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Structure and Function of Thyrotropin Receptors (Hormone Mechanism/Cholera Toxin/Luteinizing Hormone/Human Chorionic Gonadotropin/Adenylate Cyclase) BRIAN R

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Structure and Function of Thyrotropin Receptors (Hormone Mechanism/Cholera Toxin/Luteinizing Hormone/Human Chorionic Gonadotropin/Adenylate Cyclase) BRIAN R Proc. Nat. Acad. Sci. USA Vol. 73, No. 3, pp. 842-846, March 1976 Cell Biology Thyrotropin-ganglioside interactions and their relationship to the structure and function of thyrotropin receptors (hormone mechanism/cholera toxin/luteinizing hormone/human chorionic gonadotropin/adenylate cyclase) BRIAN R. MULLIN*, PETER H. FISHMANt, GEORGE LEE*, SALVATORE M. ALOJ*t, FRED D. LEDLEY*, ROGER J. WINAND§, LEONARD D. KOHN*, AND ROSCOE 0. BRADYt * Section on Biochemistry of Cell Regulation, Laboratory of Biochemical Pharmacology, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014; t Developmental and Metabolic Neurology Branch, National Institute of Neurological and Communicative Disorders and Stroke; f Centro di Endocrinologia ed Oncologia Sperimentale C.N.R., Naples, Italy; and § DNpartement de Clinique et de Semiologie Medicales, Institut de MWdecine, Universit6 de Liege, B4000 Liege, Belgium Contributed by Roscoe 0. Brady, December 18, 1975 ABSTRACT Gangliosides inhibit 1251-labeled thyrotropin since neuraminidase digestion eliminated the ability of both binding to the thyrotropin receptors on bovine thyroid plas- the purified receptor fragment and the crude solubilized re- ma membranes, on guinea pig retro-orbital tissue plasma membranes, and on human adipocyte membranes. This inhi- ceptor preparation to specifically bind TSH (1). bition by gangliosides is critically altered by the number and Recent studies have indicated that gangliosides, glyco- location of the sialic acid residues within the ganglioside sphingolipids that contain sialic acid, specifically interact structure, the efficacy of inhibition having the following with cholera toxin and that variations in the oligosaccharide order: GD1b > GTI > GM1 > GM2 = GM3 > GD1a. The inhibi- structure of the gangliosides influence this interaction (3-5). tion results from the interaction of thyrotropin and ganglios- The present report demonstrates that TSH also interacts ides, rather than the interaction of membrane and ganglios- ides. Fluorescence studies show that the inhibition is associ- with gangliosides and that the location and number of the ated with a distinct conformational change of the thyrotro- sialic acid residues on the ganglioside molecule is critical to pin molecule and that the progression from a "noninhibitory this interaction. The report further shows that there is a se- conformation" to an "inhibitory conformation" parallels ex- quence homology in the B component of cholera toxin and actly the order of effectiveness in inhibiting 125I-Yabeled thy- the ,B subunits of TSH, luteinizing hormone, and human rotropin binding. The ganglioside inhibition of 1251-labeled chorionic gonadotropin. It is suggested, therefore, that a thyrotropin binding appears to be hormonally specific in that it is not affected by a umin, glucagon, insulin, prolactin, fol- ganglioside-like structure is a basic component of glycopro- licle-stimulating hormone, growth hormone, or corticotropin. tein hormone receptors and that cholera toxin and the glyco- The possibility that a ganglioside or ganglioside-like struc- protein hormones might have a common mechanism by ture is a component of the thyrotropin receptor is suggested which their message is transmitted to the cell machinery. by the finding that gangliosides more complex than N-acetyl- neuraminylgalactosylglucosylceramide are present in bovine MATERIALS AND METHODS thyroid membranes in much higher quantities than have been previously found in extraneural tissue. The finding that TSH, 125I-labeled TSH, and thyroid plasma membranes the B component of cholera toxin, which also interacts with were bovine preparations prepared as described (6-9). 1251_ gangliosides, has a peptide sequence in common with the f, labeled TSH binding to plasma membranes was assayed by subunit of thyrotropin, suggests that thyrotropin and cholera the filtration techniques already described (8, 9) with the ex- toxin may be analogous in their mode of action on the mem- brane. ception that oxoid filters (Amersham/Searle Corp.) were re- placed by cellulose acetate filters (EHWP-02500, Millipore In previous studies that detailed the solubilization of the thy- Corp.). In addition to the agents tested for their ability to in- rotropin (TSH) receptor from bovine thyroid plasma mem- fluence binding, binding assays contained in a 130-Iu vol- branes, tryptic digestion was shown to yield a receptor frag- ume, 0.025 M Tris-acetate, pH 6.0, 0.6% bovine serum albu- ment that exhibited specific TSH binding and had proper- min, approximately 125,000 cpm (2 X 10-9 M) 125I-labeled ties similar to those exhibited by the TSH receptor prior to TSH, and 13-15 ,ug of membrane protein. The amount of solubilization (1, 2). This receptor fragment was purified by thyroid plasma membranes used was within the linear phase chromatography over TSH-Sepharose preparations (1, 2); it of binding when evaluated as a function of membrane pro- was shown to have a molecular weight of 25,000-30,000 by tein concentration. To insure that the binding and the inhi- gel electrophoresis in the presence of sodium dodecyl sulfate bition of binding measured in these assays were specific, (2), and to contain 30% carbohydrate and 10% sialic acid by control incubations containing 1.5 X 10-5 M unlabeled TSH, weight (1). The sialic acid was vital to receptor function gangliosides, or no membranes were included in each indi- vidual experiment. Gangliosides N-acetylneuraminylgalactosylglucosylcera- Abbreviations: TSH, thyrotropin; GM3, N-acetylneuraminylgalacto- mide (GM3), N-acetylgalactosaminyl-(N-acetylneuraminyl)- sylglucosylceramide; GM2, N-acetylgalactosaminyl-(N-acetylneura- galactosylglucosylceramide (GM2), and N-acetylneuraminyl- minyl)-galactosylglucosylceramide; GM1, galactosyl-N-acetylgalac- galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)- tosaminyl-(N-acetylneuraminyl)-galactosylglucosylceramide; GDla, galactosylglucosylceramide (GDLa) were obtained as pre- N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl-(N-acetyl- viously described (10). Gangliosides galactosyl-N-acetylga- neuraminyl)-galactosylglucosylceramide; GDib, galactosyl-N-ac- etylgalactosaminyl-(N-acetylneuraminyl-N-acetylneuraminyl)-ga- lactosaminyl-(N-acetylneuraminyl)-galactosylglucosylcera- lactosylglucosylceramide; GT1, N-acetylneuraminylgalactosyl-N-ac- mide (GM,), galactosyl-N-acetylgalactosaminyl-(N-ace- etylgalactosaminyl-(N-acetylneuraminyl-N-acetylneuraminyl)-gal- tylneuraminyl- N- acetylneuraminyl) - galactosylglucosylcer - actosylglucosylceramide. amide (GD1b), and N-acetylneuraminylgalactosyl-N-acetyl- 842 Downloaded by guest on October 1, 2021 Cell Biology: Mullin et al. Proc. Nat. Acad. Sci. USA 73 (1976) 843 Table 1. Effect of preincubation of membrane and ganglioside on inhibition of '25I-labeled TSH binding to thyroid plasma membranes* Inhibition Exp. Preincubation components (%) 1 None 95 2 Membranes + '25I-labeled TSH 87 3 Ganglioside + "2II-labeled TSH 97 O z 4 Ganglioside + membranes z 60 without centrifugation before assay 93 5 Ganglioside + membranes followed by centrifugation before assay 0 A M 40- z * In the control experiment where no preincubation was performed (Exp. 1), all components (membranes, gangliosides, and 1251- labeled TSH) were added within 10 sec, mixed, and incubated for 0~~~~ ~ ~ ~ ~ ~ a total of 75 min prior to filtration. In Exps. 2, 3, and 4, the noted components were preincubated in assay buffer for 15 min before the missing component, ganglioside, membranes, and 125I- labeled TSH, respectively, were added; the binding assay then 20~~~~ proceeded for 60 min before filtration or a total of 75 min from the onset of preincubation. In Exp. 5, after the ganglioside and membranes were preincubated for 15 min, the mixture was cen- trifuged at 12,000 x g for 15 min to sediment the membranes. The ~~~NANA membranes -- were then resuspended in buffer and the missing 20 40 60 component, 125I-labeled TSH, was added. The ganglioside prepa- GANGLIOSIDE ADDED (nmol) ration used in these experiments was a mixed preparation (from bovine brain) containing 47% GD1a, 25% GT1, 16% Dib, and 12% FIG. 1. Inhibition of to 125I-labeled TSH binding bovine thy- GM1; 70 nmol were added. In addition to serving as a control for roid plasma membranes by gangliosides or NANA (N-acetylneura- - Exp. 5, Exp. 2 shows that gangliosides can "chase" bound TSH off minic acid). Addition of 1 nmol of fetuin does not inhibit 1251-la- the membrane. All procedures were performed at 2-4°. beled TSH binding (data not shown); this amount of fetuin is equivalent to 13 nM of protein-bound NANA. RESULTS galactosaminyl-(N-acetylneuraminyl-N-acetylneuraminyl)- Gangliosides inhibit 125I-labeled TSH binding to thyroid galactosylglucosylceramide (GT1) were isolated from com- plasma membranes, and this inhibition is related to the car- mercial preparations (Supelco, Inc., Bellefonte, Pa.) by pre- bohydrate structure of the ganglioside (Fig. 1). The best in- parative thin-layer chromatography (11). After visualizing hibitor is GD1b, where two sialic acid residues are located on with iodine vapor, the individual gangliosides were scraped the internal galactose residue of the oligosaccharide portion from the plates, eluted with chloroform:methanol:water of the ganglioside structure. The addition of a sialic acid res- (10:10:3, vol/vol/vol), taken to dryness under a stream of ni- idue to the terminal galactose residue of the ganglioside trogen, and redissolved in 0.01 M
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