Restoration of Glucagon Responsiveness In

Proc. Nati Acad. Sci. USA Vol. 78, No. 1, pp. 182-186, January 1981 Biochemistry

Restoration of glucagon responsiveness in spontaneously transformed rat hepatocytes (RL-PR-C) by fusion with normal progenitor cells and rat liver plasma membranes glucagon receptors/adenylate cyclase/polyethylene glycol) THOMAS M. REILLY AND MELVIN BLECHER* Department of Biochemistry, Georgetown University Medical Center, Washington, D.C. 20007 Communicated by Roscoe 0. Brady, October 1, 1980

ABSTRACT Spontaneously transformed RL-PR-C rat hepa- remain stable culturally and karyotypically, during which time tocytes, unlike their normal differentiated progenitor cells, are they display a wide variety of liver functions, including the pro- insensitive to glucagon, although seemingly possessing large num- duction of many specific liver enzymes and structural proteins bers of glucagon receptors and although retaining a guanyl nucleotide regulatory protein-adenylate cyclase [ATP pyrophos- (7) and responsiveness to insulin (8), cholera toxin (9), cate- phate-lyase (cyclizing), EC 4.6.1.1] system that responds to cholamines (10), and glucagon (11). After this stable period- catecholamines, cholera toxin, and fluoride ions. Biochemical fu- i.e., between 200 and 300 population doublings-the cells sions between normal RL-PR-C hepatocytes or purified rat liver spontaneously transform and acquire the cultural and chro- plasma membranes (whose adenylate cyclases were previously ir- mosomal characteristics ofcells transformed by artificial means, reversibly inactivated with N-ethylmaleimide) with spontaneously including loss of contact inhibition and in iso- transformed hepatocytes produced hybrids whose basal and flu- tumorigenicity oride-stimulated adenylate cyclase activities reflected those of the genic animals (12), and also become insensitive to glucagon (11). parental transformed cells but that now responded to glucagon. We considered that the basis for this latter defect is unlikely to Using cholera toxin-catalyzed ADP-ribosylation of transformed be due to an alteration in the catalytic moiety of the adenylate hepatocytes to mark their guanyl nucleotide regulatory protein, cyclase, because transformed RL-PR-C cells readily produce fusing such cells with N-ethylmaleimide-treated normal hepato- 3',5'-cyclic AMP (cAMP) in response to catecholamines, chol- cytes, and examining glucagon stimulation of adenylate cyclase era toxin, and fluoride ions (10). Additionally, these cells bind activity in fusion hybrids produced results suggesting that the regulatory protein of the transformed cells is functionally normal. In large amounts ofglucagon (10, 11). We reasoned, therefore, that fusion experiments between N-ethylmaleimide-treated hepato- it might be possible to identify the lesion in transformed he- cytes and pigeon erythrocytes, we found that normal, but not patocytes by transferring to them by biochemical fusion (5) com- transformed, hepatocytes were effective in conferring glucagon ponents of the plasma membrane of their early passage, normal sensitivity upon erythrocytes. Glucagon binding data revealed progenitor cells or of purified rat liver plasma membranes that that, whereas normal RL-PR-C hepatocytes have two independent contain a normally coupled glucagon receptor-adenylate cy- classes of binding sites, one of higher and the other of lower affinity, transformed cells possess only the low-affinity receptors. clase system. In this report, we describe experiments of this From these and previous observations, it is possible to conclude type which suggest that the glucagon insensitivity of trans- that the insensitivity of spontaneously transformed RL-PR-C he- formed RL-PR-C cells is related to the absence of a high-affinity patocytes to glucagon is due to the loss, during the transformation glucagon-binding component and indicate that this hormone process, of the high-affinity glucagon receptor. receptor can be transferred between cells and between membranes and cells by a fusion process. In recent years, the heterologous cell fusion technique has been applied to the study of hormone-sensitive adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] systems in MATERIALS AND METHODS a variety of cell types. Generally, in these experiments, a donor Chemicals. Stock solutions of glucagon (Elanco) were pre- cell or membrane preparation contributes a specific hormone pared in 10 mM HC1. Biologically active "2I-labeled glucagon receptor after chemical inactivation of its adenylate cyclase. The (25I-glucagon) was prepared from carrier-free Na'25I (Amer- recipient cell, lacking that particular hormone receptor and sham) as described (13) and was checkedfor quality by thin-layer therefore unresponsive to the hormone, contributes the cata- chromatography on cellulose and binding to highly purified rat lytic function. By using fusion agents such as Sendai virus or liver plasma membranes. [a-32P]ATP was obtained from Amer- polyethylene glycol (PEG), -hormone receptor donor cells or sham, and PEG 6000 from Fischer. membranes have been fused with recipient cells or membranes Cells. Normal (70-150 population doublings) and trans- in such a manner as to confer a new hormone responsiveness formed (300-400 population doublings) RL-PR-C hepatocytes on the fusion hybrid (1-5). Such studies have provided evidence were cultured as described (8, 9). The criteria for spontaneous that hormone receptors are discrete units that retain function transformation were those of Schaeffer et al. (6, 7, 12). even after transfer to a foreign plasma membrane. Glucagon Binding Assays. Cells were seeded in 60 x 15 mm We have been studying the glucagon receptor-adenylate cy- plastic petri dishes at a density of approximately 5 x 105 cells clase system in a cloned line of differentiated rat hepatocytes, per dish. Cultures were refed on the next day and binding was designated RL-PR-C (6). These cells represent a unique model measured 24 hr later. For glucagon binding assays, cells in system for investigating hormonal regulation of adenylate cy- monolayers were washed twice with phosphate-buffered saline clase. For well over a year (over 200 population doublings) they Abbreviations: PEG, polyethylene glycol; cAMP, 3',5'-cyclic AMP; Pi/ The publication costs of this article were defrayed in part by page charge NaCl, phosphate-buffered saline; MalNEt, N-ethylmaleimide; N, payment. This article must therefore be hereby marked "advertise- guanyl nucleotide regulatory protein. ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. * To whom reprint requests should be addressed. 182 Downloaded by guest on September 24, 2021 Biochemistry: Reilly and Blecher Proc. Natl. Acad. Sci. USA 78 (1981) 183 (PjNaCl) for 5 min at 220C, and 1 ml of assay buffer was added. The adenylate cyclase of purified Neville rat liver plasma The assay buffer consisted of 100 mM Hepes buffer (pH 6.5) membranes (15) was inactivated by MalNEt essentially as de- containing 1.5 nM 125I-glucagon, 120 mM NaCi, 5 mM KCI, 1.2 scribed by Schramm (5). mM MgSO4, 15 mM Na acetate, and 1 mg of dialyzed bovine Fusion Systems. Fusions between hepatocytes were accom- serum albumin per ml. Unlabeled glucagon (10 ,uM) was added plished by the PEG 6000 method of Schramm (5). Generally, where appropriate to determine nonspecific binding. After between 1.0 and 1.5 x 107 cells of each type were used in a steady-state binding had been achieved (1 hr), the medium was fusion experiment. Light microscopy revealed that most cells aspirated, and the cells were washed quickly five times with Pj had fused, with several large aggregates of fused cells visible, NaCl (4°C) containing 15 mg of bovine serum albumin per ml. as previously noted (5). After fusion, hybrids were washed with, Cells were then dissolved in 1 ml of 2 M NaOH, the dishes were and resuspended in, 1 mM NaHCO3, and homogenized for washed with 1 ml of water, and the radioactivities of the pooled adenylate cyclase assays as described above. solutions were measured in a gamma counter. The fusion, system had to be modified for fusions between The N-ethylmaleimide (MalNEt) alkylating agent used to in- hepatocytes and pigeon erythrocytes. Pigeon erythrocyte activate the adenylate cyclase of hepatocytes (vide infra) might ghosts (17) were fused with hepatocytes in a ratio of 2.5 X 108 conceivably influence binding of glucagon, because glucagon ghosts to 2.5 X 107 hepatocytes. In addition, the ATP concen- receptors of purified rat liver membranes appear to be sulfhy- tration was increased from 2 mM to 10 mM, and the final 7-ml dryl proteins (14). Accordingly, we performed control experi- dilution of the hybrid cell suspension (5) was omitted. ments in which suspensions (MalNEt detaches the hepatocytes Fusions between hepatocytes and purified rat liver plasma from culture dishes) of RL-PR-C cells were incubated for 10 min membranes were carried out with the PEG-phospholipid- at 40 with 5 mM MalNEt, washed free of the reagent with cold ATP-stearylamine system of Schramm (5). PjNaCl, and resuspended in the standard binding assay me- In experiments testing the effects ofADP-ribosylation ofhe- dium for a 1-hr glucagon binding assay. Cell-bound labeled glu- patocytes on fusion transfers, cells were incubated with cholera cagon was separated from unbound hormone by centrifuging toxin (1 ,g per ml) for 2 hr at 37°C, washed with cold Pi/NaCl, a 200-,ul sample of the assay mixture through 200 ,ul of ice-cold and fused as above. PJNaCVbovine serum albumin solution in a Microfuge; after Protein. The assay of Lowry et al. (18) was used. aspiration of the supernatant fluid, the radioactivity in the pel- let was quantified by gamma counting. Such control experi- RESULTS ments (Table 1) revealed that, over a range of concentrations Adenylate Cyclase Responses. Adenylate cyclase activities of glucagon that encompassed those used for Scatchard analyses of homogenates prepared from normal and transformed hepa- (see below), MalNEt caused a maximal (and constant) inhibition tocytes are shown in Fig. 1. The enzyme from normal (early- of about 50%. Schramm (5) has reported that 5 mM MalNEt passage) cells responded to glucagon with a small (30%) increase did not inactivate functional glucagon receptors in purified (15) in activity and to fluoride with a 10-fold increase. Transformed rat liver plasma membranes. Thus, in the present experiments, (late-passage) hepatocytes, in contrast, did not respond to the hepatocytes pretreated with MalNEt to inactivate adenylate hormone, even at the very high concentration employed. Al- cyclase would quite likely retain high-affinity glucagon recep- though the basal cyclase activity in transformed cells was ap- tors for transfer by fusion processes. proximately halfthat observed in normal cells, the fluoride re- Adenylate Cyclase. The enzyme was assayed in a cell ho- sponse in the former was normal (almost a 10-fold increase), mogenate. Hepatocytes were washed twice with ice-cold 1 mM suggesting that the catalytic unit was intact. NaHCO3, scraped from the flask with a rubber policeman, and Treatment of normal or transformed RL-PR-C cells with homogenized in a Teflon-glass homogenizer (1000 rpm, 10 MalNEt abolished basal, glucagon-stimulated, and fluorides- strokes) in this solution. Adenylate cyclase activity was mea- timulated adenylate activities and this inhibi- sured essentially according to Salomon et al. (16). cyclase (Fig. 1), Inactivation of Adenylate Cyclase with MalNEt. Previous studies with intact cells showed that adenylate cyclase was in- 2100 activated irreversibly by brief treatment with MalNEt (1, 2, 4, 5). Accordingly, hepatocytes in suspension were treated with .0 1800 5-10mM MalNEt in PJNaCl for 10 min at 4°C and washed free a:" 1500 of the reagent with cold PJNaCl, prior to homogenizingfor ade- 0 -p4 1200 nylate assays. - o4 900 Table 1. Effect of MalNEt treatment on the ability of normal t¢ 600 4 C) r: RL-PR-C hepatocytes to bind glucagon ¢o o 300 (1.3) Binding of '251-glucagon fmol/106 cells 200 Concentration of MalNEt- % of 100 -r B G F fBF B G F 125I-glucagon, nM Control treated control EP EPMaINEt LP LPMaINEt 0.025 36 ± 5 26 ± 5 72 0.25 87 ± 14 42 ± 12 48 FIG. 1. Adenylate cyclase responses in normal and transformed 2.5 931 ± 54 551 ± 38 59 RL-PR-C hepatocytes. Incubation systems are as follows: EP, normal 25 10,722 ± 282 4910 ± 588 46 early-passage hepatocytes; EPM^1NE,, MalNEt-treated normal hepatocytes; LP, transformed late-passage hepatocytes; and LPm,,NEt, Normal RL-PR-C hepatocytes were incubated in suspension for 10 MalNEt-treated transformed late-passage hepatocytes. Adenylate cy- min at 40C in the absence (control) or presence of 5 mM MalNEt, clase responses shown represent: B, basal; G, 10 AM glucagon; and F, washed free of the reagent with cold Pi/NaCl, and resuspended in bind- 3 mM fluoride. Where applicable, the stimulation of enzyme activity ing media for 1-hr glucagon binding assays. Results are expressed as byglucagon over the basal value is given. Bars atthe topofthe columns the mean ± SD of triplicate determinations. represent standard deviations oftriplicate determinations. Downloaded by guest on September 24, 2021 184 Biochemistry: Reilly and Blecher Proc. Nad -Acad..Sci. USA 78 (1981)

._. Table 2. Membrane-cell transfer of components responsible for -> 2100 glucagon sensitivity Adenylate cyclase activity, u l1500- IT- pmol cAMP/10 min per mg protein , 1200 Fusion Glucagon- NaF, co J004w :Q 0 system Basal 10 jtM 3.0 mM CD 600 n- 0 (1-8)- LMalone 883 ± 40 2008 + 102 3633 ± 423 ,' 3001 (1.2) (2.7) T Treated LM t 200 MalNEt, 100 1|G 10 mM 8.0 ± 1.0* 2.0 + 0.2* 8.0 ± 2.0* -F rBIb-. Fl Fl BIGIFI P. EP-EP EPMaINEt- LP-LP EPMlNEt- LPMa1NEt 37C, 30 min 40 ± 3.0 7 ± 1* 40 ± 4.0 EPMaINEt LP EP LP hepatocytes 80 ± 9 75 ± 2 348 ± 17 Fusion products FIG. 2 Adenylate cyclase responses in fused cell homogenates. In- LM,,MaNEt) cubation systems are as follows: EP-EP, homologous fusion between -LPcells 46 ± 4 66 ± 3 606 ±141 normal early-passage hepatocytes; EPmjNEt-EPM.NEt, homologous LM(H.Bt) fusion between MalNEt-treated normal hepatocytes; LP-LP, -LP cells 128 ± 13 182 ± 21 1320 ± 36 homologous fusion between transformed late-passage hepatocytes, EPmwNEt-LP, heterologous fusion between MalNEt-treated normal Results are given asthe mean ± SDoftwo independentexperiments, hepatocytes and transformed hepatocytes; and LPM.INEt-EP, heterol- each performed in triplicate. Fusion between rat liver plasma mem- ogous fusion between MalNEt-treated transformed hepatocytes; and branes (LM, 450 ug) and transformed hepatocytes (LP, 1 x 107 cells) normal hepatocytes. Symbols are as described for Fig. 1. was performed at 30TC. *These values are close to the limits of sensitivity of the cyclase assay in this experiment. tion was not reversed by homologous fusion of MalNEt-treated normal cells (Fig. 2). Homologous fusions between normal hepatocytes and be- glucagon receptors. Because cholera toxin is thought to stim- tween transformed cells yielded fusion hybrids whose adenylate ulate adenylate cyclase by catalyzing the ADP-ribosylation of cyclases responded to glucagon and to fluoride ions qualitatively N from NAD+ (17, 21, 22), we reasoned that toxin treatment and quantitatively the same as did the cyclases ofunfused par- of RL-PR-C cells prior to fusion might serve to identify the ents (Fig. 2), indicating that the fusion reagents did not signif- source of this protein in the fusion hybrid. As shown in Table icantly influence cyclase activity. 3, cholera toxin markedly stimulated the adenylate cyclase ac- When transformed cells were fused with MalNEt-treated tivities ofboth normal and transformed hepatocytes. The extent normal hepatocytes, basal and fluoride-stimulated adenylate of the stimulation was much greater than the glucagon effect cyclase activities of the fusion hybrids were almost identical to in normal cells (Fig. 1). When transformed RL-PR-C hepato- those observed in the parental transformed hepatocytes (Fig. cytes were fused with MalNEt-treated normal cells previously 2), as would be expected ifthe catalytic moiety was being sup- ADP-ribosylated by cholera toxin (EPCTM.JNEt-LP), basal and plied solely by the transformed cells. The fusion hybrids, how- glucagon-stimulated adenylate cyclase activities of the fusion ever, now responded to glucagon; although the magnitude of hybrid reflected very closely those observed in the EPMaINEt the hormone effect on this type offusion hybrid varied consid- -LP fusion controls (Table 3). Clearly, the toxin treatment did erably among experiments, a hormonal effect was consistently not enhance basal or glucagon-stimulated activity under these observed. conditions (i.e., MalNEt treatment and fusion). When, how- Fusion hybrids ofthe reverse type, that is, formed by fusion ofnormal hepatocytes with MalNEt-treated transformed cells, Table 3. Effect of cholera toxin pretreatment on adenylate cyclase exhibited a basal adenylate cyclase activity reflective, as ex- responses in RL-PR-C hepatocytes pected, of the normal parental cell, but a glucagon response Adenylate cyclase activity, that was somewhat greater (1.8-fold vs. 1.3-fold) than normal pmol cAMP/10 min (Fig. 2). per mg protein We also attempted to reconstitute glucagon responsiveness in transformed hepatocytes by fusing such cells with plasma Glucagon, membranes from a source other than RL-PR-C cells. Purified Hepatocytes Basal 10 AM Neville rat liver plasma membranes (15) contain a tightly cou- Controls pled glucagon receptor-adenylate cyclase system (19) that is EP 194 ± 3 256 + 13 inactivated either by treatment with MalNEt or by incubation EPCT 1340 + 82 1440 ± 90 at 37°C (Table 2). When membranes treated by either method LP 86+ 6 86± 6 were fused with glucagon-unresponsive transformed RL-PR-C LPCT 691 + 58 677 ± 28 hepatocytes, a product was created whose adenylate cyclase responded to glucagon (Table 2) and did so to a degree similar Fusion hybrids to that observed in normal RL-PR-C cells (Fig. 1). EP(MalN.Et)-LP 104 + 4 128 ± 14. Cholera Toxin Treatment. The aforementioned experi- EP(CTMaINEt)-LP 102 ± 4 132 ± 4 ments do not,.ihowever, identify the component supplied by the EP(MalNF~t)-LP'(CT) 256 ± 22 292± 4 normal hepatocytes or plasma membranes, and therefore do not Normal hepatocytes (EP), treated with 5 mM MalNEt to inactivate identify the lesion in transformed RL-PR-C hepatocytes. One adenylate cyclase, were fused with transformed hepatocytes (LP), and is that the the adenylate cyclase responses of the fusion hybrids were determined. possibility guanyl nucleotide regulatory protein (N), The subscript CT indicates thatcellswere incubated withcholera toxin which is known to be involved in coupling hormone receptors at 1 plg/ml for 2 hr at 37°C andwashed thoroughly to remove the toxin, to the adenylate cyclase (20), is altered during transformation prior to MalNEt treatment and fusion. Results are expressed as the of the cells so that it is no longer capable of coupling with the mean ± SD of triplicate determinations. Downloaded by guest on September 24, 2021 Biochemistry: Reflly andlBlecher Proc. NatL Acad. Sci. USA 78 (1981) 185 ever, MalNEt-treated normal hepatocytes were fused with Table 4. Fusions between hepatocytes and pigeon erythrocytes toxin-treated transformed cells (EPMaINEf-LPc5), a- fusion hy- Adenylate brid was produced whose basal cyclase activity was 2.5 times cyclase activity, that observed in the control experiment, reflecting toxin mod- Fusion pmol cAMP/lO min per mg protein ification of the N protein of transformed cells. The glucagon system Basal Glucagon, 10 AM effect on this fusion product was now partially masked by toxin- Erythrocytesalone 69 14 70 ± 7 induced activation of the transformed cell adenylate cyclase. *EPNU~ erythrocytes 64± 3 -98 ± 5 These results, using increased adenylate cyclase activity as a LPUm.-erythrocytes 76.± 5 73 - 7 markerfor the cholera toxin-modified N protein, suggested that Normal RL-PR-C hepatocytes (EP) and spontaneously transformed the transformed cells are the source of N in the glucagon-sen- RL-PR-C hepatocytes (LP), treated with 5 mM MalNEt to inactivate sitive hybrid, .making it unlikely that an alteration of N is the adenylate cyclase, were.fused with pigeon erythrocyte ghosts, and the lesion in transformed RL-PR-C cells. adenylate cyclase of hybrids was assayed for glucagon response. Re- Glucagon Receptors. Although there are greater numbers sults are expressed as the mean ± SD for triplicate experiments. of glucagon receptors on transformed than on normal Rb-PR- C hepatocytes (10, 11), we felt that analysis of binding characteristics might reveal the basis for the insensitivity of the trans- similar to that ofthe low-affinity sites ofnormal RL-PR-C cells formed cells to glucagon. As shown in Fig. 3, Scatchard analysis and a substantially greater binding capacity (about 300,000 sites (23) of competition binding data for normal cells produced a per- cell). curvilinear plot that could be interpreted as resulting from two Fusions-Between Hepatocytes and Pigeon.Erythrocytes. In classes ofindependent binding sites or from one class, of inter- another approach to identify the nature ofthe glucagon. recep- acting sites exhibiting negative,cooperativity (24). Because dis- torsoftransformed hepatocytes, we fused such cells with pigeon sociation experiments carried out by the method of De Meyts erythrocyte ghosts and assayed the adenylate cyclase response et al. (24) provided no evidence for negative cooperativity (un- -to glucagon in the fusion product. 'Pigeon erythrocytes contain published), we conclude that there are two classes of inde- the N protein-adenylate cyclase system (25), but do not contain pendent glucagon binding sites in normal RL-PR-C cells, with glucagon receptors or respond to this hormone. As shown by the affinity constants of 4.6 x 108 M-1 and 1.7 x 107 M-1, and dataofTable 4, EPM.INE,-erythrocyte hybrids responded to glu- binding capacities of about'10,000 and .112,000 sites per cell, cagon, whereas LPMANEt-erythrocyte hybrids did not. Clearly, respectively (Fig. 3). In contrast, the Scatchard plot for the the glucagon receptors of the transformed hepatocytes were transformed hepatocytes was linear throughout, revealing a sin- nonfunctional. gle class of sites with an constant x binding affinity (3.4 107 M-1) DISCUSSION The present work shows that glucagon sensitivity can be produced in glucagon-unresponsive spontaneously transformed Rb-PR-C hepatocytes by biochemical fusion either with normal progenitor cells or with isolated.rat liver plasma membranes. Previously (10), we had eliminated two possible explanations for the lack of sensitivity of transformed 'RL-PR-C cells to glu- 141 cagon, namely, excessively active cAMP phosphodiesterase and excessive excretion ofnewly made cAMP. In addition, a grossly altered membrane lipid structure (26, 27) in the transformed cells did not appear likely because the adenylate cyclase ofthese cells responded Transformed normally to catecholamines.and cholera toxin 310,000 sites/cell (10), responses that also.require an intact plasma membrane CD structure. x The lesion in the transformed cells most likely represents, then, a specific component ofthe hormone receptor-adenylate cyclase complex. According to current hypotheses, this system consists ofat least three proteins: hormone receptor, a regulatory guanyl nucleotide binding protein (N), and the catalytic moiety. Clearly, the transformed cells have an active catalytic unit and an N protein capable of functioning at least with epi- nephrine and cholera toxin, because the cells are highly respon- sive to' these agents (10). These observations, however, do not rule out the possibility 112,000 sites/cell that an alteration in N during the transformation process may selectively alter its ability to couple functionally with the glucagon receptor. Alterations in the coupling protein have been invoked by Gilman's group to explain the catecholamine insensi- B, fmol tivity in a S49 mouse lymphoma cell variant, UNC (28, 29). This does not appear to be the explanation in the present instance, FIG. 3. Scatchard plots of glucagon binding data for normal and however, because the transformed hepatocytes appear to be the transformed RLPR-C hepatocytes. Normal cells (133 population dou- source of a functional N protein in a glucagon-sensitive fusion blings) and transformed cells (380 population doublings) were incu- hybrid (Table 2). Apparently, neither an alteration in N nor in- bated with '25I-glucagon (1.5 nM) and 0-10 ,pM native glucagon for 1 hr at 22TC. The Scatchard plots were generated from specific binding sufficient levels of this protein can- be invoked as explanations data by theleast-squares linear regression method. K., the association for the glucagon insensitivity oftransformed RL-PR-C hepato- constant, is the slope of the curve. B and F, bound and free glucagon. cytes. Downloaded by guest on September 24, 2021 186 Biochemistry: Reilly and Blecher Proc. Nad Acad. Sci. USA 78 (1981)

The most attractive remaining hypothesis to explain the le- 2. Schramm, M., Orly, J., Eimerl, S. & Korner, M. (1977) Nature sion in transformed hepatocytes is a proposal that such cells lack (London) 268, 310-313. a functional glucagon receptor. Several key observations sup- 3. Schwarzmeier, J. & Gilman, A. G. .(1977)J. Cyclic Nucleotide Res. 3,227-238. port this proposal. The first is that sensitivity to glucagon could 4. Schulster, D., Orly, J., Seidel, G. & Schramm, M. (1978)J. Biol. be conferred upon pigeon erythrocytes by fusing them with nor- Chem. 253, 1201-1206. mal, but not with transformed, hepatocytes; clearly, the gluca- 5. Schramm, M. (1979) Proc. Natl. Acad. Sci. USA 76, 1174-1178. gon receptors in the transformed cells must not be capable of 6. Bauscher, J. & Schaeffer, W. I. (1974) In Vitro 9,286-293. coupling to an adenylate cyclase system and are, therefore, 7. Schaeffer, W. I. & Heintz, N. H. (1978) In Vitro 14, 418-427. without function. The second derives from an analysis ofgluca- 8. Petersen, B., Beckner, S. K. & Blecher, M. (1978) Biochim. Bio- phys. Acta 542, 470-485. gon binding data in both types ofhepatocytes. Normal cells con- 9. Beckner,,S. K. & Blecher, M. (1978) FEBS Lett. 95, 319-322. tain two noninteracting classes ofbinding sites, one ofhigh af- 10. Beckner, S., K., Reilly, T. M., Martinez, A. & Blecher, M. (1980) finity and the other with an affinity constant an order of Exp. Cell Res. 128, 151-158. magnitude lower, whereas transformed hepatocytes possess 11. Reilly, T. M., Beckner, S. K. & Blecher, M. (1980)J. Receptor Res. only low-affinity binding sites. Such differences can be inter- 1, 277-311. as that the receptors, which 12. Schaeffer, W. I. & Polifka, M. D. (1975) Exp. Cell Res. 95, preted indicating only high-affinity 167-175. the transformed cells lack, mediate responses to glucagon. 13. Goldstein, S. & Blecher, M. (1976) in Methods in Receptor Re- Transfer ofsuch high-affinity sites from normal sources (normal search, Part 1, ed. Blecher, M. (Dekker, New York), pp. 119-142. hepatocytes or isolated rat liver plasma membranes) to trans- 14. Tomasi, V., Koretz, S., Ray, T. K., Dunnick, J. & Marinetti, G. formed RL-PR-C cells could account for the restoration of glu- V. (1970) Biochim. Biophys. Acta 211, 31-42. cagon responsiveness in the latter. 15. Neville, D. M., Jr. (1968) Biochim. Biophys Acta 154, 540-552. There is support for this- interpretation in the literature. 16. Salomon, Y., Londos, C. & Rodbell, M. (1974) AnaL Biochem. 58, 541-548. High- and low-affinity glucagon-binding components were 17. Gill, 0. M. & Meren, R. (1978) Proc. Natl Acad. Sci. USA 75, found in purified rat liver plasma membranes (30), as well as in 3050-3054. freshly isolated collagenase-treated rat hepatocytes (31). Both 18. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. Sonne et al. (31) and Rosselin et al. (32) have observed a nonlin- (1951) J. Biol Chem. 193, 265-275. ear relationship between glucagon binding and cAMP produc- 19. Rodbell, M., Birnbaumer, L., Pohl, S. L. & Kraus, H. M. J. (1971) tion in freshly isolated rat-hepatocytes, with a near maximal rate J. Biol. Chem. 246, 1877-1882. 20. Rodbell, M. (1980) Nature (London) 284, 17-21. ofcAMP production occurringwhen only a small fraction oftotal 21. Cassel, D. & Pfeuffer, T. (1978) Proc. NatL Acad. Sci. USA 75, binding sites for glucagon were occupied by the hormone. It has 2669-2674. also been estimated (33) that 80-90% of glucagon binding sites 22. Johnson, G. L., Kaslow, H. R. & Bourne, H. R. (1978) J. Biol in rat liver plasma membranes do not take part in the activation Chem. 253, 7120-7128. process and, therefore, were presumed not to be true receptors. 23. Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 51, 660-672. Although we could not obtain accurate correlation curves be- 24. DeMeyts, P., Roth, J., Neville, D. M., Jr., Gavin, J. R., III & Lezniak, M. A. (1973) Biochem. Biophys. Res. Commun. 55, tween glucagon binding and activation of adenylate cyclase in 154-162. isolated membranes from normal RL-PR-C hepatocytes, we 25. Pfeuffer, T. & Helmreich, E. J. M. (1975) J. Biol Chem. 250, have, however, found (11) that only a small percentage of the 867-876. glucagon receptors on intact normal RL-PR-C hepatocytes 26. Pohl, S., Krans, M. J., Kosyneff, V., Birnbaumer, L. & Rodbell, needed to be occupied in order tostimulate near maximal cAMP M. (1971)J. Biol Chem. 246, 4447-4454. synthesis. These observations are compatible with the notion 27. Rubalcava, B. & Rodbell, M. (1973)J. Biol Chem. 248,6239-6245. 28. Ross, E. M., Maguire, M. E., Sturgill, T. W., Biltonen, R. L. that, in RL-PR-C hepatocytes, just as in freshly isolated rat he- & Gilman, A. G. (1977) J. Biol Chem. 252, 5761-5775. patocytes and purified rat liver plasma membranes, it is occu- 29. Schleiffer, L. S., Garrison, J. L., Sternweis, P. C., Northup, J. pancy ofthe high-affinity, rapidly saturable, low-capacity recep- K. & Gilman, A. G. (1980)J. Biol Chem. 255, 2641-2644. tors that is associated with activation of the adenylate cyclase 30. Schaltz; L. & Marinetti, G. V. (1972) Science 176, 175-177. system. 31. Sonne, O., Berg, T. & Christofferson, T. (1978)J. Biol Chem. 253, 3203-3210. These studies were supported by Grant AM-05475 END from the 32. Rosselin, G., Freychet, P., Fouchereau, M. & Broer, Y. (1974) National Institutes ofHealth to M B. Horm. Metab. Res. Suppl. 5, 78-86. 1. Orly, J. & Schramm, M. (1976) Proc. Nati. Acad. Sci. USA 73, 33. Birnbaumer, L. & Pohl, S. L. (1973) J. Biol Chem. 248, 4410-4414. 2056-2061. Downloaded by guest on September 24, 2021