoted in groups of 10 into 16-mm unsulfated octapeptide and the C-terminal tetrapeptide, but flat-bottomed polystyrene wells, each containing 0.5 ml of these forms were weaker than CCK octapeptide by factors of MBSH plus soybean trypsin inhibitor (1 mg/ml). This me- 25 and 150 (Fig. 3). These relative potencies are similar to dium was changed every 10 min for 70 min and thereafter at those observed for interaction with the CCK receptor on 5-min intervals. The removed medium was either discarded AR42J cells (30). Normal agonist interaction of CCK with its or was placed in liquid scintillation vials to which was added receptor requires C-terminal amidation (1-3). When C- 5 ml of Liquiscint cocktail. In the standard protocol, hor- terminal CCK analogs with and without amidation were mones were added at 90 min followed by carbachol at 120 compared, only the amidated form induced 45Ca2+ efflux min. Oocytes possess muscarinic receptors (28) and, in the from oocytes (data not shown). absence of a hormonal response, the response to carbachol A number of potent and selective CCK antagonists have indicated the oocytes were healthy and capable of generating recently been developed that interact with peripheral CCK a Ca2+-efflux transient. Initial experiments with carbachol receptors. One of these, CR1409 (chlorglumide), which is were used to optimize the loading procedure and additions to the medium to the oocytes responsive. To 1200 T necessary keep CCK * normalize for different amounts of 45Ca2+ in each group of 1000 oocytes, the45Ca2+ cpm in the 5-min wash immediately 0 before stimulation was set at 100% for each well. Under the 0 800 ) specified loading conditions, mean 45Ca2+ efflux during the x 85- to 90-min collection period immediately prior to stimu- 0 600 S CCh lation between 52 and 225 over 51 experiments. vi ranged cpm us. 400 Bombesin *\___ RESULTS 200 S. C*CK \ L_,_ISCOCK-oL 110-OS,)A0-//1 0 13 80 90 1 00 Oocytes Express Pancreatic Receptors. Oocytes demon- 0 I strate a time-dependent uptake and efflux of 45Ca2+, which 70 80 90 100 110 120 130 follows a multiexponential time course (29). In our experi- ments, efflux was characterized by a rapid phase that was TIME (min) largely over by 60 min and by a much slower phase with FIG. 1. Effect of CCK, bombesin, and carbachol on 45Ca2" almost constant release. Preliminary studies established that efflux from Xenopus oocytes. Oocytes were loaded with 45Ca2' for carbachol induced a 4- to 6-fold transient increase in oocyte 2.5 hr, washed, and aliquoted in groups of 10 in multiwells, and the 45Ca2` efflux from slowly exchanging stores. This was medium was collected periodically. 45Ca2+ efflux for each well was readily demonstrated after loading for 2.5 hr with45Ca2+ and normalized relative to efflux from the period 85-90 min immediately couple prior to stimulation. Open circles, oocytes injected with H20; other a washout of 70-90 min. Since muscarinic receptors symbols, oocytes injected with 50 ng of AR42J cell poly(A)+ RNA. via a guanine nucleotide-binding protein to inositol trisphos- Mean 45Ca2+ efflux over the 85- to 90-min period in cpm was as phate production, which then induces Ca2+ mobilization follows: o, 88 ± 3; e, 92 ± 5; a, 102 ± 13. All points are placed from intracellular stores, we reasoned that similarly acting midway in the collection period and are the means ± SEM of foreign receptors should also couple to this endogenous triplicate wells from a representative experiment. CCK and bombe- mechanism. sin were added at 10 nM, and carbachol (CCh) was added at 100 /AM. Downloaded by guest on September 28, 2021 Physiological Sciences: Williams et al. Proc. Natl. Acad. Sci. USA 85 (1988) 4941

2400 C- 0 3000 0 0 2000 -W oc 2500 4 x 1600 0 aN 2000 -J0 U1-) 1200 x LLIcam 3 1500 800 1000 0 u -J O z 400 +? 500 0 70 0 70 0 24 48 72 80 90 100 110 120 TIME POSTINJECTION (h) TIME (min)

FIG. 2. Dependence of CCK receptor expression in oocytes on FIG. 4. Effect of the CCK antagonist CR1409 to block CCK- time after injection. Points are means + SEM offive experiments in induced 45Ca2" efflux from oocytes injected with AR42J cell each of which the maximal increase in response to 10 nM CCK was poly(A)+ mRNA. CR1409 (10 ,uM) was added continuously begin- determined as in Fig. 1 in duplicate or triplicate wells at the time ning at 80 min. CCK was added at 2 nM and carbachol (CCh) was specified after injection of 50 ng of RNA per oocyte. added at 100 AM where indicated. All points are the means ± SEM of triplicate wells from a representative (of three) experiment. Mean selective for peripheral CCK receptors (31), was tested and 45Ca2+ efflux over the 85- to 90-min period in cpm was as follows: found to block the 45Ca2 + -efflux response of oocytes to CCK control, 96 ± 9; CR1409, 86 ± 5. (Fig. 4). As expected, atropine blocked the response to carbachol but had no effect on the response to CCK (data not poly(A)+ RNA was subjected to sucrose gradient fraction- shown). Note that in this experiment, in which CCK induced ation. Total pancreatic RNA was separated on a parallel a large increase in 45Ca2 + efflux, carbachol had no additional gradient and the fractions were subjected to agarose gel effect after CCK on oocytes injected with AR42J mRNA, electrophoresis to establish that the gradient resolved species indicating both agonists release 45Ca2+ from the same pool. from 0.3 to 9.0 kb by comparison to an RNA ladder. When Injection of mRNA from Rat Liver and Pancreas. To deter- fractions from the sucrose gradient were pooled and injected mine the generality of the expression of Ca2+-mobilizing into oocytes, a peak of CCK receptor activity was observed hormone receptors, we prepared poly(A) + RNA from normal in fractions corresponding to an average 3-kb mRNA (Fig. rat liver and injected it similarly into oocytes. These oocytes 6A). When the same mRNA fractions were translated in a responded with 45Ca2+ efflux to angiotensin II and vasopres- wheat germ system, the fractions encoding CCK receptors sin but did not respond to CCK (Fig. 5). This response was were observed to preferentially program the synthesis of observed in three experiments with 10 or 100 nM peptide proteins in the Mr 40,000-80,000 region (Fig. 6B). Amylase concentrations and was always a 150-350o increase, which (Mr, 55,000), the most prominent protein expressed by AR42J was smaller than the response to CCK in oocytes injected with cells, was expressed with peak activity in a lighter RNA AR42J mRNA. In several experiments, poly(A) + RNA from fraction (=2 kb), consistent with the known size of amylase rat pancreas was injected. A 100% increase in 45Ca2 + efflux in mRNA. response to CCK was seen in two of four experiments. Thus, mRNA for the CCK receptor appears to be either more abundant or more stable in AR42J cells as compared to rat DISCUSSION pancreas. This is not surprising in view of the high levels of Xenopus oocytes, similar to most mammalian cells, possess ribonuclease found in rat pancreas (21). calcium-mobilizing and sequestering systems. Release of se- Size Fractionation of CCK Receptor mRNA. To determine the size of the mRNA encoding the CCK receptor as well as Angiotensin II 0-0 to develop a preparation enriched for this mRNA, AR42J cell Vasopressin 0-0 CCK /-A Duu r

0 2400 Hormone CCh 0Lo 400 0 I0 L-0 V V 2000 0 C) A1\ A 300 x cJ 1600 x ., UL- -J 200 1200 UL-

0 cam U-) 0 800 100 If) 400 0 _ 0 70 80 90 100 110 120 0 10o10 10-9 10-8 10-7 TIME (min) CCK ANALOG (M) FIG. 5. Effect of angiotensin II, vasopressin, CCK, and carba- FIG. 3. Analog specificity for CCK-induced 45Ca2" efflux from chol (CCh) on 45Ca2+ efflux from oocytes injected with 50 ng each oocytes injected with AR42J cell poly(A)+ RNA. Oocytes (250) were of rat liver poly(A)+ RNA. After 90 min of 45Ca2" efflux, the injected with 50 ng of RNA each, loaded together with 45Ca2", and hormones were added at a concentration of 100 nM; CCh (100 ,uM) efflux was stimulated in groups of 10 with the specified concentration was added to all wells at 115 min. Mean 45Ca2" efflux just prior to of CCK peptide. Points are the means + SEM for the maximal stimulation in cpm was as follows: vasopressin stimulation, 220 ± increase determined in triplicate wells except for unsulfated CCK8, 15; angiotensin II stimulation, 189 ± 20; CCK stimulation, 190 ± 26. which was determined in duplicate. Representative of two experi- All points are the means ± SEM of triplicate wells from a repre- ments. sentative experiment. Downloaded by guest on September 28, 2021 4942 Physiological Sciences: Williams et al. Proc. Natl. Acad Sci. USA 85 (1988) more than compensated by its ease and lack of requirement of any specialized equipment such as is necessary to measure 0 internal Ca2+ concentration directly or the resulting electro- physiological changes. The present assay clearly has an x excellent signal/noise ratio and is applicable to a variety of -j hormone receptors. Thus, this assay will greatly simplify the detection in oocytes of receptors that interact with a Ca2+- 0 mobilizing system. LO The second aim was to begin characterization of the -i mRNA encoding the CCK receptor. We have shown that AR42J cell isolated poly(A) + RNA encodes a CCK receptor that can be functionally expressed in oocytes. This oocyte 0 3 6 9 12 15 18 21 30 receptor has properties similar to that of native CCK recep- FRACTION tors in regard to its ability to interact with CCK agonists and antagonists. Specifically, the receptor interacts with high B affinity with CCK analogs having a sulfated tyrosine and an Mr 1 2 3 4 5 6 7 8 9 1011 amidated C terminus. Size fractionation on sucrose gradients x 10o-3 indicated the CCK receptor mRNA is =3 kb. This mRNA 68 - size is slightly smaller than that encoding the cardiac musca- rinic receptor (37) and serotonergic 5HT1c receptor (20). ::' 45 - While we cannot conclude definitely that the CCK receptor ." is encoded by a single mRNA species, this seems likely. :_iu 30 - Sucrose gradient centrifugation shows a single peak encoding .. CCK receptors. Moreover, crosslinking studies on rat pan- creas have shown labeling of a single binding subunit vari- ously estimated at Mr 75,000-90,000 (10, 11). Finally, the 18- similar acting muscarinic acetylcholine, a-adrenergic, and 12 - serotonergic receptors are specified by a single mRNA. The sucrose gradient clearly separated mRNA encoding the CCK receptor from that for amylase, the most abundant protein in _ ... AR42J cells. Thus, this size-fractionation procedure should FIG. 6. Size fractionation of AR42J cell poly(A) I RNA on a 10- allow construction of a cDNA library enriched for the CCK 50%1o sucrose gradient. Thirty fractions were collected and pooled in receptor. This opens the possibility of cloning the CCK threes, and RNA was injected into oocytes or expressed in a wheat receptor by using the Xenopus oocyte as an expression germ cell-free system. (A) CCK-stimulated 45Ca2l efflux was mea- system in which clones may be screened (38, 39). sured from oocytes injected with the various fractions. Size markers were based on the separation of total rat pancreas RNA in parallel We thank Karen Perot for assistance with RNA preparation. This tubes, which were then evaluated by agarose gel electrophoresis. (B) work was supported by National Institutes of Health Grants The same mRNA fractions translated in vitro in a cell-free system and DK32994, DK35912, and GM31626. the synthesized proteins analyzed by NaDodSO4/PAGE. Molecular weight markers are indicated on the left. Lanes: 1, products of 1. Mutt, V. (1980) in Gastrointestinal Hormones, ed. Glass, fractions 1-3; 2, fractions 3-6, etc.; 11, endogenous wheat germ G. B. J. (Raven, New York), pp. 171-221. translation products in the absence of added RNA. The dense band 2. Walsh, J. H. (1987) in Physiology ofthe Gastrointestinal Tract, at Mr 55,000 was confirmed as amylase by immunoprecipitation. ed. Johnson, L. R. (Raven, New York), 2nd Ed., pp. 195-206. 3. Williams, J. A. (1982) Biomed. Res. 3, 107-121. 4. Neuropeptides intracellular Ca2 + in is initiated to various Beinfeld, M. C. (1983) 3, 411-427. questered oocytes 5. Vanderhaeghen, J. J., Signeau, J. C. & Gepts, W. (1975) degrees by fertilization, progesterone, and acetylcholine (29, Nature (London) 257, 604-605. 32, 33). This Ca2" mobilization appears to involve activation 6. Dockray, G. J., Gregory, R. A. & Hutchison, J. B. (1978) of a phospholipase C, which acts to break down inositol Nature (London) 274, 711-713. phospholipids. It has been reported that acetylcholine acting 7. Hootman, S. R. & Williams, J. A. (1987) in Physiology ofthe on endogenous oocyte muscarinic receptors brings about an Gastrointestinal Tract, ed. Johnson, L. R. (Raven, New York), increase in the inositol content while pp. 1129-1146. trisphosphate (34), 8. Sankaran, H., Goldfine, I. D., Deveney, C. W., Wong, K. Y. injection ofinositol trisphosphate into oocytes initiates release & Williams, J. A. (1980) J. Biol. Chem. 255, 1849-1853. of Ca2+ from intracellular stores (33-35). The physiological 9. Jensen, R. T., Lemp, G. F. & Gardner, J. D. (1980) Proc. Natl. function of acetylcholine acting on oocytes is not yet clear, Acad. Sci. USA 77, 2079-2083. although the increase in intracellular Ca2+ is believed to 10. Rosenzweig, S. A., Miller, L. J. & Jamieson, J. D. (1983) J. control the opening of a chloride channel that can lead to rapid Cell Biol. 96, 1288-1297. oscillations in membrane potential. Oscillations in membrane 11. Sakamoto, C., Goldfine, I. D. & Williams, J. A. (1983) J. Biol. Chem. 258, 12708-12711. potential similar to those normally induced by acetylcholine 12. Szecowka, J., Goldfine, I. D. & Williams, J. A. (1984) Regul. are initiated in oocytes by serotonin (20) or substance P (19) Pept. 10, 71-83. after injection with mRNA prepared from rat brain. 13. Lane, C. D. (1983) Curr. Top. Dev. Biol. 18, 89-119. Our work had two aims. The first was to develop a simple 14. Sumikawa, K., Houghton, M., Emtage, J. S., Richards, B. M. assay for demonstrating expression of mRNA encoding & Barnard, E. A. (1981) Nature (London) 292, 862-864. receptors that activate inositol phospholipid turnover by 15. Simmen, F. A., Schulz, T. Z., Headon, D. R., Wright, D. A., measuring one of the sequelae of Ca2+ mobilization- Carpenter, G. & O'Malley, B. W. (1984) DNA 3, 393-399. the accelerated efflux of intracellular Such 16. Ebina, Y., Edery, M., Ellis, L., Standring, D., Beaudoin, J., namely, 45Ca2". Roth, R. A. & Rutter, W. J. (1985) Proc. Natl. Acad. Sci. USA protocols were developed a number of years ago and pro- 82, 8014-8018. vided some of the first evidence for release of intracellular 17. Gundersen, C. B., Miledi, R. & Parker, I. (1984) Proc. R. Soc. Ca2+ as an intracellular messenger (36). That 45Ca2I efflux London Ser. B 221, 235-244. is somewhat an indirect measure of receptor activation is 18. Sumikawa, K., Parker, I. & Miledi, R. (1984) Proc. R. Soc. Downloaded by guest on September 28, 2021 Physiological Sciences: Williams et al. Proc. Nati. Acad. Sci. USA 85 (1988) 4943

London Ser. B 223, 255-260. 31. Neiderau, C., Neiderau, M., Williams, J. A. & Grendell, J. H. 19. Parker, I., Sumikawa, K. & Miledi, R. (1986) Proc. R. Soc. (1986) Am. J. Physiol. 251, G856-G860. London Ser. B 229, 151-159. 32. Wasserman, W. J., Pinto, L. H., O'Connor, C. M. & Smith, 20. Lubbert, H., Snutch, T., Dascal, N., Lester, H. A. & David- L. D. (1980) Proc. NatI. Acad. Sci. USA 77, 1534-1536. son, N. (1987) J. Neurosci. 7, 1159-1165. 33. Buso, W. B., Ferguson, J. E., Joseph, S. K., Williamson, J. R. 21. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. & Rutter, & Nuccitelli, R. (1985) J. Cell Biol. 101, 677-682. W. J. (1979) Biochemistry 18, 5294-5299. 34. Oron, Y., Dascal, N., Nadler, E. & Lupu, M. (1985) Nature 22. Glisin, V., Crkvinjakov, R. & Byus, C. (1974) Biochemistry 13, (London) 313, 141-143. 2633-2637. 35. Parker, I. & Miledi, R. (1986) Proc. R. Soc. London Ser. B 228, 23. Aviv, H. & Leder, P. (1972) Proc. NatI. Acad. Sci. USA 69, 1408-1412. 307-315. 24. Luthe, D. S. (1983) Anal. Biochem. 135, 230-232. 36. Mathews, E. K., Peterson, 0. H. & Williams, J. A. (1973) J. 25. Erickson, A. H. & Blobel, G. (1983) Methods Enzymol. %, 38-50. Physiol. (London) 234, 689-701. 26. Laemmli, U. K. (1970) Nature (London) 227, 680-685. 37. Kubo, T., Maeda, A., Sugimoto, K., Akiba, I., Mikami, A., 27. Colman, A. (1984) in Transcription and Translation: A Practi- Takahasi, H., Haga, T., Haga, K., Ichiyama, A., Kangawa, K., calApproach, eds. Hames, D. & Higgins, S. (IRL, Oxford), pp. Matsuo, H., Hirose, T. & Numa, S. (1986) FEBS Lett. 209, 367- 271-301. 372. 28. Kusano, K., Miledi, R. & Stinnakre, J. (1982) J. Physiol. 38. Lubbert, H., Hoffman, B. J., Snutch, T. P., Van Dyke, T., (London) 328, 143-170. Levine, A. J., Hartwig, P. R., Lester, H. A. & Davidson, N. 29. O'Connor, C. M., Robinson, K. R. & Smith, L. D. (1977) Dev. (1987) Proc. Nail. Acad. Sci. USA 84, 4332-4336. Biol. 61, 28-40. 39. Masu, Y., Nakayama, K., Tamaki, H., Harada, Y., Kuno, M. 30. Logsdon, C. D. (1986) J. Biol. Chem. 261, 2096-2101. & Nakanishi, S. (1987) Nature (London) 329, 836-838. Downloaded by guest on September 28, 2021