Relationships of the Structure and Function of the Interferon Receptor to Hormone Receptors and Establishment of the Antiviral State1

[CANCER RESEARCH 38, 4172-4185, November 1978]

Evelyn F. Grollman, George Lee, Sofia Ramos, Pedro S. Lazo, H. Ronald Kaback, Robert M. Friedman, and Leonard D. Kohn Sect/on on Biochemistry of Cell Regulation, Laboratory of Biochemical Pharmacology Â¡E.F. G., G. L., P. S. L., L. D. K.] and Laboratory of Experimental Pathology [R. M. F.], National Institute of Arthritis, Metabolism, and Digestive Diseases, NIH, and Division of Bacterial Products, Bureau of Biologies, Food and Drug Administration, Bethesda, Maryland 20014 [S. R.], and Laboratory of Membrane Biochemistry, Roche Institute of Molecular Biology, Nutley, New Jersey 07110 [H. R. K.J

Abstract between TSH and receptor involves electrostatic interac tions between positively charged residues on the TSH This report describes similarities between the structure molecule and spatially oriented negatively charged residues and function of the Interferon receptor and receptors for within the receptor binding site. It is believed that this initial glycoprotein hormones and several bacterial toxins. Spe electrostatic interaction determines the locus and nature of cifically, it describes several common molecular and supplementary short-range (hydrophobic) interactions mechanistic elements, including: (a) the presence of a which approximate the hormone more firmly within the glycoprotein as well as a ganglioside component in the receptor. These interactions exclude water and salts from receptor; (b) changes in membrane structure as a conse the 2 approximating surfaces and induce a conformational quence of Interferon action; (c) Â¡nterferon-inducedintra- change in the hormone. The receptor-induced conforma cellular cyclic adenosine 3':5'-monophosphate changes; tional change, the exact nature of which is encoded by the and (d) alterations in the flux of certain ions across the oligosaccharide determinant of the receptor, allows the Â« membrane. Since Interferon has an antiviral effect, these subunit of the hormone (or a portion of the a subunit results define a relationship between hormonal perturba similar in sequence to the nonapeptide hormones oxytocin tion of cellular events and the ability of an agent to and vasopressin) to intercalate itself within the membrane prevent or suppress viral infections of cells. Further defi bilayer. Implicit in this series of events is the notion that a nition of these relationships should be important to our receptor with a structure similar to that of the TSH receptor, understanding of the oncogenic state, of hormonal effects but containing a different oligosaccharide determinant, on the oncogenic state, and of other human diseases in might accommodate TSH, but would not trigger the appro which hormonal perturbations of non-target tissues or priate conformational change necessary for message trans cross-reactivity of receptors could be pathogenic. mission. The consequence of these events is a change in the state Introduction of the membrane, which expresses itself (a) as a change in cell surface determinants, such as receptors for other Studies over the past few years indicate that the TSH2 effectors of cell function (23-25, 27), and (b) as a change in receptor is composed of glycoprotein and ganglioside com the electrical potential across the cell membrane (18). ponents and that a key determinant within the receptor is Finally, changes in cyclic AMP levels occur as a result of an oligosaccharide moiety (23-25, 27). Current results indi direct or indirect modulation of components in the ade- cate that the glycoprotein represents a high-affinity binding nylate cyclase system. As noted above, cyclic AMP is then a component which selects TSH from structurally unrelated cellular message which effects changes in the translational effectors such as insulin, glucagon, adrenocorticotropic and transcriptional machinery of the cell in the absence of hormone, prolactin, growth hormone, etc. (2, 25). The any input of additional genetic information in the form of ganglioside, in contrast, is believed to serve as a low-affinity DMA or RNA (as in the case for fertilization or for a virus discriminator which completes the selection process by infection). distinguishing between structurally related effectors, such Evidence has accumulated which indicates that the bind as the other glycoprotein hormones (luteinizing hormone, ing of interferon to specific cell surface receptors is neces follicle-stimulating hormone, and human chorionic gonad- sary for its antiviral action (13). Other studies suggest that otropin). The ganglioside thus contributes to the TSH re the interferon receptor has a ganglioside or ganglioside- ceptor a 3-dimensional structure with the stereospecific like structure as one of its component parts and that an binding properties of an enzyme (2, 25). oligosaccharide moiety on this ganglioside or ganglioside- Current experiments suggest that the initial association like component is a critical feature for receptor function (5- 7, 62). Since cholera toxin and TSH interact with cell 1 Presented at the John E. Fogarty International Center Conference on surface receptors believed to contain a ganglioside as a Hormones and Cancer, March 29 to 31, 1978, Bethesda. Md. functional component (23-25, 27), the following possibili 2 The abbreviations used are: TSH, thyrotropin; cyclic AMP, cyclic aden osine 3':5'-monophosphate; Gsn, galactosyl-/V-acetylgalactosaminyl(N-ace- ties were raised. First, the mechanism by which these 3 tylneuraminyl)galactosylglucosylceramide; GU2, W-acetylgalactosaminyl(N- effectors and interferon transmitted their messages to the acetylneuraminyl)galactosylglucosylceramide, GM3, N-acetylneuraminylga- cell might have elements in common. Secondly, cholera lactosylglucosylceramide; TPMP". triphenylmethylphosphonium ion; EGTA, ethyleneglycol bis(/3-aminoethyl ether)-W,N'-tetraacetic acid; PHA, phytohe- toxin and TSH might be able to block the ability of interferon magglutinin. to induce an antiviral state.

4172 CANCER RESEARCH VOL. 38

Recent experiments (14, 26) have shown: (a) that cholera Preparation of Membranes, Ganglioside, and Glycopro toxin and TSH do in fact inhibit the establishment of the tein Extractions. Plasma membranes from mouse Ly and antiviral state by interferoni (b) that this action reflects an human KB-3 cells were prepared by adapting methods used effect of these agents on plasma membranes, and (c) that previously to obtain bovine thyroid plasma membranes (3, these effectors do not appear to act as simple competitive 26, 56, 57). Plasma membrane gangliosides were extracted antagonists. according to a modification of the methods of Yu and This report will summarize experiments which further Ledeen (65) and Saito and Hakomori (54). The details of characterize: (a) the structure of the interferon receptor; these procedures are described elsewhere (17). Ganglioside and (b) the mechanism by which interferon-receptor inter standards for these analyses (GM1,GN12,GM3,N - acetylneu- actions might initiate or propagate the antiviral state. In raminylgalactosyl - N - acetylgalactosaminyl[N - acetylneu- sum, the data show that the interferon receptor, like the raminyl]galactosylglucosylceramide, galactosyl-A/-ace- TSH receptor, is a 2-component (glycoprotein and ganglio- tylgalactosaminyl[/V - acetylneuraminyl - N - acetylneurami- side) receptor, that both components are necessary for nyljgalactosylglucosylceramide, and W-acetylneuraminyl- message transmission, that membrane changes are a con galactosyl - N - acetylgalactosaminyl[A/ - acetylneuraminyl - sequence of the interferon-receptor interaction, and that N - acetylneuraminyljgalactosylglucosylceramide) were ob phosphorylated intermediates such as cyclic AMP may be tained as previously described in (40-43, 46, 47). Identifica involved in mediating the response to interferon. In addi tion of the gangliosides in the mouse Ly and human KB-3 tion, this report describes several interferon-induced effects cell membranes was based on content of sialic acid and on ion fluxes across the membrane. terminal galactose residues, sensitivity to neuraminidase, In the context of this conference, we believe these studies and Chromatographie properties. are important in 2 respects. First, they relate hormonal Surface-exposed terminal galactosyl residues on glyco- interactions with cells to the interaction of an agent known lipids and glycoprotein components of membranes were to produce an antiviral state. Secondly, we believe that they labeled with tritium according to modifications of described offer additional insight into the mechanism by which inter procedures (16, 44). Sialic acid residues on the ganglio feron can prevent oncogenic viruses from transforming sides and glycoproteins were tritiated after mild periodate cells. oxidation (44). Details of these procedures are described elsewhere (17). Materials and Methods The glycoprotein component of the interferon receptor was solubilized by extracting membrane preparations with Cells and Viruses. Mouse Ly cells are a strain of inter- 0.1 M lithium diiodosalicylate in analogy with procedures feron-sensitive mouse fibroblasts originally obtained from used to isolate the glycoprotein component of the TSH Dr. J. S. Younger, University of Pittsburgh School of Medi receptor from thyroid plasma membranes (17, 56, 58). cine. Human KB-3 cells are the same strain used in our Centrifugation at 100,000 x g for 2 hr pelleted less than 2 to previous report (26). They are insensitive to mouse or 5% of the binding activity at concentrations up to 1 mg human interferon, but bind both interferons to their cell protein per ml. Protein was measured colorimetrically (38) surface membranes (26). Mouse Ly and human KB-3 cells with the use of bovine serum albumin as a standard and were grown as monolayers in Eagle's minimum essential prior treatment with 0.1 N NaOH. medium with 10% fetal calf serum, except during treatment Binding Studies and Liposome Preparations. Bovine with interferon, when medium without serum was used. TSH and 125l-labeledTSH were prepared according to meth Interferon and Interferon Assays. The mouse interferon ods analogous to those described previously (28, 56, 63). used was prepared by Dr. K. Paucker, Medical College of Cholera toxin was purchased from Schwarz/Mann, Orange- Pennsylvania, Philadelphia, Pa., according to the method burg, N. Y., and radioiodinated in the same manner as that of Ogburn ef al. (49). The preparation had a specific activity described for TSH (46). 125l-labeled cholera toxin or 125I- of at least 2 x 107 mouse Interferon reference units per mg labeled TSH binding to plasma membranes was assayed of protein and had the chemical and physical properties with the use of a filtration technique previously detailed (3, usually ascribed to mouse interferon. Interferon was as 17, 18, 56, 57). In addition to the agents tested for their sayed by a cytopathic effect inhibition assay in mouse Ly ability to influence binding, the standard incubations con cells; vesicular stomatitis virus was used as the challenge tained, in a 100-jLtl volume, 20 mM Tris-acetate (pH 6.0), virus. 0.6% bovine serum albumin, 50,000 to 100,000 cpm of 125I- For establishment that interferon treatment was effective, labeled cholera toxin (2 nM) or TSH (7.5 nM), and a concen monolayers of cells in microtiter dishes were washed 5 tration of plasma membranes or liposomes known to be times with medium and infected with virus at a virus:cell within the linear phase of binding when related to mem multiplicity of 1. After 16 hr the cells were frozen and brane protein concentration. To ensure the specificity of thawed, and the culture fluids of at least 6 wells were binding and the specificity of inhibition, control incubations pooled. These were assayed for virus yields by 10-fold serial contained unlabeled TSH (10 /Â¿M)orcholera toxin (5 Â¿Â¿M) dilutions in microtiter plates which were observed for 72 hr with or without membranes. after infection for viral cytopathic effects. Liposomes containing the glycoprotein component of the Interferon treatments sufficient to establish maximal an receptor were formed according to a modification of a tiviral protection in mouse Ly cells were performed as procedure used to make liposomes containing gangliosides previously detailed (14, 26). Human KB-3 cells received the (1, 17). The final liposome pellet was resuspended in 500 same treatment where appropriate. IJL\of 0.01 M Tris:acetate (pH 7.0) and stored at 4Â°.Lipo-

NOVEMBER 1978 4173

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1978 American Association for Cancer Research. E. F. Grollman et al. somes were quantitated with the use of a Model ZB1 Coulter the following observation. The relative potency and revers counter, with lower and upper thresholds set at 10 and 100 ibility of TSH and cholera toxin as inhibitors of the antiviral /urn, respectively. They were diluted prior to the assay to state in vivo correlates with the relative ability of interferon yield 5 x 105particles/50 /Â¿I. to inhibit reversibly the binding of these agents in vitro (14, Uptake of [3H]TPMP-. [3H]TPMP (bromide salt) uptake 26). was measured by filtration through Millipore Cellotate filters Analyses of a similar nature show that human KB-3 cells (0.5 Ã•JLM)(18).Incubations were carried out at 20Â°in 100-^1 bind mouse and human interferon. although neither agent reaction mixtures (total volume). Unless otherwise noted, induces an antiviral effect (26). One interpretation of this reactions were initiated by diluting membrane vesicle prep result suggests that the species specificity of interferons in arations suspended in 0.25 M sucrose containing 0.01 M these systems is not related to a lack of binding, but rather Tris-HCI (pH 7.4) 10-fold into reaction vessels containing to an inability to initiate message propagation. The studies 0.05 M NaCI, KCI, or Tris-HCI (pH 7.4), 0.125 M sucrose, 0.4 summarized below indicate that the ability of interferon to mM [3H]TPMP- (120 Ci/mol), and other additions as indi bind to human KB-3 cells can be accounted for by a cated. Uptake was terminated by addition of 5 ml of ice- glycoprotein component of the receptor that is similar on cold 0.8 M NaCI containing 0.1 M Tris-HCI (pH 7.5), imme both mouse Ly and on human KB-3 cells. The studies also diate filtration, and washing with an additional 5 ml of the show that the inability of interferons to induce an antiviral same solution. Dilution, filtration, and washing were com state in human KB-3 cells correlates with a ganglioside plete within 20 sec, and the filters were immediately re deficiency in these cells. moved from the suction apparatus and assayed for radio The 2 predominant gangliosides in the plasma mem activity by liquid scintillation spectrometry. Corrections for branes of interferon-sensitive mouse Ly cells are G\l2 and nonspecific adsorption were made by diluting the reaction GM:,(Chart 1/\; Table 1). Gangliosides with the Chromato mixtures with wash buffer before addition of [3H]TPMP~. graphie characteristics of GN11,N-acetylneuraminylgalacto- followed by filtration and washing, and this value was syl-A/-acetylgalactosaminyl[/V-acetylneuraminyl]galactosyl- subtracted from the experimental values presented. glucosylceramide, galactosyl-N-acetylgalactosaminyl[/V- [3H]TPMP' (bromide salt) was prepared by the Isotope acetylneuraminyl-W-acetylneuraminyllgalactosylglucosyl- Synthesis Group at Hoffman-La Roche Inc., Nutley, N. J., ceramide, W-acetylneuraminylgalactosyl-N-acetylgalactos- under the direction of Dr. Arnold Liebman. aminyl[N-acetylneurammyl-A/-acetylneuraminyl]galactosyl- Vesicle Preparations. Thyroid cell suspensionswere ob glucosylceramide are also detectable in these membranes, tained by gentle scraping of the culture flasks after a 10- but in amounts less than 5% of that of either G\,2or Gu.,.GN,2 min incubation with 3 mM EGTA in Ca2 - and Mg2*-free has been implicated as a receptor component or receptor phosphate-buffered saline (0.138 M NaCI; 2.7 mM KCI; 8.1 analog in studies concerned with the ability of gangliosides mivi Na.HPO.,; 1.47 HIM KH,,P04). Cells were recovered by to inhibit the antiviral action of interferon (5-7); GM:1has centrifugation at 200 to 500 x g, washed twice in the same been found to be as effective as GM2in adsorbing human medium without EGTA or with an isotonic (0.25 M) sucrose interferon in affinity Chromatographie studies (C. B. Anfin- solution containing 0.02 MTris-HCI (pH 7.4), and suspended sen, personal communication). in one or the other of these media prior to use. Mouse Ly The total ganglioside content of the mouse Ly cell mem and human KB-3 cells were obtained in the same manner, brane, expressed as nmol of lipid-bound sialic acid, is 1.5 except that EGTA was omitted and replaced with phos nmol per mg membrane protein (Table 1). Interferon treat phate-buffered saline, pH 7.4. ment of mouse Ly cells does not alter the total ganglioside Purified plasma membrane vesicle preparations from content of the membranes (Table 1) nor does it alter the these cells were obtained by nitrogen cavitation as de content of any individual ganglioside as measured by quan scribed by Lever (35, 36). The final vesicle preparation was titative densitometric analyses of resorcinol-stained thin- suspended in 0.25 M sucrose containing 0.01 M Tris-HCI layer chromatography plates (Chart 1; Table 1). (pH 7.5), unless otherwise stated, and stored at a concen Human KB-3 cell plasma membranes contain approxi tration of 4 to 20 mg of protein per ml in liquid nitrogen. mately one-tenth of the total amount of ganglioside per mg Vesicle preparations were the same in character as those membrane protein as found in interferon-sensitive mouse previously described for thyroid vesicle preparations (18) as Ly cells (Table 1). In terms of specific gangliosides, they far as marker enzyme analyses were concerned. contain only small amounts of GU3(Chart 2; Table 1), no Miscellaneous Assays. The internal pH was measured ganglioside comigrating with GN,2(Chart 2; Table 1), and with [14C]methylamine according to the procedure de small amounts of an unidentified disialyl ganglioside, called scribed by Rottenberg eia/. (53). G,)s(see below) (Chart 2; Table 1). As with mouse Ly cells (see above), there is no difference in the total amount of Results and Discussion gangliosides extracted from the cell membranes of human The Structural Components of the Interferon Receptor. KB-3 cells which had or had not been treated with inter The ability of mouse interferon to establish an antiviral state feron (Table 1). in mouse Ly cells correlates with an ability of mouse These results indicate that the failure of human KB-3 cells interferon to bind to plasma membranes from these cells to develop antiviral activity correlates with a ganglioside (14, 26). Although binding is measured indirectly as the deficiency and with the absence of the ganglioside GM2in ability of interferon to inhibit I25l-labeled TSH and 125I- the membranes of these cells; GN12hasbeen implicated as a labeled cholera toxin binding to mouse Ly cell plasma receptor or receptor component by Besancon and Ankel (5, membranes, the relevance of this measure is supported by 6) Besancon eÃal. (7) and is one of the predominant

4174 CANCER RESEARCH VOL. 38

STANDARD LY CELLS STANDARD with their interferon sensitivity and that they could recon stitute" interferon sensitivity by incubating gangliosides A. with ganglioside-deficient cells. The observation by Vengris ef a/. (62) that commercial preparations of mixed brain gangliosides which do not appear to contain Gx,_,or GM:,can reconstitute the antiviral state is of interest in the light of the data showing that Qvl and CM.;are the major gangliosides in an interferon-sensitive mouse Ly cell strain. It should be noted, however, that a minor ganglioside component of thyroid membranes (0.015% of membrane gangliosides) has the most potent inhibitory activity toward thyrotropin binding (48) and that Goia very small levels of G\n are necessary to produce cholera toxin sensitivity in reconstitution experiments (45).Also, GTr grossly detectable amounts of G\n are not present in chol era toxin-sensitive fat cells (12, 21). Origin - Origin It has been reported previously that TSH receptors on bovine thyroid plasma membranes can be solubilized with lithium diiodosalicylate (56. 58, 64) and that tryptic diges tion of the solubilized receptor preparation yields a receptor fragment with a molecular weight of 24,000 which retains B. specific TSH binding activity (56, 58, 64). Analysis of a RADIOACTIVITY (cpm x 1O") RADIOACTIVITY (cpm x 1(H) purified preparation of this receptor fragment indicates that 1.6 1.2 0.8 0.4 0 0 0.4 0.8 1.2 it is.a glycoprotein containing 30% carbohydrate and 10% sialic acid by weight (56, 58, 64). As noted above, more recent studies (23-25, 27) indicate that gangliosides may be ,GMS an important component of the thyrotropin receptor and that the role of gangliosides in transmitting the hormonal message to the cell machinery may be analogous to their role in transmitting the message of cholera toxin to cells exposed to this bacterial product (4, 23-25, 27). The 2 components of thyroid plasma membranes known \ Ã›M2 to interact with TSH,/.e., the glycoprotein with specific TSH binding activity and the gangliosides of the thyroid mem

NO 1 2 INTERFERON branes, segregate differently when membranes are solubi INTERFERON TREATED lized with lithium diiodosalicylate (41). Thus, whereas 80% TREATMENT of the TSH binding activity can be recovered in the super Chart 1. A. thin-layer chromatography of gangliosides extracted from mouse Ly cell membranes. The extraction procedure is described in 'Mate natant solution resulting from the lithium diiodosalicylate rials and Methods." The chromatography solvent was chloroform:meth- procedure, less than 10% of the membrane gangliosides anol:0.25%CaCI2 (60:35:8, v/v/v). Gangliosides are visualized with resorcinol are in this supernatant preparation (41). spray. The standards in both cases are gangliosides extracted from bovine Since the data above (Charts 1 and 2; Table 1) show that brain. Data are further detailed in (17). B radioscans of sialic acid-labeled human KB-3 cells contain negligible amounts of ganglio gangliosides extracted from the membranes of mouse Ly cells which had been treated with Interferon (30 units/ml) for 14 hr (Lane 2) and gangliosides sides compared to mouse Ly cells, no G\|2, and only trace extracted from the membranes of mouse Ly cells which had not been amounts of G\n, the possibility was raised that interferon, exposed to Interferon (Lane 1). Gangliosides were labeled by sequential exposure of the cell membranes to periodate and [3H]sodium borohydride TSH, and cholera toxin binding to the human KB-3 mem (see "Materials and Methods" and Table 1). Labeling (130,000 and 104,000 branes could be accounted for by a glycoprotein receptor cpm) was applied to Lanes 1 and 2, respectively, i.e., 2.2 and 1.9 nmol lipid- bound sialic acid. After radioscans were performed the gangliosides on each component in these membranes. This hypothesis was plate were visualized with resorcinol (see 'Materials and Methods"). The tested when membranes of human KB-3 cells and mouse Ly altered ratio of GM3to GV2labeling before and after inferieron is evident not cells were extracted with lithium diiodosalicylate, and the only from the different relative peak heights, but also from measurements of the area under each peak. Data are further detailed in (17). G,â€žâ€ž,lithium diiodosalicylate supernatant of both membrane A/-acetylneuraminylgalactosyl-N-acetylgalactosaminyl[N-acetylneuram- preparations was incorporated into artificial membranes myljgalactosylglucosylceramide: G ,,. galactosyl-N-acetylgalactosammyl- (/Vâ€¢¿acetylneuraminyl-/V-acetylneurammyl]galactosylglucosylceramide: (liposomes). As seen in Chart 3, liposomes containing the G,,, N - acetylneuraminylgalactosyl - /V- acetylgalactosaminyl[W - acetylneu- lithium diiodosalicylate supernatant from either membrane raminyl - N â€¢¿acetylneuraminyljgalactosylglucosylceramide. preparation bind 125l-labeledTSH (Chart 3,4), and binding is specific (Chart 36), it is inhibited by interferon at concentra gangliosides in the Interferon-sensitive mouse Ly cells. tions similar to those necessary to inhibit TSH binding to These data are thus consistent with the hypothesis that plasma membranes of both mouse Ly and human KB-3 cells gangliosides are components of the interferon receptor and (Chart 3C; Ref. 14), and it has a similar temperature sensi that their absence prevents antiviral message transmission. tivity to that exhibited by the plasma membranes (Chart In addition, the data support the work of Vengrisef al. (62), 3D). who showed that the ganglioside content of cells correlates That this binding activity is unrelated to gangliosides is

NOVEMBER 1978 4175

Table 1 Ganglioside content of the membranes from mouse Ly cells and human KB-3 cells treated with inferieron by comparison to the membranes from mouse Ly cells and human KB-3 cells never exposed to interferon Cells were treated overnight with sufficient interferon to ensure maximal antiviral protection (see 'Materials and Methods"). The effectiveness of the interferon treatment was established by challenging aliquots of these cells with virus (see "Materials and Methods"). Individual membrane gangliosides'' (nmol lipid-bound sialic Total membrane gangliosides acid/mg membrane protein) lipid- lipid- bound bound sialic boundgalactose1' terminal sialic acid acid'' residues (nmol/mg (cpm/mg residues (cpm/ membrane membrane mg membrane protein")1.5 protein)71 protein)2,400 M0.65 GDX0.03 G,)lb GT1 Mouse Ly cells Interferon-treated ,500 Â±5,000' Â±500 0.03 0.03 UntreatedLipid- 1.5Tritiable89,300 Â±5,000Tritiable5,000 Â±500GÂ»,0.650.65G 0.65GMI0.030.03GDI/ 0.03 0.03 0.03 Human KB-3 cells Interferon-treated 0.12 20,300 Â±1,500 1,100 Â±200 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 0.05 Untreated 0.12 27,200 Â±1,500 1.300 Â±200 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 0.05 " Protein was measured colorimetrically with the use of bovine serum albumin as the standard. '' Measured after membranes were sequentially treated with periodate and [3H]sodium borotritide (see "Materials and Methods" and Ref.'' Measured 17). after membranes were sequentially treated with galactose oxidase and [3H]sodium borotritide (see "Materials and Methods" and Ref. 17). d Determined from densitometry scans of resorcinol-stained thin-layer plates analogous to those in Chart '\A. e G,)la,A/-acetylneuraminylgalactosyl-A/-acetylgalactosaminyl(A/-acetylneuraminyl)galactosylglucosylceramide; G,,,,,,galactosyl-A/-acetylgalactosaminyl(A/-acetylneuraminyl-A/-acetylneuraminyl)galactosylglucosylceramide; GM, /V-acetylneuraminylgalactosyl-rV-acetylgalactosaminyl(A/-acetylneuraminyl-A/-acetylneuraminyl)galactosylglucosylceramide. ' The values given are for the particular experiment presented; the error values represent the extreme range of values seen in multiple separate experiments.

RADIOACTIVITY (cpm x 10-3) RADIOACTIVITY (cpm x 10-3)

6.0 4.5 3.0 1.5 0 0 1.5 3.0 4.5

GDX 10 20 30 40 50 0.1 02 0.3 0.4 2.5 5 10 20 30 LIPOSOMES ADDED UNLABELED TSH INTERFERON TEMPERATURE NO INTERFERONTREATMENT INTERFERONTREATED ( Â¿Â¿(-assay) (mg ml) (units x 10Â°) Chart 2. Radioscans of sialic acid-labeled gangliosides extracted from the Chart 3. '"l-labeled TSH binding to the glycoprotein receptor component membranes of KB-3 cells which had been treated with interferon (30 units/ isolated from mouse Ly cells or human KB-3 cells. After the glycoprotein ml, right) and which had not been exposed to interferon. The gangliosides component was obtained by solubilization of membrane preparations with had been labeled by sequential exposure to periodate and [3H]sodium lithium diiodosalicylate (see "Materials and Methods"), it was incorporated borohydride (see "Materials and Methods." Table 1. and Chart 16). The into artificial membranes, i.e., liposomes (see "Materials and Methods"). extraction and chromatography procedures are the same as in Chart 10 and "Materials and Methods." The radioscans were performed before the plates Binding is examined as a function of liposome concentration lAi. in the presence of unlabeled TSH (B) or unlabeled interferon (C), and as a function were stained with resorcinol. Approximately 0.16 nmol of lipid-bound sialic of temperature (D). Binding was performed as described in "Materials and acid extracted from the interferon-treated and untreated KB-3 cell mem Methods." In all cases. 50 Â¿JÂ°fliposomes contained 3 x 105particles. In B. branes was applied to the plate, i.e., approximately 27,000 cpm and 36,000 C, and D. the assays contained 40 /x, human KB-3 or mouse Ly cell lithium diiodosalicylate ex a disialyl ganglioside, may be G,,, [A/-acetylgalactosaminyl-/V-(acetylneuraminyl-/V-acetylneuraminyl)-galactosylglucosylceramide], for which no stan tracts were used (Chart 3). The binding activity of the dard was available. The solvent in this experiment was chloroform:metha- nol:ammonium hydroxide (see "Materials and Methods" and Ref. 17). membranes from both cells is trypsin sensitive (Table 3), and the binding activity of liposome preparations contain ing either the human KB-3 or mouse Ly cell lithium diiodo evident (a) from the ganglioside content of the lithium salicylate extracts increase in direct proportion to their diiodosalicylate extracts (Table 2); (b) from previous (1) and incorporation of carbohydrate-labeled components from current results showing that this amount of ganglioside the lithium diiodosalicylate extracts (Table 4). material does not bind TSH when incorporated into a The binding by the liposomes is not restricted to TSH. liposome; and (c) from the equivalent results, whether the Thus, both types of liposomes bind cholera toxin (17),

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Table 2 cholera toxin binding has the same temperature sensitivity Ganglioside content of lithium diiodosalicy/ate extracts of human as do plasma membranes (17). and interferon will inhibit KB-3 and mouse Ly cell membranes cholera toxin binding to the liposomes (17). Ganglioside These data are of interest with respect to the species content (nmol/ specificity problem and to interferon receptor function in mg protein) general. The ability of human KB-3 cells to bind TSH. Human cholera toxin, and interferon in the absence of specific KB-3 Mouse hormone or toxin effects or induction of an antiviral state cells Ly cells (26) is shown to correlate with the presence in these Total membrane gangliosides (see Table 1) 0.12 1.5 Lithium diiodosalicylate extract 0" 0.35'' membranes of the glycoprotein component of the receptor. The glycoprotein nature of this component is indicated by " No gangliosides were detectable chromatographically, even its trypsin sensitivity and the correlation of binding with a with the use of lithium diiodosalicylate extracts from membranes carbohydrate-containing moiety released from the mem labeled with tritium by mild periodate oxidation. Extracts equiva lent to 30 to 100 mg of membrane protein were analyzed. branes by lithium diiodosalicylate treatment (Table 4). Stud '' In liposome preparations with gangliosides, the usual liposome ies already reported by this laboratory in preliminary form preparation was made with 10 /Â¿molof phosphatidylcholine and (30) have further established this point by identifying frag between 1.0 and 0.5 /Â¿molof gangliosides. No TSH binding was ments of the glycoprotein component of the receptor and detectable when less than 0.05 /*mol ganglioside was used. This would be over 100-fold less per mg membrane protein used in the by showing that the intact glycoprotein component, as well liposome preparation (see "Materials and Methods" and Ref. 17). as the glycopeptide fragments, cross-react with an antibody directed against the glycoprotein component of the TSH Table 3 receptor on bovine thyroid membranes. In short, "nonpro Effect of trypsin on binding of '"/-/aoe/ed TSH or '25/-/at>e/ed ductive" but "hormonally" specific binding can occur on cholera toxin to membranes from mouse Ly and human KB-3 cells the human KB-3 cell membranes, presumably because they Medium was removed from the cells, and the cells (5 mg mem brane protein/flask) were washed 3 times with 5-ml volumes of contain the glycoprotein component of the receptor; mes phosphate-buffered saline (0.138 M NaCI; 2.7 PIM KCI; 8.1 mw sage transmission is presumably blocked because of the Na,HPO4; 1.47 mw KH,PO4, pH 7.4). Cells were then exposed to ganglioside deficiency. This result is similar to the situation (5 ml) 0.025% trypsin (Grand Island Biological Co.. Grand Island, with the TSH receptor, for which previous studies have N. Y.) in phosphate-buffered saline (pH 7.4) for 20 min at room demonstrated simultaneous protease inactivation of bind temperature. Control cells were incubated for 20 min at room tem ing and adenylate cyclase stimulation by TSH in cultured perature in the same buffer without trypsin. The trypsin or buffer was removed by aspiration, and cells were washed 3 times with thyroid cells (63) and loss of a functional TSH response in a 5-ml volumes of phosphate-buffered saline (pH 7.4). Cells were thyroid tumor, the membranes of which are ganglioside scraped and suspended in 5 ml of 0.01 M Tris-acÃ©tatebuffer (pH 7.0), and membrane preparations were prepared (see "Materials deficient (40. 43). Each component thus contributes to the and Methods"). Membrane preparations were stored at -90Â°until function of the intact plasma membrane receptor in that assayed, with the use of the standard binding conditions described message transmission across the membrane requires the (see "Materials and Methods" and Ref. 17). presence of both a glycoprotein and glycolipid component protein)'"l-labeled (cpm bound/5 ^9 membrane in the receptor structure. Previous studies (8) suggest that interferon inhibits mu Ly cell KB-3 cell rine leukemia virus at a late stage in virus replication. The membranesNo membranesNo expression of at least 2 major viral proteins as well as the trypsin+trypsin10,700 trypsin+trypsin15,800 sucrose gradient patterns of the viral RNA is unaffected by TSH 2,300 4,900 Â¡nterferontreatment in these same experiments. In view of '"l-labeled cholera 12,400 7,300Human 15,000 4.300 the effect of interferon on the binding of thyrotropin and toxinMouse cholera toxin, the presence of a glycoprotein component in

Table 4 Correlation of carbohydrate uptake from the lithium diiodosalicylate preparations into liposomes with the ability of the liposomes to bind '"/-labeled TSH and 125/-/abe/edcholera toxin Lithium diiodosalicylateprotein sialicacidadded tophosphatidylcholine:cholesteroladded tophosphatidylcholine:cholesterol sialicacidincorporated(cpm)64,000137,000253,000Protein:sialicacidtoxinbound/2 105particles''x x105particles lipidfilm" lipidfilm'' ratio(cpm//*g)9401,062960125l-labeledTSHbound/4(cpm)9,50020,80044,200'"l-labeledcholera (Mg)5001,0002,000Tritiated(cpm)500.0001.000,0002,000,000Proteinincorporated(Mg)68129263Tritiated (cpm)18,50035.40074,200

" See "Materials and Methods" for details of method by which protein was added to the previously dried phosphatidylcholine:cholesterol film in the tear-shaped flask. '' Sialic acid-containing components in the membrane were labeled with tritium as detailed in "Materials and Methods." The tritiated membranes were solubilized with lithium diiodosalicylate as described (see "Materialsand Methods" and Ref. 17). Radioactivity was both trichloroacetic acid precipitable and neuraminidase sensitive. In this experiment, a mixture of unlabeled lithium diiodosalicylate to tritiated lithium diiodosalicylate (3:1) was used. Mixtures of 1:1, 1:3, and use of only the tritiated lithium diiodosalicylate preparation gave the'' same Particles result. determined by Coulter counter analysis. Size distribution was the same, independent of the protein content.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1978 American Association for Cancer Research. ÃŠ. F. Grollman et al. thÃ¨ Interferon receptor, and the relationship between gly- indicated in the observation that interferon treatment re coproteins and intramembranous particles, the block in sults in a 20% decrease in tritiable sialic acid and a 50% virus production by interferon may involve an alteration of decrease in tritiable terminal galactose residues in the a membrane-associated glycoprotein. This may. in turn, gangliosides of mouse Ly cell membranes in the absence of result in an impairment of virus release through an as yet a difference in total ganglioside content (Table 1). Radio- undefined mechanism (9). These findings may also explain scans of gangliosides extracted from the membranes of the decrease in infectivity of murine leukemia viruses pro mouse Ly cells before and after treatment with interferon duced by interferon-treated cultures of TB cells (8). The are consistent with this conclusion. Analyses of glycopro decrease may be caused by defective viral envelope com teins (17) and neutral glycolipids (17) obtained from mouse ponents derived from the interferon-altered host plasma Ly cell membranes show, however, that the membrane membrane (9). The mechanism of inhibition under these changes are not restricted to alterations in the exposure of circumstances is under further investigation. gangliosides on the surface of the cell. Thus, interferon Membrane Changes as a Consequence of Interferon treatment results in decreased exposure of the terminal Action. Previous work from this and other laboratories has galactose residue in oligosaccharides of at least 4 glycopro implicated cell surface alterations in the mechanism of tein components of the membrane and in either decreased action of interferon. Mouse interferon enhances the expres or increased exposure of several neutral glycolipids of sion of cell surface histocompatibility antigens in mouse Ly mouse Ly cell membranes. 1210 cells (37) and increases the net negative charge on the Interferon-insensitive human KB-3 cells do not exhibit the surface of mouse Ly cells (22). Interferon treatment also same changes in cell surface exposure of gangliosides, decreases the capacity of mouse Ly cell membranes to bind glycoproteins, and glycolipids after interferon treatment the glycoprotein hormone thyrotropin (26) and alters the (17). Thus, although interferon treatment causes a 25% cell surface binding of cholera toxin (14, 26). The latter decrease in the amount of tritiable lipid-bound sialic acid effect which involves an initial enhancement and subse (Table 1), other data are not either quantitatively or qualita quent inhibition of binding is best explained by postulating tively identical with the results obtained with mouse Ly cells an altered orientation of the cholera toxin receptor second (17). For example, in contrast to results with membranes ary to interferon-induced changes in the membrane. from interferon-sensitive mouse Ly cells, interferon treat Recent studies of interferon-induced alterations in the ment does not cause a detectable shift in the labeling of cell membrane (9, 17) correlate these changes with the individual ganglioside components, the sialic acid residues development of the antiviral state. The oligosaccharide of which had been tritiated (Chart 2). No change is detecta chains of glycoproteins in RBC membranes serve as recep ble in the labeling pattern of neutral glycolipids in the tors for mitogens such as PHA and concanavalin A (Con A) membranes of human KB-3 cells before or after treatment (50) as well as for myxoviruses such as influenza virus (59). with interferon (17), and only one difference is notable in Preincubation of interferon-sensitive cells with PHA blocks the labeling pattern of the glycoproteins (17). interferon action. Since PHA receptor sites are associated Cyclic AMP and the Interferon Response. The studies with intramembranous particles observed in freeze-fracture discussed above suggest that there are structural similari electron microscopic studies of the cell membrane (11. 39), ties between the TSH and interferon receptors. In view of interferon treatment should affect the appearance or distri these analogies, the possibility that Interferon action may bution of these intramembranous particles. In experiments be cyclic AMP-mediated was considered. In this regard, it is in which interferon-sensitive AKR/C~ cells are used, the well known that glycoprotein hormones stimulate the ade- number of intramembranous particles on both faces is, in nylate cyclase activity of cell surface membranes and that fact, found to be increased after interferon treatment for the resultant elevation in cellular cyclic AMP plays an 48 hr. For example, on the fracture face adjacent to the important role in linking the informational input of the cytoplasm, the particle number per unit area in the control effector at the cell surface to the transcriptional and trans- AKR/C cells is 337 particles/sq Â¿Â¿m;inthe interferon- lational machinery of the cell. treated cells, it increases to 665/sq /Â¿m(9, 17). Mouse interferon does induce an increase in cyclic AMP The increase in the number of intramembranous particles levels in interferon-sensitive mouse Ly cells, but not in is associated with a parallel increase in resistance to viral interferon-insensitive human KB-3 cells (42), despite the infection (9, 17). In addition, there is a coincident decrease ability of human KB-3 cells to bind mouse interferon to their in both intramembranous particles and resistance upon cell membrane. The interferon-induced elevation in cyclic removal of interferon (9, 17), and maximal particle density AMP precedes the induction of antiviral activity (42). Al is achieved after 8 hr of interferon treatment when maximal though interferon does not stimulate adenylate cyclase antiviral action is not yet evident (9,17). These data are thus activity in mouse Ly cell membranes, interferon does stim clearly indicative of a relationship between intramembra ulate adenylate cyclase activity in mouse thyroid cells (42). nous particle number and establishment of the antiviral These results cannot, however, be interpreted as an indi state and also suggest that the increased particle number cation that cyclic AMP mediates the induction of the anti may precede its establishment. viral state, since a previous observation indicates that dibutyryl cyclic adenosine 3':5'-monophosphate poten Interferon treatment of cells also appears to alter the accessibility of the oligosaccharide moiety of membrane tiates, but does not mimic, interferon activity (15). gangliosides to surface labeling procedures (Table 1). The In the case of interferon, the elevated cyclic AMP levels ability of interferon to alter the accessibility of membrane may be related to the formation of phosphorylated interme gangliosides to surface labeling procedures was initially diates demonstrated to exist (51, 52) in experiments in

4178 CANCER RESEARCH VOL. 38

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1978 American Association for Cancer Research. Interferon Receptor which the cytoplasm of interferon-treated cells is used to Tune imn study in vitro, RNA-dependent protein synthesis. These 6 10 1530 0 4 12 20 phosphorylated cytoplasmic intermediates may be related to cyclic AMP-dependent kinase activities which can affect thetranscriptional-translational processes induced by interferon. Effects of Interferon on Ion Fluxes across Cell Mem branes and the Relationship of These Changes to the Establishment of an Antiviral State. Current views of glycoprotein hormone interactions with cells invoke cyclic AMP as the "second" functional component of message transmission, i.e., the hormone is the "first message" and the cyclic AMP is the "second message." The observation that dibutyryl cyclic AMP potentiates, but does not mimic, interferon activity (see above) raises the possibility that cyclic AMP is not necessarily a "second message," but merely one of several messages responsive to interferon- induced changes in the cell membrane. The following observations in this regard should be noted. Similarities in receptor structure and function between interferon and glycoprotein hormones extend to the receptor structure and function of bacterial toxins such as cholera and tetanus toxins (19, 24, 25, 33). The primary effect of tetanus toxin is an alteration in neuronal transmission (29). Cholera toxin 100 i causes a dramatic loss of electrolytes and water through the intestinal epithelium (60), TSH increases the rate and Chart 4. TSH-stimulated TPMP' uptake by intact bovine thyroid cells (A), extent of iodide uptake by thyroid cells (64), and ouabain, trypsinized bovine thyroid cells (B). plasma membrane vesicles prepared the classic inhibitor of Na - and K -stimulated ATPase, from bovine thyroid cells (C), and TSH-stimulated TPMP' uptake in bovine blocks the antiviral action of Â¡nterferon (31). thyroid vesicles relative to TSH-stimulated adenylate cyclase activity in the Further, a recent report (18) shows that cultured thyroid same vesicles (D). \nA andS, the experiments were carried out as described cells accumulate the lipophilic cation triphenylmethylphos- in Ref. 18 with a cell protein concentration of reaction mixture (500 tig/Ã¬QQ fj.\). Where indicated. TSH and human chorionic gonadotropin were added phonium, indicating that there is an electrical potential at final concentrations of 100 nM (i.e., about 7 milliunits per 100 Â«Â¿Iofassay volume) and 100 HM. respectively, and carbonylcyanide-m-chlorophenylhy- (interior negative) across the plasma membrane (Chart drazone (CCCP) was used at a final concentration of 2.5 Â¿Â¿M(dashed 4).TSH stimulates the uptake of the lipophilic cation 3-fold, arrows). In C. vesicles were diluted into reaction mixtures containing 0.05 M and the proton conductor carbonylcyanide-m-chlorophen- NaCI, KCI. or Tris:HCI(pH 7.4), as described in Ref. 18, and the concentration ylhydrazone causes efflux of TPMP' accumulated in the of membrane protein was 115 ^g per 100 ^l of assay. In the experiments shown in D, vesicles suspended in 0.25 M sucrose containing 0.01 MTris:HCI presence or absence of TSH. The stimulatory effect of TSH (pH 7.4) were diluted 1:10 into medium containing 0.05 M NaCI:0.125 M on TPMP accumulation is not mimicked by human cho- sucrose:! mw MgCI2:1 rnw ATP:200 nm TSH; the final concentration of membrane protein was 115 /Â¿gPer 100 /il of assay. Incubations were rionic gonadotropin, a glycoprotein hormone with a similar performed in quadruplicate, and duplicate samples were evaluated for structure (the target organ of which is not the thyroid), and TPMP' uptake and for adenylate cyclase activity as described in Ref. 18. the effect is abolished when the TSH receptor activity of the cells is destroyed by treatment with trypsin (Chart 4). Analogous effects are observed with thyroid plasma mem vesicles have been used for these studies, since they allow brane vesicles which are essentially devoid of mitochondrial manipulation of ion gradients across the membrane in the and soluble enzyme activities. TSH-stimulated TPMP' up absence of cellular metabolic events. take in the vesicle preparations reaches a quasi-steady state Effect of Interferon on the Membrane Potential. Inter within 3 min; in contrast, TSH-stimulated adenylate cyclase feron at low concentrations can stimulate the uptake of activity is negligible during this period of time, becomes TPMP' in mouse Ly cell vesicles (Chart 5/4). However, this measurable after about 4 min, and is optimal after 12 to 15 hyperpolarization phenomenon is influenced by a host of min (Chart 4). factors, only some of which are currently delineated. At In sum, it would appear possible that another primary high interferon concentrations, for example, the interferon action of each of these agents is an effect on the membrane can cause a decrease in TPMP' uptake (Chart 5A) relative which results in altered ion fluxes across the membrane to vesicles treated with low doses of interferon. Also, and in altered electrochemical ion gradients vital to the prolonged preincubation of the vesicles with interferon can transport of sugars and amino acids into the cell. In addi cause a concentration of interferon which hyperpolarizes tion, it would appear that this effect is not necessarily the cell to become one which depolarizes it (Chart 56). mediated by cyclic AMP. Similar concentration-dependent effects have been noted Recent studies concerning the receptor structure and with TSH in physiological studies and more recently in our mechanism of action of interferon support the interpreta own continued studies of TSH effects on thyroid vesicles. tion that interferon affects the movement of ions across Another factor which can alter the response is the mode of membranes as a primary mode of action. Plasma membrane vesicle preparation. Temperature during interferon pre-

NOVEMBER 1978 4179

Chart 5. Effect of interferon on TPMP' uptake by plasma membrane vesicles pre pared from mouse Ly cells. Methodology and vesicle preparation are the same as in Chart 4. Vesicle preparations were the same in A and B\ conditions differed only in the preincubation with effector (B) before TPMP1 uptake was measured.

TIME Iminules) treatment, calcium concentration during vesicle prepara L cell vesicle KB cell vesicle tion, the stage of growth of the cells (log or confluent) from Control Interferon 1 1.30 which vesicles are prepared all appear to play a role in J a> determining the ultimate potential response. Nevertheless, TSH (data not shown) and cholera toxin (Chart 5,A and 0), X_ 0.65 Interferon Control 2 agents which also bind to the membranes of these cells, it- Â°* do not induce the hyperpolarization response noted with interferon. Effect of Interferon on Proton Permeability across Cell 0.0 ' 10 0 2 10 Membranes. When a pH gradient is artificially imposed across mouse Ly cell membrane vesicles ([H']^ > [H*]oul), TIME (minutes) Chart 6. The effect of interferon on ['4C]methylamine hydrochloride up the gradient dissipates faster when the membranes are first take by mouse Ly cell vesicles (A) and human KB-3 vesicles (B). The experiments were carried out with the use of vesicles prepared as described treated with interferon. This phenomenon has been assayed in Materials and Methods," except that instead of Tris-chloride, the vesicle with the use of methylamine uptake as a measure of internal preparation was in 20 mM of AMris(hydroxymethyl)methyl-2-aminoethanesul- pH. Amines can be used to study the hydrogen ion concen fonic acid (Calbiochem. San Diego, Calif.), titrated to pH 6.4 with KOH. For tration across membranes when the [H']â€ž,> [H â€¢¿],â€žâ€ž.since the experiments shown, 5 /il of vesicles (17 mg protein per ml) were suspended in 0.25 M sucrose containing 0.02 M/V-tris(nydroxymethyl)methyl- the membrane is permeable to the neutral species only and 2-aminomethanesulfonic acid:NaOH (pH 8.6) and 7 nmol [14C]methylamine hydrochloride (56 Ci/mol). Interferon treatment was at 0Â°for 30 min at 100 since the impermeable ionized species will be concentrated units/Ml prior to assay. Incubations were at 37Â°at the times indicated. on the more acidic side of the membrane (53). Uptake of ["CJmethylamine hydrochloride was measured by filtration as As seen in Chart 6/4. when membrane vesicles, isosmotic described in Materials and Methods." The base-line value was determined with sucrose and prepared in 20 mM A/-tris(hydroxy- by performing the same experiments after suspending the vesicles in 20 mM rV-tris(hydroxymethyl)methyl-2-aminomethanesulfonic acid at pH 6.4, i.e., methyl)methyl-2-aminoethanesulfonic acid at pH 6.4, are under conditions in which no gradients could form. resuspended in the same sucrose buffer at pH 8.5, the uptake of [14C]methylamine is 1.37 nmol/mg protein within vesicles from interferon-sensitive cells, but has no effect on 1 min and reaches a value of 0.87 nmol/mg protein by the vesicles from interferon-insensitive cells. end of 2 min. Between 2 and 10 min (data not shown), the Two final points concerning proton concentration and uptake gradually declines toward the base line established permeability in mouse Ly and human KB-3 cells or vesicles by experiments performed by resuspending the vesicles at should be made. First, when present in a buffered medium pH 6.4 where no pH gradient exists. at pH 7.2, mouse Ly cells accumulate methylamine, but do Different results are obtained when vesicles from mouse not take up dimethyloxazolidine-2,4-dione as measured by Ly cells are first treated with interferon and then resus either filtration or centrifugation of whole cells. In contrast, pended in the pH 8.5 buffer. The initial methylamine uptake human KB-3 cells accumulate dimethyloxazolidine-2,4- is significantly lower (0.87 nmol/mg protein) and, although dione, but do not accumulate methylamine. Dimethyloxa- at 2 min the uptake approaches that of the untreated zolidine-2,4-dione is a weak acid, the ionized form of which vesicles, a small but consistent difference remains (Chart is concentrated on the more basic side of a membrane. It 6/4). Between 2 and 10 min (data not shown), this small has been used to measure proton gradients when the difference is maintained as the values decline in parallel to internal pH of a cell or vesicle is more basic than the outside the base line. (53). The difference between mouse Ly and human KB-3 The effect of interferon seems specific, since neither TSH cells in their accumulation either of a weak acid or an amine nor cholera toxin depresses methylamine uptake by mouse at an external pH of 7.2 suggests that there is a significant Ly vesicles and since interferon does not have a similar difference in the internal pH of these 2 cell types. Whether effect on methylamine uptake by human KB-3 cell vesicles this difference is related to interferon sensitivity or to mode (Chart 65), i.e., interferon increases proton permeability in of action of the interferon is under investigation.

4180 CANCER RESEARCH VOL. 38

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1978 American Association for Cancer Research. Interferon Receptor revolve about changes in electrochemical ion gradients 3 distinct, although closely related, classes of proteins (the across the membrane and in membrane transport. The short neurotoxins containing 60 to 62 residues, the long possibility exists, therefore, that this common sequence on neurotoxins containing 71 to 74 residues, and the cytotox- the Â«subunits of the glycoprotein hormones is not only the ins). site of their membrane-active event, but also that their Unlike the actions of the pituitary glycoprotein hormones modification of membrane changes is similarly induced. and the nonapeptide hormones, the activity of the neuro Computer searches (32) have now identified similar se toxins and cytotoxins is not thought to involve the induction quence regions on neurotoxic and cytotoxic peptides found of cyclic AMP. Their biological activity involves hyperpolar- in cobra venom (Chart 10). This superfamily is composed of ization or depolarization of the cell membrane resulting

Fragment3 2 12345678901234567890123456S 789012M CC4 5 6 7 8 9 Glycoprotein hormonea chain TSH(bovine)LH P D G E f T QG aC K L K Â£ N (human)LH VQD EC C T L 0 Â£ N (pig)LHand TSH T: MQG E: CKLKEN (sheep)Cholera P N G O f T M QGP a NACKLKE toxin A1 fragmentGlycoproteinCholeratoxin A, chain ?ZP H /HAt GGK :B: PRCI hormone0 chain (First region) hCG(human)LH KCS ATLAVE EG vc TVNTT (human)TSH R E11IPLRPRCRP//P LRPWCHP / /l/ A / L A V E K EGR v CI T V W T T (human)(Second F C 1 P TiT : Y M T H /EY Rf1 :Y: THCLT/*T region) TSH(human)hCG RTVE PG LC VAPYF (human)LH F E S /RF LPG R: GVNPVV (human)Nonapeptide E S /RC L PGF R: GVDPVVG hormones (mammals)Arg-vasopressinLys-vasopressin YC QH K: (mammals)Ocytocin YC FQN RC G raffish)Vasotocin(mammals, Yc QN L: G (vertebrates)Mesotocin yc QN RG lungfishei).Isotocin(birds, reptiles,amphibians, v1c QN :/ G [Aspartocin(bony fishes) yC SN :/ G dogfish)Glumitocin(spiny YC NN :L G ...:.Valitocin(rays) YC SNo. : a G dogfish)Cytotoxins(spiny YIP ND :v: GN

Cytotoxincobra)Cytotoxin 1(029-050) (bandedEgyptian S T\K VKRGC VN K SALVK cobra)Cytotoxin4(029-050) (Mozambique K M\K 1 P V K R G C V :K NSALVK cobra)Cytotoxin1(029-050) (forest STA PVKRGC DV :KSSLLVK cobra)Cytotoxin1(029-050) (Mozambique P M\M IP V K R G C DV :KS S L L 1 K (ringhals)Cytotoxin1(030-051) PKP PI K R G C TOA SSLLVK â€¢¿TYC( N R G :K cobra)Long 2(030-051) (forest K K1S A ATA :K: SSLLVKK neurotoxins Longneurotoxinkrait)Long 1(035-056) (Formosanbanded R GtS V VE L G C AT s KPYEE cobra)Longneurotoxin 1(033-054) (forest R GfSAGAM CV/ELGC VAT :K V K P Y Â£Q cobra)Longneurotoxin 1(033-054) (king (RVDLGC AAT :/ V K P G V Â£ cobra)Longneurotoxin 1(032-053) (Cape R GtQ (RVDLGC AAT :K VKPGVN mamba)Longneurotoxin 1(033-054) (black R GtQ. (RVELGC AAT :K VKAGVE mamba)Longneurotoxin 1(033-054) (WestAfrican green R GtQ f R E E L G C k A T V K A G VG mamba)Shortneurotoxin 1(033-054) (Jameson's :K R GtH (RCâ€¢ VE L G A/iTG :K: VKTGVÂ£V neurotoxins Câ€¢1 1 E Ã±G Shortmamba)Short neurotoxin 1(030-049) (black R G1H i / E a Gc' -- K K P GVGV cobra}Shortneurotoxin 1(031-050) {Cape R G1D G-- K K G VG 1 1 E R G C : s snake)Shortneurotoxin 1(sel.pos.) (broad-bandedbluesea F R G1H G-- V K P G 1 N â€¢¿R/C' E R G :T neurotoxin 1(030-049) (commonseasnake) R G1G G --T :a: KGV K P G 1 Prolactins Prolactin001-012(bovine)Prolactin PV N PGBCQ (sheep)Prolactin001-012 TPV :NGPGDCO (pig)CytochromesCytochrome001-012 L P11 :s: G0C A VN C K G K K 1 F D VlG MK - K G K K 1 F s a HT V E K (pig)FerredoxinsFerredoxinC001-022 D VLG F:-KCKKV VQK : A a C H T V Â£K lM DV V QKA : Aa: BGCHTVZ

(AHIH V 1 S D E v KCG A c ST( p T A / E E G E 1 CA 1 T D E : i s c GA c A A E t.: p v E A / H f G T C (011-028) tClostridÃ¬umKidi-uricii1 Y V 1 N f AC:: i s c G/AcSearchD P Â£l:: p v^3D A / S u G DS

Chart 10. Proteins which contain sequences resembling the test sequences, i.e., the region of sequence similarity between the a subunit of the glycoprotein hormones and the Q chain of cholera toxin. As noted in (31), in each case the protein exhibits a relationship which is in the top 0.1% of the population examined, i.e., a population exceeding 100,000 known proteins or fragments of known proteins. Numbers at the top of the chart are arbitrary; they are used simply as references for comparing the different sequences presented. Numbers in parentheses next to each protein or peptide fragment refer to its actual placement within the native protein or peptide. Residues in boldface type or boldface italic type are common to the test sequences or can be considered isomorphic sequence substitutions. The isomorphic substitutions considered to exist are as follows: (a) B, N, D, E, O, Z, K, and Ft (charged residues); (b) F and Y (phenylalanine and tyrosine); and (c) A. L, I. V, G, and M (hydrophobic residues). Intrafamily homologÃasare preserved (32, 34). Residues in normal italic type define groups of sequences common to more than one group of the several protein families.

NOVEMBER 1978 4183

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1978 American Association for Cancer Research. E. F. Grollman et al. from a change in membrane permeability and the conse 21. Kanter, J.N.. Carter, T. P..and Katzen, H. M. Lipolytic Action of Cholera quent alteration of transport across the membrane. These Toxin on Fat Cells. J. Biol. Chem..257: 7610-7619, 1976. 22. Knight, E., Jr., and Korant, B. D. A Cell Surface Alteration in Mouse L effects are at least qualitatively similar to the peripheral Cells Induced by Interferon. Biochem. Biophys. Res. Commun., 74: 707- effects of tetanus toxin (61), which has recently been shown 713.1977. 23. Kohn, L. D. Characterization of the Thyrotropin Receptor and Its Involve to share certain biochemical characteristics and actions of ment in Graves' Disease. In: Quagliariello (ed.), Horizons in Biochemistry TSH (33) and to have thyroid-stimulating ability in vivo (19). and Biophysics, Vol. 3, pp. 123-163. Reading. Mass.: Addison-Wesley In sum, for each of these effectors (toxin, glycoprotein Publishing Co., 1977. 24. Kohn, L. D. Relationships in the Structure and Function of Cell Surface hormone, or interferon) some element in their action may Receptors for Glycoprotein Hormones, Bacterial Toxins, and Interferon. well be an alteration in the physical and electrical state of Ann. Rept. Med. Chem. 27: 211-222, 1977. the membrane which is independent of the activation of 25. Kohn, L. D. Relationships in the Structure and Function of Receptors for Glycoprotein Hormones, Bacterial Toxins, and Interferon. In: P. Cuatre adenylate cyclase. The relationship of this effect to the casas and M. F. Greaves (eds.), Receptors and Recognition, Series A, bioactivity of these agents will certainly be a common and Vol. 5, pp. 134-212. London: Chapman & Hall, 1978. active thread in studies of their mechanism of action. It is 26. Kohn, L. D., Friedman, R. M.. Holmes, J. M., and Lee, G. Use of Thyrotropin and Cholera Toxin to Probe the Mechanism by Which hoped that they will contribute to our understanding of how Interferon Initiates Its Antiviral Activity. Proc. Nati. Acad. Sei. U. S., 73: diverse hormones effect the oncogenic state. 3695-3699, 1976. 27. Kohn, L. D., Lee, G., Grollman, E. F., Ledley. F. D.. Mullin, B. R., Friedman, R. M., Meldolesi, M. F., and Aloj, S. M. Membrane Glycolipids References and Their Relationship to the Structure and Function of Cell Surface Receptors for Glycoprotein Hormones. Bacterial Toxins, and Interferon. 1. Aloj. S. M.. Kohn, L. D., Lee. G.. and Meldolesi. M. F. The Binding of In: R. E. Harmon (ed.), Cell Surface Carbohydrate Chemistry, pp. 103- Thyrotropin to Liposomes Containing Gangliosides. Biochem. Biophys. 133. New York: Academic Press, Inc., 1978. Res. Commun., 74: 1053-1059, 1977. 28. Kohn, L. D.,and Winand, R. J. Relationship of Thyrotropin to Exophthal- 2. Aloj, S. M.. Lee, G.. Consiglio, E., Formisano, S., Minton. A. 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Cancer Res 1978;38:4172-4185.

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