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Tissue-specific expression of Na,K-ATPase β-subunit Does β2 expression correlate with tumorigenesis?
Volume 289, number I, 8-10 FEBS 10095 %r 1991 Federation of European Biochemical Societies OOlJ5793/91/S3.50 ADONIS
September
199 1
0014579391007995
Tissue-specific expression of Na,K-ATPase Does p2 expression correlate N.S. Akopyanz’,
P-subunit
with tumorigenesis?
N.E. Broude’, E.P. Bekman’.
E-0. Marzen
and E.D. Sverdlov’
‘Shen~wkitl Institute of Bioorgmic Chemistry. USSR Acdetqv of Sciences. MOKOW. USSR. ‘hutitirte of Lywolog_v, Sibesim Depurtnmt of USSR Acc~dcr~~yof Sciences, Irkutsk, USSR and “All- Uuiorl Scieutifc Researci~ hstitlrte qf Biotechnology, USSR Mitlisir_v qf’ Medical ad Biologicd hdrstr~~. MOSCOWUSSR Received 24 June 1991 We have found a substantial decrease in the level of Na.K-ATPascBt-subunit in nude culture. cell line was the
mRNA in xcnografts of human renal. lung hepatoccllular carcinomas mice as compared with corresponding normal tissues, as well as in the ncuroblastoma cell line as compared with the neuron primary cell The level ofbl mRNA is dccrcascd in kidney and lung tumor cells, but is unchanged in hcpatocellular carcinoma. In the ncuroblastoma the level of /?I subunit mRNA was found to be higher then in neuron primary cell culture. The level of CLI mRNA in investigated tumors same as in normal tissues. These results may give evidence of the involvement ofp2-subunil in the process of tumorigenesis as was shown for some other adhesion molecules. Na.K-ATPase:
P-subunit:
Adhesion
I. INTRODUCTION Na,K-ATPase in the plasma membrane of animal cells is responsible for tfle active transport of Na’ and K’ ions against their electrochemical gradients by means of energy created by ATP hydrolysis. The enzyme consists of 2 subunits, a and p. The a-subunit is a catalytic one. the functional rote of the P-subunit remains a matter of discussion. It was supposed that the /?-subunit serves as an anchor for the insertion into the membrane of the a-subunit [I]. The discovery of the fl2-isoform as an adhesion molecule on gfia (AMOG) which was found in astrocytes, clearly showed the participation of the P2-subunit in interccffufar interactions. that is in specific neuron-gfiaf contacts [2.3]. By using the tcchniquc of mofccufar cloning. scvcraf laboratories have discovered gene famifics related to the a- and P-subunit of Na.K-ATPase [4-61. We [7,8] and otflers [9.!0] have shown the tissue-specific manner of expression ofdiffcrent gents that cncodc isoforms ofthe Na.K-ATPuse a-subunit. Throughout thcsc investigations WCstudied the level of mRNAs encoding the a- and p-isoforms of Na,KATPasc in normal ccfls and tumor ccffs. WC hove found a dccrcascd of/32 mRNA in all tumor cclfs studiccf,
molecule: Tumorigcncsis
2. MATERIALS
AND METHODS
All tissues wcrc obtained by autopsy 4-6 h after death from 25-40year-old donors who died in accidents. Human tumors transplanted into nude mice wcrc obtained from Dr E. Rcvasova. Total cellular RNA was isolated from I g of lissuc or from SxlOb- IO’ cells according to the method of Fcranrisco ct al. [I I]. The level of Y- and /?-subunit isoform specific mRNAs was dctcrmined by USC of PCR-amplitication. PCR was used in a semi-quantitative modification: a cDNA first strand was synthesized using I fig of total RNA and a random primer. This cDNA WIS then llscd in all subscqucnt amplilication reactions by adding corresponding spccilic primers. l/IO of the cDNA was amplified in a Pcrkin-Elmer-Cctus thermal cyclcr: 94°C. 30 s. 60°C. 30 s. 72°C. 30 s. 36 cycles. 111control cspcrimcnls WChave shown that under thcsc conditions there is ;I proportional relationship bctwccn RNA concentration and the slrcngth of the signal (by taking pictures of agarosc gels stained by EtBr in W-light). The following primers wcrc used iIs LXI -specific: S- AAGAACTGCCTGGTGAAGAACCTGGAC-3’ and 5’.AGCTGAAGTCTTGTCAAAAGAGAC-3’; ;IS /II-spccilic: Y-ACTGAAATTTCCTTTCGTCCTAAT-3’ and 5’-ATCACTGGGTAAGTCTCCA-3’: and as /3!-specific: S’-CTTGATGTCATTGTCAATGTCAGT-3’ and STCGATGTTGCCGTTGGCGGGGAAC-3’. AS iI control WC amplilird ;I f’ragmrnt of hunlan p-actin gene. The amplilication products wrc milyzcd by gel clcctrophorcsis hi a 2% ;lpilrOSC pcl containing 0.S mg/mI cthidium bromide. The pictures from ~hc gels wcrc taken in UV-lipbt using Polaroid 065 film and ncgativcs wcrc scannedon the Apple xxnncr (AMS. England).
3.
RESULTS
AND DISCUSSION
Figure I shows the rcsufts of the amplification of Nit.K-ATf’asc at. PI- ilnd P2-specific n~RNA frag mcnts in normal Il\lnliln kidney. Iuu~. fivcr tissues and corresponding tumor ccfls (other isoforms ot’ Na.K-
8
Volume 289, number 1
FEBS LETTERS
September 1991
._C
;3 d
Act in Fig. I. PCR analysis of Na.K-ATPase mRNAs distribution in normal and tumor human tissues. The lcngh of amplified fragments is indicated to the right. Lanes 1.2.3.4.5.6. PCR from RNA isolated from human kidney normal tissues. renal tumor. lung normal tissue. lung tumor. liver normal tissue and hepatic tumor. rcspcctively. Lane 7. markers pUC19/Swt3A.
ATPase a-subunit are not expressed or are expressed in very small amounts in these tissues [12]). Figure 2 illustrates the results of the analysis of expression of the isoforms in the primary culture of neurons compared with the neuroblastoma cells, line lMR32 (neuroblastlike (N) and fibroblast-like (S) [ 12.131). As a control the results of the amplification of human j%cytoplasmic actin mRNA are given. These show that all probes contained a roughly equal amount of RNA and also demonstrate the standard conditions of amplification. Figure 3 presents the quantitative estimation of results of Figs 1 and 2. It follows from Figs 1 and 2 that the level of Na,KATPase oil mRNA does not change significantly when normal and tumor cells are compared. The level of bl mRNA in renal and lung cell carcinomas was decreased compared with normal tissues, but in hepatocellular tissues it was the same as in normal tissues. The level of/?1 in the ncuroblastoma cell line was higher than in neurons (Fig. 2). In contrast we observed a decreased level of/32 mRNA in all tumors investigated (Fig. 1 and 2). The decrease in the level of the p2 mRNA in cancer cells as compared with normal tissues deserves special attention bccausc rcccnt results give evidence that differcnt adhesion molcculcs play an important role in processes of tumorigencsis [14-161. Recently it was shown that the P2-subunit of Na,K-ATPase is an adhcsion molcculc on glia (AMOG) [2,3]. The rcsu1t.s of our investigation give cvidcncc that this molcculc may play sonic role in tumor formation. Howcvcr. the possibility that the cffcct obscrvcd is only an accompanying one cannot bc cxcludcd.
Fig. 2. PCR analysis ot’ Na.K-ATPase p mRNAs distribution in human neurons and neuroblastoma IMR32 cells. Lanes 1.2.3, PCR from RNA of neurons. neuroblastoma cells (N-type). neuroblastoma cells (S-type), respectively. Lane 4, size markers, pUCI9/.%7r,3A.
Numerous studies have noted the similarity in amino acid seqcences between cell surface glycoproteins which are supposed to be involved in the neoplasia formation. Therefore we have searched in a protein sequence data bank (National Biological Research Foundation Protein Identification Resource (USA) release number 22) for proteins which share homology with P-subunits of Na,K-ATPase. Our preliminary results revealed two fragments homologous with different members of the Ig-superfamily (data not shown). Therefore the possibility that P-subunits and the proteins of this superfamily have a common origin but diverged early in evolution cannot be excluded. The results of the homology search will be published elsewhere. If AMOGIfl2 is involved in the process of transformation the question then arises as to whether it exerts its function as part of Na.K-ATPase or apart from it. Despite the fact that our results do not enable us to make final conclusions they give some evidence to suggest that the P-subunit of Na,K-ATPase and 82 in parA
Fig. 3. Histograms quantifying the lcvcl of Na,K-ATPesc mRNAs in diffcrcnt huwn cells. (A) Corresponds tp the results prcscntcd in Fig. I. (B) Corresponds to the results prcscntcd in Fig. 2. t1.u. arbitrary units.
9
Volume 289, number 1
FEES LETTERS
titular may function apart from the a-subunit. In fact, Figs 1 and 2 show that in tumor cells there is no balance in the content of the 01and p mRNAs in comparison with normal tissues: while the level of al mRNA is relatively constant, both /?l and p2 mRNAs are significantly reduced in renal and lung tumors. When different types of cells were compared [9], cases were observed where there was no co-regulation, at least at the RNA level, of a- and p-subunits. The authors suppose that if the synthesis of the Q- and p-subunits of Na,K-ATPase is coordinately regulated, then the mechanism is dependent upon cell ‘lype [9]. Marxer et al. [17] have not found, Wa,K-ATPase activity in the brush border *membrane of the distal colon although a B-subunit of Na,K-ATPase or P-like protein has been detected. The proposal regarding the possibility of separate functions for P-subunits was also put forward by Good et al. [18] who found a nervous system-specific isoform of p-subunit, 83, expressed during early development of X0lopus luevis. In summary, the rcsuits obtained allow the formulation of several questions of general and particular value: (i) is there a causal-consequence relation between 82 expression and tumorigenesis? (ii) what molecular mechanisms provoke a decrease in the level of p2 in cases of tumor formation? 2nd (iii) do P-subunits exert other functions apart from ion transport? One can thus hope that answering these questions will shed light on the mechanism of Na,K-ATPase functioning as weil as on the mechanisms of tumorigenesis. A~k,lo,~‘k~~lgp,,~errrs~ We thank Dr E.S. Rcvasova for generously providing us with samples of human tumors. WC also thank Dr Yu. Balabanov for the neuron primary cell culture and Dr N. Vinogradova for neuroblastoma cells culti*,stion.
September 1991
REFERENCES [I] Zamofing,D., Rossier. B.C. and Geering, K. (1988) J. Membr. Biol. 104. 69-79. [2] Pagliusi, S., Antonicek. H., Gloor, S., Frank, R., Moos, M. and Schachner. M. (!989) J. Neurosci. Res. 22, I 13-l 19. [3] Gloor. S.. Antonicek. I-i.. Sweadner, K.J.. Pagliusi, S., Frank. R.. Moos, M. and Schachner, M. (1990) J. Cell. Biol. I IO, 165-174. [4] Sverdlov. E.D.. Monastyrskaya, G.S.. Broude, N.E.. Ushkaryov, Yz. A., Allikmets, R.L.. Melkov, A.M., Smimov. Yu. V.. Malyshev, L.V.. Dulubova. I.E.,Petrukhin, K.E..Grishin,A.V., Kijatkin, N.I.. Kostina, MB., Sverdlov. B.E.. Modyanov, N.N. and Ovchinnikov. Yu. A. (1987) FEBS Lett. 217. 275-278. [S] Shull. G.E., Greeb, J. and Lingrel, J.B. (1986) Biochemistry 25. 8125-8132. [6] Shull. M.M. and Lingrel, J.B. (1987) Proc. Natl. Acad. Sci. USA 84. 40394043. [7] Sverdlov. E.D., Akopyanz, N.S., Pctrukhin. K.E.. Broude. N.E., Monastyrskaya. G.S. and Modyanov. N.N. (1988) FEBS Lett. 239. 65-68. [P] Broude. N.E.. Modvanov. N.N., Monastyrskava. G.S. and Sverdlov, E.D. (1989) FEBS Lett. 257. l-9. _ _ 191Young. R.M. and Lingrel. J.B. (1987) Biochem. Biophys. Res. Commun. 145, 52-58. 1101Emanuel, J.R., Garetz, S., Stone. S. and Levenson. R. (1987) Proc. Natl. Acad. Sci. USA 53, 9030-9034. [ill Feramisco, J.R., Helfman. D.M., Smart. J.E.. Burridge, K. and Thomas, G.P. (1982) J. Biol. Chem. 257. 11024-l 1031. iI21 Akopyanz. N.S.. Broude, N.E., Vinogradova. N.G., Balabanov, Yu. A.. Monastyrskaya. G.S. and Sverdlov. E.D. (1991) in: The Sodium Pump: Recent Developments, The Rockefel!er University Press. in press. [I31 Ciccarone, V.. Speng!er. B.A.. Meyers, M.B.. Bicdler. J.L. and Ross. R.A. (1990) Cancer !?es. 49. 219-225. Giawotti, F.G. and Ruoslahti (IY90) Cell 60. 849-859. t::; Fearon,E.R..Cho, K.R.. Nigro. J.M.. Kern.S.E..Simons. J.W., Ruppert. J.M., Hamilton. SK.. Preisinger, AC.. Thomas. G.. Kinzlcr, K.W. and Vogelstein, B. (1990) Science 247. 49-56. [I61 Inghirami. G.. Grignani. F.. Sternas. L., Lombardi. L., Knowles, D.M. and Dalla-Favera. R. (1990) Science 250. 682-686. [I71 Msrxer. A., Stieger. B.. Quaroni. A.. Kashgdrian, M. and Hauri. H.-P. (1989) J. Cell. Biol. 109. 1057-1069. [IS] Gcod, P.J., Richter. K. and David, I. (1990) Proc. Natl. Acad. Sci. USA 87. 9082-9092.
September
199 1
0014579391007995
Tissue-specific expression of Na,K-ATPase Does p2 expression correlate N.S. Akopyanz’,
P-subunit
with tumorigenesis?
N.E. Broude’, E.P. Bekman’.
E-0. Marzen
and E.D. Sverdlov’
‘Shen~wkitl Institute of Bioorgmic Chemistry. USSR Acdetqv of Sciences. MOKOW. USSR. ‘hutitirte of Lywolog_v, Sibesim Depurtnmt of USSR Acc~dcr~~yof Sciences, Irkutsk, USSR and “All- Uuiorl Scieutifc Researci~ hstitlrte qf Biotechnology, USSR Mitlisir_v qf’ Medical ad Biologicd hdrstr~~. MOSCOWUSSR Received 24 June 1991 We have found a substantial decrease in the level of Na.K-ATPascBt-subunit in nude culture. cell line was the
mRNA in xcnografts of human renal. lung hepatoccllular carcinomas mice as compared with corresponding normal tissues, as well as in the ncuroblastoma cell line as compared with the neuron primary cell The level ofbl mRNA is dccrcascd in kidney and lung tumor cells, but is unchanged in hcpatocellular carcinoma. In the ncuroblastoma the level of /?I subunit mRNA was found to be higher then in neuron primary cell culture. The level of CLI mRNA in investigated tumors same as in normal tissues. These results may give evidence of the involvement ofp2-subunil in the process of tumorigenesis as was shown for some other adhesion molecules. Na.K-ATPase:
P-subunit:
Adhesion
I. INTRODUCTION Na,K-ATPase in the plasma membrane of animal cells is responsible for tfle active transport of Na’ and K’ ions against their electrochemical gradients by means of energy created by ATP hydrolysis. The enzyme consists of 2 subunits, a and p. The a-subunit is a catalytic one. the functional rote of the P-subunit remains a matter of discussion. It was supposed that the /?-subunit serves as an anchor for the insertion into the membrane of the a-subunit [I]. The discovery of the fl2-isoform as an adhesion molecule on gfia (AMOG) which was found in astrocytes, clearly showed the participation of the P2-subunit in interccffufar interactions. that is in specific neuron-gfiaf contacts [2.3]. By using the tcchniquc of mofccufar cloning. scvcraf laboratories have discovered gene famifics related to the a- and P-subunit of Na.K-ATPase [4-61. We [7,8] and otflers [9.!0] have shown the tissue-specific manner of expression ofdiffcrent gents that cncodc isoforms ofthe Na.K-ATPuse a-subunit. Throughout thcsc investigations WCstudied the level of mRNAs encoding the a- and p-isoforms of Na,KATPasc in normal ccfls and tumor ccffs. WC hove found a dccrcascd of/32 mRNA in all tumor cclfs studiccf,
molecule: Tumorigcncsis
2. MATERIALS
AND METHODS
All tissues wcrc obtained by autopsy 4-6 h after death from 25-40year-old donors who died in accidents. Human tumors transplanted into nude mice wcrc obtained from Dr E. Rcvasova. Total cellular RNA was isolated from I g of lissuc or from SxlOb- IO’ cells according to the method of Fcranrisco ct al. [I I]. The level of Y- and /?-subunit isoform specific mRNAs was dctcrmined by USC of PCR-amplitication. PCR was used in a semi-quantitative modification: a cDNA first strand was synthesized using I fig of total RNA and a random primer. This cDNA WIS then llscd in all subscqucnt amplilication reactions by adding corresponding spccilic primers. l/IO of the cDNA was amplified in a Pcrkin-Elmer-Cctus thermal cyclcr: 94°C. 30 s. 60°C. 30 s. 72°C. 30 s. 36 cycles. 111control cspcrimcnls WChave shown that under thcsc conditions there is ;I proportional relationship bctwccn RNA concentration and the slrcngth of the signal (by taking pictures of agarosc gels stained by EtBr in W-light). The following primers wcrc used iIs LXI -specific: S- AAGAACTGCCTGGTGAAGAACCTGGAC-3’ and 5’.AGCTGAAGTCTTGTCAAAAGAGAC-3’; ;IS /II-spccilic: Y-ACTGAAATTTCCTTTCGTCCTAAT-3’ and 5’-ATCACTGGGTAAGTCTCCA-3’: and as /3!-specific: S’-CTTGATGTCATTGTCAATGTCAGT-3’ and STCGATGTTGCCGTTGGCGGGGAAC-3’. AS iI control WC amplilird ;I f’ragmrnt of hunlan p-actin gene. The amplilication products wrc milyzcd by gel clcctrophorcsis hi a 2% ;lpilrOSC pcl containing 0.S mg/mI cthidium bromide. The pictures from ~hc gels wcrc taken in UV-lipbt using Polaroid 065 film and ncgativcs wcrc scannedon the Apple xxnncr (AMS. England).
3.
RESULTS
AND DISCUSSION
Figure I shows the rcsufts of the amplification of Nit.K-ATf’asc at. PI- ilnd P2-specific n~RNA frag mcnts in normal Il\lnliln kidney. Iuu~. fivcr tissues and corresponding tumor ccfls (other isoforms ot’ Na.K-
8
Volume 289, number 1
FEBS LETTERS
September 1991
._C
;3 d
Act in Fig. I. PCR analysis of Na.K-ATPase mRNAs distribution in normal and tumor human tissues. The lcngh of amplified fragments is indicated to the right. Lanes 1.2.3.4.5.6. PCR from RNA isolated from human kidney normal tissues. renal tumor. lung normal tissue. lung tumor. liver normal tissue and hepatic tumor. rcspcctively. Lane 7. markers pUC19/Swt3A.
ATPase a-subunit are not expressed or are expressed in very small amounts in these tissues [12]). Figure 2 illustrates the results of the analysis of expression of the isoforms in the primary culture of neurons compared with the neuroblastoma cells, line lMR32 (neuroblastlike (N) and fibroblast-like (S) [ 12.131). As a control the results of the amplification of human j%cytoplasmic actin mRNA are given. These show that all probes contained a roughly equal amount of RNA and also demonstrate the standard conditions of amplification. Figure 3 presents the quantitative estimation of results of Figs 1 and 2. It follows from Figs 1 and 2 that the level of Na,KATPase oil mRNA does not change significantly when normal and tumor cells are compared. The level of bl mRNA in renal and lung cell carcinomas was decreased compared with normal tissues, but in hepatocellular tissues it was the same as in normal tissues. The level of/?1 in the ncuroblastoma cell line was higher than in neurons (Fig. 2). In contrast we observed a decreased level of/32 mRNA in all tumors investigated (Fig. 1 and 2). The decrease in the level of the p2 mRNA in cancer cells as compared with normal tissues deserves special attention bccausc rcccnt results give evidence that differcnt adhesion molcculcs play an important role in processes of tumorigencsis [14-161. Recently it was shown that the P2-subunit of Na,K-ATPase is an adhcsion molcculc on glia (AMOG) [2,3]. The rcsu1t.s of our investigation give cvidcncc that this molcculc may play sonic role in tumor formation. Howcvcr. the possibility that the cffcct obscrvcd is only an accompanying one cannot bc cxcludcd.
Fig. 2. PCR analysis ot’ Na.K-ATPase p mRNAs distribution in human neurons and neuroblastoma IMR32 cells. Lanes 1.2.3, PCR from RNA of neurons. neuroblastoma cells (N-type). neuroblastoma cells (S-type), respectively. Lane 4, size markers, pUCI9/.%7r,3A.
Numerous studies have noted the similarity in amino acid seqcences between cell surface glycoproteins which are supposed to be involved in the neoplasia formation. Therefore we have searched in a protein sequence data bank (National Biological Research Foundation Protein Identification Resource (USA) release number 22) for proteins which share homology with P-subunits of Na,K-ATPase. Our preliminary results revealed two fragments homologous with different members of the Ig-superfamily (data not shown). Therefore the possibility that P-subunits and the proteins of this superfamily have a common origin but diverged early in evolution cannot be excluded. The results of the homology search will be published elsewhere. If AMOGIfl2 is involved in the process of transformation the question then arises as to whether it exerts its function as part of Na.K-ATPase or apart from it. Despite the fact that our results do not enable us to make final conclusions they give some evidence to suggest that the P-subunit of Na,K-ATPase and 82 in parA
Fig. 3. Histograms quantifying the lcvcl of Na,K-ATPesc mRNAs in diffcrcnt huwn cells. (A) Corresponds tp the results prcscntcd in Fig. I. (B) Corresponds to the results prcscntcd in Fig. 2. t1.u. arbitrary units.
9
Volume 289, number 1
FEES LETTERS
titular may function apart from the a-subunit. In fact, Figs 1 and 2 show that in tumor cells there is no balance in the content of the 01and p mRNAs in comparison with normal tissues: while the level of al mRNA is relatively constant, both /?l and p2 mRNAs are significantly reduced in renal and lung tumors. When different types of cells were compared [9], cases were observed where there was no co-regulation, at least at the RNA level, of a- and p-subunits. The authors suppose that if the synthesis of the Q- and p-subunits of Na,K-ATPase is coordinately regulated, then the mechanism is dependent upon cell ‘lype [9]. Marxer et al. [17] have not found, Wa,K-ATPase activity in the brush border *membrane of the distal colon although a B-subunit of Na,K-ATPase or P-like protein has been detected. The proposal regarding the possibility of separate functions for P-subunits was also put forward by Good et al. [18] who found a nervous system-specific isoform of p-subunit, 83, expressed during early development of X0lopus luevis. In summary, the rcsuits obtained allow the formulation of several questions of general and particular value: (i) is there a causal-consequence relation between 82 expression and tumorigenesis? (ii) what molecular mechanisms provoke a decrease in the level of p2 in cases of tumor formation? 2nd (iii) do P-subunits exert other functions apart from ion transport? One can thus hope that answering these questions will shed light on the mechanism of Na,K-ATPase functioning as weil as on the mechanisms of tumorigenesis. A~k,lo,~‘k~~lgp,,~errrs~ We thank Dr E.S. Rcvasova for generously providing us with samples of human tumors. WC also thank Dr Yu. Balabanov for the neuron primary cell culture and Dr N. Vinogradova for neuroblastoma cells culti*,stion.
September 1991
REFERENCES [I] Zamofing,D., Rossier. B.C. and Geering, K. (1988) J. Membr. Biol. 104. 69-79. [2] Pagliusi, S., Antonicek. H., Gloor, S., Frank, R., Moos, M. and Schachner. M. (!989) J. Neurosci. Res. 22, I 13-l 19. [3] Gloor. S.. Antonicek. I-i.. Sweadner, K.J.. Pagliusi, S., Frank. R.. Moos, M. and Schachner, M. (1990) J. Cell. Biol. I IO, 165-174. [4] Sverdlov. E.D.. Monastyrskaya, G.S.. Broude, N.E.. Ushkaryov, Yz. A., Allikmets, R.L.. Melkov, A.M., Smimov. Yu. V.. Malyshev, L.V.. Dulubova. I.E.,Petrukhin, K.E..Grishin,A.V., Kijatkin, N.I.. Kostina, MB., Sverdlov. B.E.. Modyanov, N.N. and Ovchinnikov. Yu. A. (1987) FEBS Lett. 217. 275-278. [S] Shull. G.E., Greeb, J. and Lingrel, J.B. (1986) Biochemistry 25. 8125-8132. [6] Shull. M.M. and Lingrel, J.B. (1987) Proc. Natl. Acad. Sci. USA 84. 40394043. [7] Sverdlov. E.D., Akopyanz, N.S., Pctrukhin. K.E.. Broude. N.E., Monastyrskaya. G.S. and Modyanov. N.N. (1988) FEBS Lett. 239. 65-68. [P] Broude. N.E.. Modvanov. N.N., Monastyrskava. G.S. and Sverdlov, E.D. (1989) FEBS Lett. 257. l-9. _ _ 191Young. R.M. and Lingrel. J.B. (1987) Biochem. Biophys. Res. Commun. 145, 52-58. 1101Emanuel, J.R., Garetz, S., Stone. S. and Levenson. R. (1987) Proc. Natl. Acad. Sci. USA 53, 9030-9034. [ill Feramisco, J.R., Helfman. D.M., Smart. J.E.. Burridge, K. and Thomas, G.P. (1982) J. Biol. Chem. 257. 11024-l 1031. iI21 Akopyanz. N.S.. Broude, N.E., Vinogradova. N.G., Balabanov, Yu. A.. Monastyrskaya. G.S. and Sverdlov. E.D. (1991) in: The Sodium Pump: Recent Developments, The Rockefel!er University Press. in press. [I31 Ciccarone, V.. Speng!er. B.A.. Meyers, M.B.. Bicdler. J.L. and Ross. R.A. (1990) Cancer !?es. 49. 219-225. Giawotti, F.G. and Ruoslahti (IY90) Cell 60. 849-859. t::; Fearon,E.R..Cho, K.R.. Nigro. J.M.. Kern.S.E..Simons. J.W., Ruppert. J.M., Hamilton. SK.. Preisinger, AC.. Thomas. G.. Kinzlcr, K.W. and Vogelstein, B. (1990) Science 247. 49-56. [I61 Inghirami. G.. Grignani. F.. Sternas. L., Lombardi. L., Knowles, D.M. and Dalla-Favera. R. (1990) Science 250. 682-686. [I71 Msrxer. A., Stieger. B.. Quaroni. A.. Kashgdrian, M. and Hauri. H.-P. (1989) J. Cell. Biol. 109. 1057-1069. [IS] Gcod, P.J., Richter. K. and David, I. (1990) Proc. Natl. Acad. Sci. USA 87. 9082-9092.