Proc. Nati. Acad. Sci. USAVol. 73, No. 11, pp. 4060-4064, November 1976Cell Biology

Relationship of gangliosides to the structure and function ofthyrotropin receptors: Their absence on plasma membranes of athyroid tumor defective in thyrotropin receptor activity

(adenylate cyclase/adenosine 3':5'-cyclic monophosphate/glycosyl transferases/cholera toxin/glycoprotein hormones)

MARIA F. MELDOLESI*t, PETER H. FISHMANf, SALVATORE M. ALOJ*t, LEONARD D. KOHN*,AND ROSCOE 0. BRADYt* Section on Biochemistry of Cell Reulation, Laboratory of Biochemical Pharmacology, National Institute of Arthritis, Metabolism, and Digestive Diseases,National Institutes of Health, Bethesda, Maryland 20014; t Centro di Endocrinologia ed Oncologia Sperimentale del C.N.R., Naples, Italy; and * Developmentaland Metabolic Neurology Branch, National Institute of Neurological and Communicative Disorlers and Stroke, National Institutes of Health, Bethesda,Maryland 20014

Contributed by Roscoe 0. Brady, July 28, 1976

ABSTRACT Plasma membranes derived from a rat thyroidtumor (1-8R) which is unresponsive to thyrotropin but isresponsive to dibutyryl adenosine 3':5'-cyclic monophosphatebind less than 20% of the [25#Ithyrotropin which can be boundto plasma membranes from normal rat thyroids under condi-tions which optimize tumor membrane binding relative tonormal thyroid membranes. In addition, the binding is differentfrom thyrotropin binding to normal thyroid membranes bothin its altered sensitivity to changes in hydrogen ion concentra-tion and in a decreased sensitivity to competition by unlabeledthyrotropin. This reduced capacity to bind ['25I]thyrotropincannot be attributed to degradation of the hormone by mem-brane-associated proteases. Although the supernatant phase ofthe thyroid tumor homogenates contains a soluble componentwhich inhibits [125Isthyrotropin binding to thyrotropin receptorson plasma membranes, its level is the same as in homogenatesof normal thyroid tissue. Trypsin digestion does not exposethyrotropin receptors in a manner analogous to that seen innormal thyroid tissue. The major ganglioside in the tumormembranes is N-acetylneuraminylgalactosylglucosylceramideand the membranes lack the N-acetylgalactosaminyltrans-ferase required for the synthesis of more complex gangliosides.In contrast, the normal rat thyroid membranes contain morecomplex gangliosides such as galactosyl-N-acetylgalactosam-inyl[N-acetylneuraminyljgalactosylglucosylceramide andN-acetylneuraminylgalactosyl-N-acetylgalactosaminyl-[N-acetylneuraminylJgalactosylglucosylceramide as well as theglycosyltransferase activities required for their syntheses.Galactosyl-N-acetylgalactosaminyl[N-acetylneuraminylJga-lactosylglucosylceramide can also be detected in normalmembranes, but not in tumor membranes, by selective labelingwith galactose oxidase (D-galactose:oxygen 6-oxidoreductase,EC 1.1.3.9) and [3H]sodium borohydride. These results supportthe hypothesis that gangliosides are important structural orfunctional components of thyrotropin receptors on thyroidplasma membranes.

Recently we reported that gangliosides could inhibit the bindingof thyrotropin (TSH) to bovine thyroid membranes (1) andsuggested that the mechanism by which TSH transmits itsmessage to the thyroid cell has certain analogies to that ofcholera toxin (1-4). Both TSH and cholera toxin interact withspecific gangliosides; both molecules undergo conformationalchanges upon binding to these gangliosides; both molecules arecomposed of dissimilar subunits, one subunit which binds to

Abbreviations: TSH, thyrotropin; cAMP, adenosine 3':5'-cyclicmonophosphate; GM3, N-acetylneuraminylgalactosylglucosylceramide;GM2, N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglu-cosylceramide; GM1, galactosyl-N-acetylgalactosaminyl-[N-acetyl-neuraminyll-galactosylglucosylceramide; GDla, N-acetylneura-minylgalactosyl-N-acetylgalactosaminyl-'[N -acetylneuraminylI]-ga-lactosylglucosylceramide.

surface receptors and the other which activates cellular ma-chinery, presumably by stimulation of adenylate cyclase [ATPpyrophosphate-lyase(cyclizing), EC 4.6.1.1]; correspondingsubunits of both molecules have certain homologies of aminoacid sequence; and, finally, both molecules can compete forbinding to thyroid membranes.To evaluate further the importance of gangliosides as

structural or functional components of the TSH receptor, weexamined thyroid cells which have a defective TSH receptorbut a cell machinery normally responsive to altered levels ofadenosine 3':5'-cyclic monophosphate (cAMP). Macchia et al.(5-8) have characterized the defects of several thyroid tumorsof rats. One of these, thyroid tumor line 1-8, was shown to beunresponsive to TSH but to be responsive to dibutyryl cAMP.More important, Mandato et al. (9) showed that the plasmamembranes of these tumors were unable to bind 125I-labeledTSH, but contained an adenylate cyclase activity normallyresponsive to fluoride and prostaglandins. They concluded thatthe thyroid cells of this tumor had a nonfunctional or nonexis-tent TSH receptor as its "genetic defect" and that studies of theplasma membranes from this tumor would be important indefining the structure of the TSH receptor as well as its couplingto the adenylate cyclase system.

In the present report we demonstrate that the marked de-crease in TSH binding to the 1-8R thyroid tumor plasmamembranes can be correlated with the absence in these plasmamembranes of gangliosides like galactosyl-N-acetylgalactos-aminyl- [N-acetylneuraminyl-N-acetylneuraminyl] -galacto-sylglucosylceramide, N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl- [N-acetylneuraminyl-N-acetylneura-minyl]-galactosylglucosylceramide, and galactosyl-N-acetyl-galactosaminyl- [N-acetylneuraminyl]- galactosylglucosyl-ceramide (GM1), which are capable of interacting with TSH andare present in normal thyroid plasma membranes (1-4).

MATERIALS AND METHODSBovine TSH and 125I-labeled TSH were prepared as previouslydescribed (10-12). '25I-labeled TSH binding was routinely as-sayed by the filtration techniques already described (1, 10, 13).Unless otherwise stated, binding assays contained in a volumeof 100 ,l the following components: 0.025 M Tris-acetate at pH6.0, 0.6% bovine serum albumin, approximately 125,000 cpmof 125I-labeled TSH (7.5 nM), and 7-30 ,ug of membrane pro-tein. To insure that the 125I-labeled TSH binding measured wasspecific binding, we incubated controls which included 15 ,uMunlabeled TSH as well as all other components of that particularassay.

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El.10

z

0comI

1-UZ

2

3 4 5 6 7 8 9 10

pH

FIG. 1. Binding of 1251-labeled TSH as a function of pH tomembranes from rat tissue (0, &) and to membranes from thyroidtumor 1-8R tissue (0, A). Binding was at hormone concentration of7.5 nM. The buffer in all cases was at 0.02 M concentration; thetemperature during the incubation was 0°. The buffers used were

acetate (0, 0) or Tris-acetate (A, A). Assays of the tumor 1-8Rmembranes contained 15.4 jsg of membrane protein. Assays of normalthyroid plasma membranes contained 3 gg of membrane protein.

The thyroid tumor line used through these studies was one

of a series of thyroid tumors originally developed in Fischer ratsby Wollman (5-9, 14) and designated as line 1-8 in Wollman'sclassification. The tumor used in these studies was kindly sup-plied by Jacob Robbins (National Institutes of Health, Bethesda,Md.) and is designated 1-8R accordingly, to define its source.All of the tumors used were derived from a single tumor thatwas carried by subcutaneous implantation through seven

generations of male Fischer rats during the course of this study.The tumor did not appear to undergo significant alteration infunction during this time. The tumors were collected ap-proximately 2 months after subcutaneous implantation whenthey had attained an average weight of 5 g, and they were keptat 1° until used. Data concerning the histology, the growth rate,and some metabolic properties of this tumor have been pre-viously reported (5-9). Control thyroids were taken fromnontumor-bearing Fischer rats.

Plasma membranes were prepared from normal thyroid andrat thyroid tumors with procedures described for the isolationof bovine thyroid plasma membranes, all procedures beingperformed at 0-2° (10, 13). Cell sap is the 105,000 X g super-natant after a 90 min centrifugation from either normal thyroidor the 1-8R tumor homogenized with nine volumes of ice-cold0.01 M Tris-acetate buffer at pH 7.0 (15).

Gangliosides were extracted from normal and tumor thyroidmembranes, purified, separated by thin-layer chromatography,and visualized with resorcinol reagent as previously described(1). The procedure for labeling ganglioside in intact membraneswith galactose oxidase (D-galactose:oxygen 6-oxidoreductase,EC 1.L3.9) and NaB[3H]4 has been described in an earlier re-

port (2). Activities of glycosyltransferases involved in gan-glioside biosynthesis were determined either in whole ho-mogenates or partially purified membrane preparations bymeans of established procedures (16, 17).

Table 1. I 2I5 I-Labeled TSH binding activity to membranepreparations from normal and 1-8R tumor thyroids underconditions which optimize binding to each preparation

Specific 1 25I-labeled TSHbound* (cpm/7 ,4g ofmembrane protein)

Conditions t

Membranes pH 6.0, 2-4° pH 7.5, 23°

Rat thyroid 20,980 38,046Tumor 1-8R 4,029 1,616

* As noted in Materials and Methods, specific binding is the dif-ference between binding in the presence and absence of a 10,000-fold excess of unlabeled TSH.

t The binding assay was performed for 1 hr at 4° in 0.02 M Tris-acetate buffer and 0.6% bovine serum albumin at pH 6.0, or for40 min at 230 in 0.02 M Tris-HCl buffer and 0.6% bovine serumalbumin at pH 7.5. The former conditions optimize binding totumor membranes; the latter conditions optimize binding to nor-mal membranes.

RESULTSComparative Properties of '25I-Labeled TSH Binding to

Plasma Membranes of the Normal-Rat Thyroid and PlasmaMembranes of the Rat Thyroid Tumor 1-8R. In the initialcharacterization of the receptor defect in the plasma mem-branes of this tumor by Mandato et al. (9), the binding of'25I-labeled TSH to the membranes used conditions whichmaximized the ability of TSH to stimulate the adenylate cyclaseactivity in the thyroid tumor membranes. Recent reports haveshown, however, that optimal '25I-labeled TSH binding tobovine thyroid plasma membrane preparations in vitro occursunder distinctly different conditions (10, 18-20). To insure thatthe defective TSH receptor activity of the rat thyroid tumor1-8R was not reversed under optimal in vitro binding condi-tions, we evaluated the properties of 125I-labeled TSH bindingto normal and tumor membranes in greater detail.The pH optimum of 125I-labeled TSH binding to the plasma

membranes from the two preparations was significantly dif-ferent (Fig. 1). 125I-Labeled TSH binding to the normal thyroidplasma membranes was biphasic and showed pH maxima atboth 5.5 and 7.8. In contrast, the tumor binding showed nomaxima at 7.8, but did improve relative to the normal thyroidmembranes at higher hydrogen ion concentrations. As reportedfor bovine thyroid membranes (10, 20), 125I-labeled TSHbinding to the thyroid tumor membranes at pH 7.5 was im-proved at 40 rather than 230 (data not shown). The 125I-labeledTSH binding to the normal rat thyroid plasma membranes hada different temperature optimum, i.e., had improved bindingat 230 rather than 40 (Table 1). Nevertheless, even under con-ditions which optimized the 125I-labeled TSH binding to thetumor membranes relative to normal membranes (a 1 hr in-cubation at 40 in 0.02 M Tris-acetate at pH 6.0), '25I-labeledTSH binding to the tumor membranes was significantly re-duced (Table 1). Binding of TSH to both normal and tumormembranes were inhibited by mono- and divalent cations;however, binding to tumor membranes was more sensitive todivalent cations.

Binding of 125I-labeled TSH to normal rat thyroid mem-branes was readily blocked by unlabeled TSH, whereas, in ac-cord with previous findings (9), the low level of 125-IlabeledTSH binding to the thyroid tumor was less inhibited under thesesame experimental conditions (Table 2). l25I-Labeled TSHbinding to the normal and tumor rat thyroid membranes both

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Table 2. Inhibition of 25 I-labeled TSH binding to plasmamembranes from normal rat thyroid and the 1-8R rat

thyroid tumor by unlabeled TSH

% Inhibition of 25I-labeledTSH binding by unlabeled

hormonet withfinal concentration of theunlabeled TSH added*

Membranes None 1.0 2.0

Rat thyroid 0 50 95Tumor 1-8R 0 10 40

* A final concentration ofTSH of 0.2 mg/ml is approximately equi-valent to a 7 MM solution.

t Membranes were preincubated for 1 hr at 2-4° in the presence of0.02 M Tris-acetate buffer at pH 6.0, and 0.6% bovine serum al-bumin with or without nonradioactive TSH. The binding assaywas started by adding l251-labeled TSH (7.5 nM).

yielded nonlinear Scatchard plots analogous to those obtainedwhen 125I-labeled TSH binding was measured under identicalassay conditions using bovine thyroid plasma membranes (20);the Scatchard analysis of 125I-labeled TSH binding to tumormembranes at 00 and pH 6.0 confirms that there is a decreaseof the number of binding sites available per mg of membraneprotein (9).

Because the inability of the thyroid tumor membranes tobind TSH might be due to degradation of TSH by mem-brane-associated proteases, unlabeled TSH was preincubatedin the presence of 0.25% bovine serum albumin (to make theratio of soluble protein to membranes equivalent to that of thebinding assay) with the 1-8R tumor membrane or with normalbovine thyroid membranes or Tris-acetate buffer at pH 7.0, ascontrols. After centrifugation at 25,000 X g for 10 min, thesupernatant phase containing the cold hormone was collectedand used to inhibit the '25I-labeled TSH binding to bovinethyroid plasma membranes. All three TSH preparations dis-played almost identical inhibitory action.

As previously shown with bovine thyroid membranes (20),treatment with trypsin causes a loss in receptor binding activity

Table 3. Trypsin treatment of tumor 1-8R and normal ratthyroid plasma membranes

% of 125I-labeled TSHbound withoutpreincubation

Source of membranes Buffer Trypsin

1-8R tumor 86 61Normal rat thyroid 86 128Normal bovine thyroid 83 142

Membranes were exposed to L-1-tosylamino-2-phenylethyl-chloromethyl ketone trypsin at room temperature for 2 hr at a14.8:1 ratio of membranes to trypsin (wt/wt) in Tris-acetate bufferat pH 7.0. At the end of the incubation period, the membranes weremixed with an amount of cold soybean trypsin inhibitor 5-fold in ex-

cess of the trypsin concentration, and the suspension was chilled to2-4° (13, 20). The suspension was immediately assayed for bindingactivity in optimal assay conditions at pH 6.0 and at 7.5 nM 1251-labeled TSH concentration. After the binding assay was performed,4% polyethylene glycol 6000 was added and the suspension was

filtered. Binding in the presence of polyethylene glycol measuresthe membrane receptor activity plus the receptor activity solubi-lized by the preincubation conditions (13).

Table 4. Inhibition of "25I-labeled TSH binding to bovinethyroid and guinea pig retro-orbital tissue plasmamembranes by tumor 1-8R cell sap* inhibitor

Cell sap from Cell sap fromnormal thyroid tumor 1-8R

Cell '25I-Labeled Cell 12I-Labeledsap TSHt sap TSHt

added bound added boundAssay membranes (ul) (cpm) (Al) (cpm)Bovine thyroid 0 16,000 0 16,000

2 9,600 2 9,2005 6,200 5 4,200

20 3,700 20 2,90040 1,900 40 1,300

Guinea pig 0 26,100 0 26,100retro-orbital 2 25,500 2 24,700tissue 5 22,300 5 15,200

20 12,500 20 11,200

* See Materials and Methods.t Binding conditions were the same as in Tables 2 and 3.

on the plasma membranes but releases receptor binding activityinto the supernatant phase. Trypsin treatment of normal rat ornormal bovine thyroid membranes results in the release ofsoluble receptor activity into the supernatant phase and in anincrease in total receptor activity as assayed using a techniquewhich will simultaneously measure both membrane-bound andsoluble binding activity (Table 3). In contrast, the trypsinizationof 1-8R tumor membranes (a) causes a loss of membrane-as-sociated binding activity (Table 4) and (b) does not releasesoluble binding activity into the supernatant phase by com-parison to identical incubations without trypsin. These datashow that trypsin digestion of 1-8R tumor membranes does notexpose or release TSH receptors in a manner analogous to thatseen in normal thyroid tissue.The supernatant phase of the thyroid homogenates contains

a soluble TSH binding component which inhibits 125I-labeledTSH binding to TSH receptors on plasma membranes bothfrom bovine thyroid and from guinea pig retro-orbital tissue(15). As shown in Table 4, the supernatant phase of the 1-8Rtumor homogenates contained such a soluble TSH bindingcomponent; however, no striking differences in the amount ofthe soluble binding component present in the cell sap werenoted between tumor and normal TSH binding prepara-tions.

Ganglioside Composition and Ganglioside Synthesis inMembrane Preparations from Normal Rat Thyroid and from1-SR Tumor Thyroids. Gangliosides were extracted fromnormal rat and tumor thyroid membranes, purified, and ana-lyzed by thin-layer chromatography (Fig. 2). The major gan-gliosides isolated from the tumor membranes had the samemobility as authentic N-acetylneuraminylgalactosylglucosyl-ceramide (GM3) (Fig. 2A). There were only trace amounts ofgangliosides with slower mobilities on the chromatogram.In contrast, gangliosides corresponding to GM3, GM1, andN-acetylneuraminylgalactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide (GMa) wereisolated from normal rat thyroid membranes (Fig. 2B). Whenganglioside fractions isolated from normal and tumor mem-branes were incubated with purified bacterial neuraminidase(21) and chromatographed, over 95% of the GM3 from thetumor membranes had disappeared, and bands correspondingto lactosylceramide had appeared on the chromatogram. Thisprocedure further identified the major ganglioside in the tumor

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A

GM3 I

GM2GMl

GDla

4-_

1 2 1 2FIG. 2. Ganglioside pattern of normal rat thyroid and rat tumor

1-8R membranes. Normal and tumor membranes were each extractedwith chloroform:methanol (2:1, vol/vol and 1:2, vol/vol) and gan-gliosides were purified from the combined extracts by column chro-matography on DEAE-Sephadex, Sephadex G-25, and Unisil (1).Gangliosides were separated by thin-layer chromatography on silicagel 60 and detected with resorcinol reagent (1). Plate A: lane 1-gangliosides isolated from rat thyroid tumor 1-8R membranes rep-resenting 5 mg of protein; lane 2-authentic ganglioside standards.Plate B: lane 1-authentic ganglioside standards as in plate A; lane2-gangliosides isolated from normal rat thyroid membranes whichrepresent 8.5 mg of protein [arrows ('-) indicate resorcinol positivebands in this lane].

membranes as GM3. After neuraminidase treatment the onlyganglioside remaining in the normal thyroid extract corre-

sponded to GM1. This result supported the identification of thepredominant gangliosides in these membranes as GM3, GM1, andGDla (1).

In a previous report we showed that the terminal galactoseresidue of the carbohydrate moiety in the ganglioside GM1 ofbovine thyroid membranes can be tritiated by sequentialtreatment with galactose-oxidase and 3H-labeled sodiumborohydride (2). When normal rat thyroid membranes were

incubated with galactose oxidase and exposed to 3H-labeledsodium borohydride, tritium was incorporated into GM1 resi-dues (Fig. 3). In contrast, when the 1-8R tumor membraneswere similarly treated, no radioactive peaks corresponding toganglioside GM1 were detected after scanning the chromato,-graphic plates (Fig. 3).

As shown in Table 5, the lack of gangliosides more complexthan GM3 in the thyroid tumor plasma membranes was relatedto a defect of synthesis of N-acetylgalactosaminyl-[N-acetyl-neuraminyl]-galactosylglucosylceramide (GM2) from GM3caused by the absence of UDP-GalNAc:GM3 N-acetylgalacto-saminyltransferase. Concurrent with this enzyme defect, otherenzymatic activities such as CMP-N-acetylneuraminyl:lacto-sylceramide sialyltransferase and the UDP-Gal:GM2 galactos-yltransferase were depressed, whereas sialyltransferase II whichcatalyzes the synthesis of GDla from GM1 was drastically en-

hanced (Table 5). All of these enzyme activities were readilydetected in the normal rat thyroid, a result consistent with ob-served presence of gangliosides GM1 and GDIa in the normalmembranes. There was a significant enrichment of these en-

zyme activities in the membrane preparations compared to the

GM3 '=

GM2 ° r

GM1 =

GD1a =

ORIGIN

RAT THYROID NORMAL RATTUMOR THYROID

FIG. 3. [3H]Ganglioside pattern in rat thyroid and tumor 1-8Rplasma membranes after treatment with galactose oxidase and 3H-labeled sodium borohydride. Ten milligrams of membrane proteinwas treated with galactose oxidase (100 units) for 2 hr in phosphatebuffered saline, pH 7.4, at 250 (2). One unit of activity equals an in-crease in absorbance of 1.00 per min when assayed according to themanufacturer, Worthington Biochemical Corp. The membranes werewashed three times with 0.05 M Tris-acetate at'pH 7.8, resuspendedin the same buffer, and then treated with 5 mCi of 3H-labeled sodiumborohydride for 30 min. The labeled membranes were washed threetimes in 0.05M Tris-acetate atpH 7.8, and the gangliosides were ex-tracted (2). The gangliosides were subjected to thin-layer chroma-tography, and the plates were scanned using a Varian radioscanninginstrument (graphs). Simultaneous chromatography of authenticgangliosides allowed the identification of the peaks of radioactivi-ty.

total homogenate. Subcellular fractionation of bovine thyroidgland indicates that these enzymes are localized in the Golgiapparatus, as there was a 20- to 50-fold enrichment of thevarious ganglioside glycosyltransferase activities (manuscriptin preparation).

DISCUSSIONIn the present report we show that even under conditions whichoptimized 125I-labeled TSH binding to the 1-8R tumor mem-branes, TSH receptor activity was significantly decreased inthe tumor membranes, and what little 125I-labeled TSH bindingactivity could be detected was abnormal in its sensitivity to pHand cations and in its resistance to inhibition by unlabeled TSH.Further, the data showed (a) that the decreased receptor ac-tivity could not be attributed to degradation of TSH by mem-brane-associated proteases during the incubation period; (b)that trypsin digestion neither exposed TSH receptors whichmight have been buried in the plasma membrane nor releasedsignificant receptor activity into the supernatant phase; and (c)that the tumor preparation was not contaminated by the in-creased presence of a soluble TSH binding component presentin the cytosol.The rat tumor thyroid membranes lacked gangliosides more

complex than GM3 and the N-acetylgalactosaminyltransferasenecessary for their synthesis. In contrast, the normal rat thyroidmembranes contained more complex gangliosides such as GM1and GDla and the requisite ganglioside biosynthetic enzymes.Deficient ganglioside biosynthesis has been observed in many

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Table 5. Activity of enzymes involved in the biosyntheticpathway for ganglioside synthesis in normal and

tumorthyroid tissue

Enzymatic activity (pmol of productformed/mg of protein per hr)

Rat thyroid* Tumor 1-8R*

Homog- Mem- Homog- Mem-Enzymet enate branest enate branest

Sialyltrans-ferase I 281 598 23 133

N-acetylgalac-tosaminyltrans-ferase 21 69 0 9

Galactosyl-transferase 225 470 103 93

Sialyltrans-ferase II 63 89 1138 1973

* Enzymatic activities were assayed as previously described (16, 17).Homogenates and membranes preparations were in 0.25 M sucrose,1 mM EDTA, 0.1% 2-mercaptoethanol (vol/vol), and 0.01 M Tris-acetate buffer at pH 7.0.

t Reactions catalyzed are: lactosylceramide - GM3; GM3 , GM2;GM2 GM1; and GM1 - GD1a-t The crude membrane preparation was a 20,000 X g sediment ofthe total homogenate which had bee; washed three times with thebuffer used to homogenize the tissues.

malignant cells (22), and a specific deficiency in this N-ac-etylgalactosaminyltransferase activity and gangliosides morecomplex than GM3 has been found in transformed mouse (22),hamster (23), and rat cells (24).

In previous reports, we indicated that ganglioside could in-hibit the binding of TSH to bovine thyroid membranes and thesubsequent activation of adenylate cyclase and suggested thatgangliosides or ganglioside-like structures may be part of theTSH receptor on these membranes (1, 2). In addition, wedemonstrated that cholera toxin and TSH could compete withone another for binding (presumably to GM1 receptors) (2).These GM1 molecules were exposed on the surface of mem-branes as they could be tritiated by the galactose oxidase-[3H]sodium borohydride procedure and specifically protectedfrom tritiation by cholera toxin (2). In a similar manner, we nowshow that GM1 residues are exposed on the membranes fromnormal rat thyroid but not from a rat thyroid tumor which isdefective in TSH binding. Thus, at least one of the gangliosidesimplicated in TSH binding is absent from this tumor.

Previous studies indicated that a glycopeptide that has TSHbinding ability could be released by trypsin or detergents fromthyroid membranes (13, 20, 25). Loss of this receptor compo-nent from thyroid membranes paralleled loss of TSH binding,adenylate cyclase stimulation, and TSH activity (13, 20, 25).The material released by trypsin contains approximately 30%carbohydrate and 10% sialic acid; disc gel electrophoreticanalysis indicated that the major protein and carbohydratestaining component has a molecular weight of 24,000 (13,25).The present studies suggest that this TSH receptor component

is either absent or defective in the tumor membranes sincecomparable material is not released from these membranes bytrypsin (Table 3). Preliminary data concerning the solubilizationof 1-8R thyroid tumor membranes by lithium diiodosalicylateconfirm this conclusion (unpublished data). The relationshipbetween the above glycopeptide TSH receptor component andthe gangliosides that are found in normal thyroid membranesand that bind TSH in vitro thus has yet to be determined. One

possibility is that after binding to the glycopeptide componentof the receptor, the TSH molecule interacts with a specificganglioside and undergoes a conformational change whichultimately leads to changes in the state of the membrane, al-terations in ion transport, and activation of adenylate cyclase.Absence of one or both of these receptor components wouldresult in thyroid membrane unresponsive to TSH as observedin the rat thyroid tumor that we have investigated. We believethat additional studies of this tumor and its membranes shouldenhance still further our knowledge of the biosynthesis of TSHreceptor components, of their integration in the membranebilayer, and of the mechanism by which the receptor transmitsits message through the membrane bilayer to cause adenylatecyclase stimulation.

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