THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

VOl. 264, No. 20, Issue of 'July 15, PP. 11901-11903,1969 Printed in U.S.A.

Expression of the Thyroid Sodium/Iodide Symporter in Xenopus laevis Oocytes*

(Received for publication, January 27, 1989)

Franck Vilijn and Nancy CarrascoS From the Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461

Poly(A+) RNA isolated from FRTL-5 cells (a contin- uous line of cultured and fully functional rat thyroid cells (Ambesi-Impiombato, F. S . , Parks, L. A. M., and Coons, H. G . (1980) Proc. Natl. Acad. Sei. U. S. A. 77, 3455-3459)) was injected into Xenopus laevis oocytes, and the expression of the Na+/I- symporter in the plasma membrane was assayed by measuring the Na+- dependent C10;-sensitive uptake of 12% Expression of the Na+/I- symporter was detected as a 7-fold average increase in transport over background, 5-6 days after injection. Poly(A+) RNA was subsequently fractionated by sucrose gradient centrifugation, and fractions were assayed for their ability to induce I- transport activity. The poly(A+) RNA encoding the Na+/I- symporter was found in a fraction containing messages of 2.8-4.0 kilobases in length.

The Na+/I- symporter of the thyroid gland, which is re- sponsible for the active Na+-dependent accumulation of I-, is a membrane protein located in the basolateral end of the epithelial cells of the thyroid follicles. I- transport into these cells is the first step in the synthesis of the iodine-containing thyroid hormones T3 and T4. After I- is taken up, it is oxidized and incorporated into tyrosyl residues on the thyro- globulin molecule; subsequently, iodinated thyroglobulin is stored extracellularly in the follicular colloid. Thyroid hor- mones T3 and T4 result from lysosomal cleavage of endocy- tosed iodinated thyroglobulin in the follicular cells (2). The general properties of the 1"concentrating system of the thy- roid were elucidated from early experiments performed in intact animals (3, 4), thyroid slices (5), and primary cell cultures (6, 7). The Na+/I- symporter has recently been further characterized in FRTL-5 cells (1, 8-12) and hog thyroid membrane vesicles (13). In summary, I- uptake in thyroid tissue and thyroid-derived cells has been found to be: (a) an active transport process that occurs against an electro- chemical gradient; ( b ) driven by an inwardly directed Na+ gradient generated by the ouabain-sensitive Na+/K+ ATPase; (c) sensitive to anions which act as competitive inhibitors, such as SCN- and C10;; and ( d ) induced by TSH' from the anterior pituitary by activation of the adenylate cyclase- CAMP-dependent protein kinase system (14). Despite the Na+/I- symporter's fundamental importance in mammalian physiology and its significance in light of iodine's relative

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$To whom correspondence should be addressed. Tel.: 212-430- 3523.

The abbreviations used are: TSH, thyroid-stimulating hormone; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.

scarcity in the environment, little is currently known about its structure, biochemistry, and molecular properties. Hence, we have initiated studies on the properties of the transporter and its cognate mRNA by expressing it in Xenopus l aev i s oocytes and identifying the size of the mRNA that encodes the carrier protein. In the absence of probes such as antibodies or oligonucleotides based on protein sequence data, this func- tional approach provides a feasible route for the cloning and structural analysis of the Na+/I- symporter. X . lueuis oocytes faithfully translate heterologous mRNA and carry out post- translational modifications, including processing of precursor molecules (15), phosphorylation (16, 17), and glycosylation (18-20). Appropriate targeting (21) also takes place in this expression system.

EXPERIMENTAL PROCEDURES

Materials-The following materials were obtained from the indi- cated sources: guanidinium thiocyanate, Fluka; CsC1, International Biotechnologies, Inc.; oligo(dt)-cellulose type 11, Collaborative Re- search; sucrose, Bethesda Research Laboratories; Leibovitz medium, penicillin, and streptomycin, GIBCO; N a Y , Amersham Corp.

Isolation of Poly(A+) RNA-Total RNA was prepared by a modifi- cation of the method of Chirwin et al. (18). FRTL-5 cells were harvested with a rubber blade and washed with phosphate-buffered saline. The cell suspension was centrifuged at 500 X g for 3 min, resuspended in 10 volumes of extraction buffer (4 M guanidinium thiocyanate, 1 M 2-mercaptoethanol, 0.1 M sodium acetate (pH 5.0), 0.01 M EDTA), homogenized with a Brinkmann Polytron homoge- nizer, and stirred for 15 min in the presence of 1 g/ml CsC1. The homogenate was layered on top of a saturated CsCl solution and centrifuged at 140,000 x g for 16 h at 18 "C. After centrifugation, the RNA band was aspirated, extracted with chloroform:butanol, 4:1, and precipitated with ethanol. Poly(A+) RNA was isolated by affinity chromatography on an oligo(dT)-cellulose type I1 column, according to Aviv and Leder (19).

Size Fractionation of Poly(A+) RNA-Poly(A+) RNA was size- fractionated in a sucrose gradient. Poly(A+) RNA (150 pg) was heated to 65 "C, immediately cooled, and then sedimented through a 5-25% (w/w) sucrose gradient containing 10 mM Hepes (pH 7.5), 2 mM EDTA, and 0.1% lithium dodecyl sulfate in an SW 41 rotor at 85,000 X g for 17 h at 5 "C. Fractions (450 pl) were collected, precipitated with ethanol, and dissolved in water.

Microinjection of PoEy(A+) RNA into X. laeuis Oocytes-X. laevis oocytes were microinjected according to the general methods of Col- man (20). Female X. h u i s frogs (Nasco, Fort Atkinson, WI) were anesthetized for 25 min in 1.8 mg/ml3-aminobenzoic acid ethyl ester. Part of the ovary was dissected, excised, and placed in a Ca*+-free ND 96 solution (96 mM NaCl, 2 mM KC1, 1 mM MgCl,, 5 mM Hepes (pH 7.5), 2.5 mM pyruvate, 100 units/ml penicillin, 0.1 mg/ml strep- tomycin). The tissue was digested with 2 mg/ml collagenase in the same Ca2+-free solution for 2 h. Oocytes were then transferred to 1.8 mM Ca2'-containing ND 96 solution and dissected manually to re- move the follicular layer. Dissected oocytes were incubated overnight a t 19 "C in Hepes-buffered Leibovitz medium containing penicillin and streptomycin. Stage V-VI oocytes were selected for microinjec- tion. Fifty nl of water containing 0 or 50 ng of poly(A+) RNA as indicated in the legends was microinjected as described (21). Microin- jected oocytes were maintained at 19 "C in Leibovitz medium, which was changed daily.

11901

11902 Expression of the Sodium/Iodide Symporter in Oocytes Transport Assays-Microinjected oocytes (5/assay) were incubated

for 45 min (unless otherwise noted) in 100 pl of a solution containing 50 pM NaiZ5I (specific activity, 100 mCi/mmol), 10 mM Hepes (pH 7.5), 1 mM MgC12,l mM CaCl2, 2 mM KC1 with either 100 mM NaCl, 100 mM NaCl, 10 pM NaC104, or 100 mM choline chloride. Reactions were terminated by the addition of 5 ml of ice-cold quenching buffer (100 mM choline chloride, 10 mM Hepes (pH 7.5),1 mM methimazole) followed by rapid filtration through nitrocellulose filters. Filters were washed twice with an additional 5 ml each of the same quenching buffer.

Northern Blot Analysis-Poly(A+) RNA was denatured in form- amide, fractionated by agarose gel electrophoresis in the presence of 2.2 M formaldehyde (21), transferred to nitrocellulose, and hybridized at room temperature with [32P]poly(dT) in 6 X SSC.

RESULTS AND DISCUSSION

As shown in Fig. 1, noninjected X . laevis oocytes exhibit a modest Na+-dependent and C1O;-sensitive I- transport activ- ity (0.5 pmol of lZ5I- uptake/oocyte in 45 min). The Na+ dependence is explored in all experiments by the substitution

A B C D E F G H I J K

- 28s

18s

injected non H20 TSH(+) TSH(-)

FIG. 1. Expression of I- transport in X . laevis oocytes. 00- cytes were microinjected with either RNA-free water or 50 ng of poly(A+) RNA from FRTL-5 cells maintained in the presence or absence of TSH. Five days after injection, oocytes were assayed for transport of NalZ5I as described under “Experimental Procedures” in the presence of either 100 mM NaCl (open bars), 100 mM NaCl, 10 p~ NaC104 (hatched bars), or 100 mM choline chloride (diagonal bars). Reactions were terminated as described under “Experimental Procedures.” Radioactivity retained in the oocytes was quantitated in a y counter. Transport values are presented as the means obtained with five oocytes, with respective S.E. values.

al x 0 0

c

\

c 0 P *

0 25 50 75 100

TIME ( rnin )

FIG. 2. Time course of I- transport in X . laevis oocytes. Oocytes were microinjected and assayed for I- transport in the presence of 100 mM NaCl (O), 100 mM NaCl, 10 p M NaC104 (O), or 100 mM choline chloride (A) as indicated under “Experimental Pro- cedures,” except that the reactions were terminated at the times shown.

FIG. 3. Northern blot analyses of size-fractionated poly(A+) RNA. Poly(A+) RNA from FRTL-5 cells was fractionated in a 5- 25% sucrose gradient, collected, and precipitated with ethanol. An aliquot containing 3% of the total poly(A+) RNA from every third fraction was electrophoresed in a 1.5% agarose formaldehyde-con- taining gel, transferred onto nitrocellulose, and probed with 32P- labeled poly(dT). For reference, positions of 28 and 18 S ribosomal RNA are indicated in the autoradiogram.

H20 1 2 3 4 5 6

GROUP NUMBER

FIG. 4. Expression of I- transport activity from size-frac- tionated poly(A+) RNA. Sucrose gradient-fractionated poly(A+) RNA aliquots were pooled into six different groups (group 1, fractions

group 6, 24-30), microinjected, and assayed as described in Fig. 1. Inset, poly(A+) RNA from group 2 was subsequently divided into three different subgroups (2a, fractions 7 and 8; 2b, 9 and 10; 2c, 11 and 12), microinjected, and assayed as described in Fig. 1.

1-6; POUP 2, 7-12; group 3, 13-15; POUP 4 , 16-18; POUP 5, 19-23;

of equimolar concentrations of choline chloride for NaCl in the medium. No significant difference in activity is detected in oocytes microinjected with water or with poly(A+) RNA from FRTL-5 cells maintained in the absence of TSH for 8 days. In contrast, microinjection of poly(A+) RNA from FRTL-5 cells maintained in the presence of TSH results in a marked enhancement of I- transport activity (an average of 3.5 pmol of ‘251-/00cyte in 45 min or a 7-fold increase) which retains the properties of transport in intact cells, i.e. Na+ dependence and sensitivity to ClO;. Fig. 2 depicts the time course for I- uptake in oocytes showing that saturation is

Expression of the Sodiumllodide Symporter in Oocytes 11903

attained in about an hour. Expression was consistently ob- served 5-6 days after injection.

Total poly(A+) RNA from FRTL-5 cells maintained in the presence of TSH was resolved into 30 fractions on a 5 2 5 % sucrose gradient. Aliquots from every third fraction were electrophoresed in a formaldehyde-containing agarose gel, transferred onto nitrocellulose, and probed with 32P-labeled poly(dT) (Fig. 3). The fractionated poly(A+) RNA was sub- sequently pooled into six different groups for microinjection into oocytes. Fig. 4 shows the results of the respective trans- port assays, indicating that group 2 contains the poly(A+) RNA responsible for expression of I- transport activity. Upon analysis of three smaller subgroups of group 2 (2a, 2b, and 2c), the corresponding poly(A+) RNA was traced to subgroup 2b (see Fig. 4, inset), whose migration in electrophoresis under denaturing conditions corresponds to sizes between 2.8 and 4.0 kilobases.

FRTL-5 cells, which are derived from normal rat thyroids, depend on the presence of TSH for growth. However, they can be maintained in the absence of TSH for at least 10 days and still remain viable (22). Significantly, it has been shown that withdrawal of TSH from the culture medium and main- tenance of these cells in non-TSH-containing medium are followed by a rapid decline in intracellular CAMP content and a concomitant decrease in I- uptake (9). This observation is consistent with our reported finding that no I- transport activity is expressed in oocytes injected with poly(A+) RNA from FRTL-5 cells maintained in the absence of TSH, while expression is observed only in oocytes injected with poly(A+) RNA from cells maintained in TSH-containing medium.

These results strongly suggest that the injected 2.8-4.0- kilobase poly(A+) RNA corresponds to the thyroid Na+/I- symporter. However, it is possible that the poly(A+) RNA responsible for expression may be directing the synthesis of a different thyroid protein with a stimulatory effect on the oocytes’ endogenous Na+/I- symport activity. While this pos- sibility seems unlikely (23, 24), it cannot be ruled out at present.

In conclusion, as no antibodies or oligonucleotides based on protein sequence data are yet available for conventional screening of a prospective cDNA library, a system for oocyte expression cloning of the Na+/I- symporter’s cDNA has hereby been generated, based on the poly(A+) RNA 2.8-4.0- kilobase fraction. This development can be expected to lead to the molecular characterization of the protein and to a better understanding of both the mechanism of I- transloca- tion in the thyroid and its regulation by TSH.

Acknowledgments-We would like to thank Dr. Evelyn Grollman for providing the FRTL-5 cells, Dr. Leslie Kushner for showing us the microinjection technique, and Dr. Charles Rubin for critical reading of the manuscript.

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