Quantitative Aspects of the Insulin-Receptor Interaction in Liver Plasma Membranes*

Transcription

1 THE Joumar. OF Broma~ca~ CHEMISTRY Vol. 249, No. 7, Issue of April 10, pp , 1974 Printed in U.S.A. Quantitative Aspects of the Insulin-Receptor Interaction in Liver Plasma Membranes* C. RONALD KAHN, PIERRE FREYCHET,$ AND JESSE ROTH (Received for publication, September 28, 1972, and in revised form, September 20, 1973) From the Diabetes Section, Clinical Endocrinology Branch, National Institute of Arthritis, Digestive Diseases, National Institutes of Health, Bethesda, Maryland DAVID R/I. SEVILLE, JR. Metabolism, and From the Section on Biophysical Chemistry, Laboratory of hteurochemistry, National Institute of Mental Health, Bethesda, Maryland SUMMARY The interaction of insulin with a purified and well characterized preparation of liver plasma membranes has been studied. The time course of binding of 1251-insulin and displacement of tracer at the plateau of binding was measured as a function of temperature and ionic strength. Hormone tracer concentrations were in the low physiologic range. The reaction is complex and includes reversible binding of insulin to its receptor and degradation of both insulin and receptor. The degrading reactions were minimized by using low to medium membrane concentrations and were corrected for by measuring the intact insulin remaining. When the binding reaction was studied over the range of 0.06 to 1000 ng per ml of insulin at 30, the data fit a model with a minimum of three classes of receptor sites: a high affinity low capacity site with a K of 2.0 x log M- and a capacity of 5 X lo-l4 moles per mg of membrane protein; a low affinity-high capacity site with a K of 2.1 x loa M- and a capacity of 1.5 X lo-l2 moles per mg of membrane protein; and a very low affinity-high capacity site ( nonspecific site ) representing about 5% of the total tracer binding. Both the affinity and binding capacity are influenced by temperature and ionic strength. Kinetic data for insulin receptor complex dissociation are also consistent with heterogeneity of receptor sites. Discrepancies in the literature between the number of reported classes of insulin receptor sites and their affinity constants are discussed in terms of the variables of this complex reaction. There is an increasing body of evidence that the primary step in the action of insulin and other polypeptide hormones is binding to * This work was presented in part at the 32nd annual meeting of the American Diabetes Association in Washington, D.C., June 24 to Present address, Unite de Recherche de Diabetologie (Institut Nationale de la Sante et de la Recherche Medicale), Antoine, Paris, France. Hopital Saint- a specific site on the plasma membrane of the target cell known as the receptor site (1). This reaction has been studied in several laboratories and in a variety of receptor preparations including purified membrane preparations (2-7), isolated fat cells (3, 8-ll), lymphocytes and fibroblasts (12-15), particulate fractions of fat and liver (16, 17), and solubilized fractions of liver (B), fat (18), and lymphocytes (19, 20). These studies have demonstrated that this reaction is rapid and reversible, that the site is specific for insulin, and that the ability of an insulin analogue to compete with native insulin for this site is directly proportional to its biological activity in vitro. Several investigators have also attempted to measure kinetic and equilibrium constants of the insulin-receptor interaction (4-11, 13, 15, 17). Some investigators have concluded that the receptor population is homogeneous (8-ll), while others have found that it is heterogenous (4-7, 13, 15) ; values for the equilibrium constants of the reaction have varied over a range of several orders of magnitude. In an attempt to resolve some of these differences and to establish a basis for the comparison of insulin-receptor interaction in different physiologic states, we have studied the quantitative aspects of insulin interaction with its receptor in a highly purified plasma membrane preparation from rat liver (21). We have directed our attention to the methods of analysis of the data and to the complexity of the reaction. In this preparation we find a population of receptor sites which is heterogenous with respect to affinity constants. The data which we present can be explained by a model which has three orders of receptor sites: a high affinity-low capacity site, a low affinity-high capacity site, and a nonspecific site. The affinity constants and the binding capacities are influenced by both temperature and ionic strength. In addition, under the incubation conditions used, there is degradation of both the hormone and the receptor site. In our analysis we have attempted to make appropriate corrections for these competing reactions. We have also tried to explain how varying analysis of the data may have resulted in some of the differences observed in the existing literature. MATERIALS AND METHODS The methods employed in the binding studies have been previously described in detail (2). In brief, Y-insulin (specific 2249

2 activity 150 to 200 &i per pg) was incubated with purified plasma membranes of rat liver (21) in a Krebs-Ringer phosphate buffer, ph 7.5. The incubation mixture also contains 1.5 % bovine serum albumin and concentrations of hormone and membrane as indicated in the legends to the figures and t)ables. The membrane-bound hormone was separated by centrifugation of duplicate or triplicate samples through 200 /*l of chilled buffer. The top of the pellet was washed with 200 ~1 of 10% sucrose, the tip of the microfuge tube excised, and the radioactivity in the pellet was counted in a Nuclear Chicago Autogamma spectrometer at 55% efficiency. Degradation of the unbound insulin in the supernatant was measured by loss of ability to bind to antiinsulin antibodies or to fresh aliquots of membrane (22). Purified plasma membranes and 1251-insulin were prepared as previously described (21, 22). The source of all other materials has been given (2, 21, 22). Mathematical methods for the treatment of data obtained in radioimmunoassays, competitive protein binding assays, and other types of binding of small molecules by macromolecules have been the subject of a number of papers (23-27). Although these methods often do not fit the data perfectly to an idealized model, they do provide constants which are useful in describing the displacement curves observed (25). These methods require several important assumptions which are also made in the treatment of the data presented in this paper. 1. The hormone is present in a homogeneous form, insulin monomer in this case. 2. Labeled and unlabeled hormone behave identically. 3. Full equilibrium is achieved. 4. Round and free hormone can be perfectly separated without perturbing the equilibrium. 5. No cooperative effects exist between binding sites. 6. The hormone is univalent; that is, one hormone molecule can react with only one binding site. If these assumptions are made in the analysis of the interaction between a hormone and a single class of receptor sites, it is possible to give a series of equations analogous to t.hose derived by Scatchard (24) in the study of ion binding by proteins, or Berson and Yalow (23) in their study of insulin and insulin antibody reactions : - WI + [RI F? IfIR1 k, 1~~1 K = i& = [H][R] where [ti] is the concentration of free hormone; [RI, the conccn tration of receptor sites; [ZiR], the concentration of hormoncreceptor complex; k, and kd are the association and dissociation rate constants, respectively; and K is the equilibrium constant or affinity constant. If the total receptor concentration is denoted by [PI, such that [R] = [R ] - [HR] then, in the form of the familiar Scatchard equation, or Bound Hormone - = K([RO] - [HRI). Free Hormone 1 These data may also be fitted to models in which both receptor and insulin molecules either polymerize or isomerize, as noted in Reference 28. I I I IO ,000 5 IO [INSULIN] (ng/mll INSULIN BOUND hg/ml) FIG. 1. I,eft, per cent of total 251-insulin bound to liver membranes as a function of total insulin branes (0.6 mg per ml) were incubated concentration. 1,iver memwith lz51-insulin (166 PM) at 30 for 60 min. Bound and free hormones were separated as described under Materials and Methods. night, Rcatchard plot of the data on t.he left uncorrected for nonspecific binding and degradation. Using this formulation, plobting bound/free hormone against bound hormone should result in a straight line with a slope of -K and the intercept on the bound axis is [PI. When there are two or more classes of receptor sites with different affinity constants, the data will no longer give a linear function. This is the situation which occurred in the analysis of the interact.ion of insulin with its receptor (Fig. 1). These data may be fitted with any number of classes of binding sites with different affinity constants. The minimum model which best fits these data consists of three orders of receptor sites: a high affinity-low capacity site, a low affinity-high capacity site, and a site which has been previously termed by us and others as a nonspecific site. The nonspecific sites are those which appear unsaturable over the range of concentration of insulin used in these studies. In this analysis then [HR] = K1[H]([R,o] - [H&l) + K~[Hl([W - [ff&i) + Kdffl where K1 and K2 are the equilibrium constants and RI and R9 the receptor concentrations for the high and low affinity sites, respectively. Ka is a constant for the nonspecific binding. By subtracting the nonspecific binding from each value of bound hormone, a Scatchard plot may be made in which [HRI - KM WI = K,([@'l - [HI&]) + K&L01 - [HRd Close approximations for KI, KS, RIO, Rs" may now be determined by manual curve fitting as follows. A least squares straight line is drawn to fit the low affinity site using data from insulin concentrations from 10 ng per ml to 1000 ng per ml. The contribution of this site is then subtracted from the points obtained at lower insulin concentrations and the resultant points fitted by a second least squares line. The slope of the latter line is the high affinity constant and the intercept on the bound axis is the number of high affinity sites; the slope of the former line is the low affinity constant and the intercept is the total number of sites (RI" + Ri'). The number of low affinity sites is obtained by subtracting the number of high affinity sites from the total. Using this method of curve fitting the high affinity constant reported is an underestimate of KI by about 10% and the low affinity constant an overestimate of K2 by 1.5- to 2-fold (25). It is important to note that this model is the minimum solution for the data obtained in these experiments. It is possible to produce an infinite number of more complex models, but for the purpose of the present study, any additional orders of sites would be indist.inguishable except as part of these larger groups.

3 2251 TIME ( mid FIG. 2. Effect of membrane concentration on time course of binding and hormone degradation. A, time course of binding of 12SI-insulin to liver membranes at 30 and 60 mm sodium. Membrane concentrations were 0.2 (O---O), 0.4 (m---m), and 0.8 (A-A) mg per ml. 2SI-insulin concentration was 20 PM. I?, degradation of the free 51-insulin as a function of membrane concentration. Aliquots of supernatant of the incubation tubes used TIME (min) -.- TIME ( mid in Purl A were taken at the indicated time points and intact W- insulin was measured by its ability to rebind to a fresh aliquot of membranes (22). C, time course of binding as a function of membrane concentration. The data of Part A were corrected for degradation of Part B by calculating the per cent of intact hormone bound as described under Materials and Met hods. Although the assignment of a nonspecific site may be a poor choice of terms, since it is not clear that this binding site is nonspecific in a biological sense, it is a useful simplification in the analysis of the data. Since this site behaves as unsaturable over the range of hormone concentrations experimentally practical, binding to this site may be represented by a constant percentage of the tracer 1251-insulin. For our data, nonspecific binding usually constitutes about 5 y0 of the total tracer binding. Thus, Bound - NS = [ml - &[Hl Free WI where Bound, Free, and NX referred to the total percentage of the 12SI-insulin bound to membranes, the percentage free in the supernatant, and the percentage nonspecifically bound to membranes, respectively. The ability of the liver membrane to degrade Y-insulin must also be considered. We have previously discussed this problem in detail (22) and pointed out three important aspects as far as the quantitative calculations are concerned: (a) free insulin is degraded on exposure to membranes at 30 into fragments which do not bind to the liver membranes; (b) bound insulin is protected from degradation; and (c) the rate of insulin degradation is a function of membrane concentration. With the relatively low membrane concentrations used in these reactions, insulin degradation usually does not exceed 15y0 of the total free hormone. To correct for this additional reaction, however, the fraction of intact free hormone in each incubation tube was determined by its ability to bind to fresh liver membranes or anti-insulin antibodies (22). Finally, therefore, Bound Bound - NS ~ (corrected) = Free Free X fraction intact RESULTS Time Course of Binding-At 30 the binding of %insulin to liver membranes was a rapid reaction, even with insulin concentrations as low as 20 PM (Fig. 2A). By 15 min the binding had reached almost 90% of its maximum value. Over a wide range of membrane concentration the binding curve showed a similar pattern, reaching a peak at about 30 to 45 min followed by a gradual decline. This has been observed in other binding studies with liver membranes (29) and in binding studies with isolated fat cells (11) and lymphocytes (13), but has been unexplained. A gradual decline in binding could be due to insulin degradation (ll), receptor degradation (tide injra), or both. When the data are corrected by taking insulin degradation (Fig. 2B) into 30 I TIME COURSE OF BINDING i 1; IL 24 3b TIME (hr) FIG. 3. Time course of binding of rz51-insulin to liver membranes at 4 and 30. Membrane concentration was 0.33 mg per ml and 1251-insulin concentration was 10 PM. Sodium concentration was 0.15 M. No correction for hormone degradation has been made. account, the binding curves show a react,ion which reaches its maximum at 60 min and an apparent equilibrium then persists for at least 2 hours (Fig. 2C). At high membrane concentrations, because the correction is large, there is considerable scatter in the data. All subsequent experiments were therefore done at relatively low membrane concentrations, and when equilibrium constants have been derived, the data have been corrected for hormone degradation. Both the amount of binding and the rate of binding were also temperature-dependent (Fig. 3). After 30 min of incubation at 4, the percentage of lzsi-insulin bound was about half that observed at 30. The reaction was likewise very slow to reach steady state, requiring 12 to 24 hours. Using identical concentrations of membrane and %insulin, the maximum value of receptor bound insulin was 2.5 to 3 times higher at 4 than observed at 30. As we have previously shown (22)) insulin degradation is significantly decreased at this temperature and accounts for some of the increase in binding and the long steady state observed; but under these dilute conditions of reactants, this alone was not sufficient to explain the difference in binding. An increase in binding at lower temperatures has been observed by Gavin et al. in a study of insulin binding to lymphocytes, but has not been observed by others (2, 10, 16) studying the insulin-receptor interaction in fat and liver over much shorter periods of time. This

4 FIG. 4. Displacement of 1251.insulin by native insulin from the liver membranes. Liver membranes (0.33 mg per ml) were incubated with 1251-insulin (12 PM) in a buffer containing 60 rnm sodium. The incubation time at 30 was 60 min and at 4 was 24 hours. Solid squares (m) and open squares (0) represent data from separate experiments. The best curves drawn were based on the Scatchard analysis in Fig. 5 and the model described under Materials and Methods. increase in binding is important since it implies that at the lower temperature there is a significant increase in either the equilibrium constant or the number of binding sites, or both. In studies of glucagon binding to liver (30) and adrenocorticotropic hormone binding to adrenal (31), decreased binding has been observed at low temperatures. Equilibrium Studies-In our previous studies (2, 3, 29) of the insulin-receptor interaction, we demonstrated two essential features of this binding reaction : (a) unlabeled insulin at concentrations found in portal blood displaces the labeled hormone from the receptor; and (b) the ability of insulin analogues to compete with labeled insulin is in direct proportion to their bioactivity in vitro. An example of a competitive displacement of 251-insulin by unlabeled insulin is depicted in Fig. 4. When the data from these experiments are corrected for degradation as described above, they may be treated as equilibrium data and analyzed by the modified Scatchard method given under Materials and Methods. The resultant points are found to fit the previous model with two orders of specific receptor sites: a high affinity-low capacity site and a low affinity-high capacity site (Fig. 5). At 30, the high affinity site has a K of 2 x log M-I and a binding capacity of 6 x IO-l4 moles per mg of membrane protein (360 pg of insulin per mg of membrane protein) ; the low affinity site has a K about an order of magnitude lower and a binding capacity about 25 times higher (9 ng per mg of membrane protein) (Table I). These values for the equilibrium constants are very similar to those obtained by Hammond et al. (7) studying fat and Gavin et al. (13) studying human peripheral lymphocytes. When the equilibrium study is conducted at 4 a much higher percentage of 1251-insulin is bound at each point in the displacement curve (Fig. 4). This increase in insulin binding at 4 was due to an increase in both the affinity constants and in the number of receptor sites, especially those of higher affinity (Fig. 5 and Table I). These changes were measured after differences in insulin degradation were taken into account. An increase in receptor sites at low temperatures has not been previously reported and may be due to some conformational change in the membrane or FIG. 5. Modified Scatchard analysis of data in Fig. 4 (see Materials and Methods ) plotting bound-free hormone versus hormone bound. All data were corrected for per cent of free hormone degraded as measured by inability of 251-insulin to bind to antiinsulin antibodies. The best fit line for the 4 data includes 2 points at insulin concentrations higher than those shown which did approach saturation. See Table I for equilibrium constants. TABLE: EJect of temperature 01~ equilibrium constants Per cent change is calculated as the (constant at 4 - constant at 30 ) X loo/(constant at 30 ). Constant Site Affinity con- High affinity stant (M-l) Low affinity Binding capac- High affinity ity (moles/ Low affinity md - I 300 Temperature I 2.3 X lo9 3.3 x X lo8 4.5 x x x 10-l 1.5 x lo- * 3.1 x 10-l Per cent change +44oj, +95% + 220% + 107% due, in part, to decrease degradation of the receptor at this lower temperature (vide infra) A similar increase in receptor sites at lower temperatures is observed in insulin binding to cultured lymphocytes.2 High ionic strength media such as 2 M NaCl have been reported to increase insulin binding to the receptor (16). We also observed t,his increase at both 30 and 4 (Fig. 6). At least two factors account for this increase. First, in 2 M NaCl, insulin degradation is decreased by about half (22). Scatchard analysis of this data, however, after taking into account this change in degradation, reveals that there is also an increase in the affinity constants of both receptor sites (Fig. 7, Table II). This increase in affinity constants is observed at 30 and 4. In contrast to reports by others (16)) we observed no significant increase in the number of receptor sites after the reaction had come to equilibrium at 60 min except those of the lowest affinity which we have included in the nonspecific binding. However, since high ionic strength results in increased receptor degradation (vide infra), if one measured the reaction at an earlier time point, the number of receptors may be increased. Kinetic Studies-In an attempt to confirm the heterogeneity of receptors, kinetic studies were performed. If there were a homogeneous population of independent receptor sites, one would 2 J. Gavin, personal communication

5 2253 predict that the dissociation would follow first order kinetics. At 30, the dissociation of *%insulin from its receptor did not follow simple first order kinetics (Fig. 8). After 10 min, 35% of the specifically bound insulin was dissociated and over was dissociated in 2 hours. The same pattern of dissociation was observed for the insulin bound to total, specific, and nonspecific sites and was observed whether the dissociation was done by 2M [No] dilution or by addition of a large excess of unlabeled hormone (data not shown). If one proposes a model with two specific sites, it is possible to treat the dissociation of the specifically bound insulin as the sum of two separate first order dissociation rates (Fig. 8B) (23) and resolve fast and slow dissociating components of bound insulin. In previous reports, we (32) and others (13) have ascribed the rapid dissociating component to the low affinity site and the slow component to the high affinity site. This is almost certainly not the case, however, since this interpretation would suggest that about 44 y0 of the insulin is bound to the high affinity site, and this is not the case under the conditions of this experiment. Thus, although such a curve is compatible with a model with heterogeneity of receptor sites, it is not possible to resolve the over-all dissociation into two components which could be accurately ascribed to the high and low affinity sites. As a simple approximation, one can calculate an over-all dissociation rate of 4 x lo-* min-i assuming a mean half-time of dissocia- [INSUUN],ng,m,, FIG. 6. Displacement of 251-insulin by native insulin as in legend to Fig. 4, but sodium concentration was 2 M b/. 0 ZM bl IL--.-J so TIME hh) TIME hh) FIG. 8. A, dissociation of W-insulin from liver membranes. Membranes (0.6 mg per ml) were incubated at 30 for 30 min with 0.25 nm lz51-insulin in a volume of 1 ml. complex was separated by centrifugation The membrane-hormone and washed two times in 5 ml of cold buffer. The membrane-hormone complex was resuspended in 2 ml of buffer containing no insulin. binding was conducted in the same manner with The nonspecific the addition of 50 pg per ml of unlabeled insulin in the original incubation. Aliquots were removed at appropriate intervals and binding meas- BOUND hghg msmbrme protein, ured as described under Materials and Methods. B, resolution FIG. 7. Modified Scatchard analysis of data in Fig. 6. Equ- of the dissociation of the specifically bound lzsi-insulin from Part librium constants are given in Table II. A into two components. loi TABLE Efect of sodium concentration equilibrium constants II Site Temperature Sodium concentration 0.06 M 2M Per cent changea Affinity constant (M-I) Binding capacity (moles/mg: High affinity Low affinity High affinity Low affinity x lo9 3.6 X lo x x x IO* 3.3 x x X x x lo x x lo x x lo x IO x lo- 2 % $ (1 Per cent change is calculated as (constant at 2 M - CoBStant at 0.06 M) X IOO/(constant at 0.06 M).

6 TIME COURSE OF BINDING AT 30 O, \, 2M Na / //,,I \ d \ I I I I I IO er-io TIME (mid FIG. 9. Time course of binding at 30 in high and low ionic strength media. Membrane and hormone concentrations were as noted in Fig these large species and are of the same order as the forward rate constant for an insulin-insulin antibody reaction (23). Receptor Degradation-Kinetic studies over long time courses had suggested that the amount of insulin bound to the receptor decreased upon prolonged incubation of the reaction mixture (Fig. 9). This decrease could be explained in part by insulin degradation, but could not be explained entirely since in high ionic strength media the binding fell more rapidly than in low ionic strength media (Fig. 9), despite about 50% inhibition of insulin degradation (22). This suggested still another aspect of the insulin receptor interactsion, receptor degradation. To study this directly, liver membranes were incubated at 30 for varying lengths of time prior to the addition of 1251-insulin, and then the binding was allowed to proceed over the usual time course. In both low and high ionic strength media, there was a decrease in maximal insulin binding with prolonged preincubation at 30 (Fig. 10). In the low ionic strength media (Fig. loa), this decline followed a short initial increase and resulted in a 50% reduc- tion of 15 min. Gavin et al. (13) studying the dissociation of insulin from its receptor on cultured lymphocytes found an even more marked deviation from first order kinetics. Gammeltoft and Gliemann (8) and Cuatrecasas (9) on the other hand, find that the dissociation of insulin from fat cells fits a single first order rate constant. The problem of measuring association rate constants when a heterogeneous population of receptors exists is even more difficult and has been discussed by Berson and Yalow (23). Several attempts at measuring these constants for this reaction directly using the methods which they had described for a mixed pool of antibodies suggested heterogeneity but yielded data which were not of sufficient precision for calculation of kinetic constants. House (4), however, studying insulin binding to liver membranes using a trichloroacetic acid precipitation method was able to st,udy the association with multiple points over the first 5 min, and observed an oscillatory pattern suggesting binding to heterogeneous sites. Based on our mean dissociation rate of 4 X lo- min-i, estimates of the association rate constants for the two sites described here would be 8 X 107 and 8 X lo6 minp M-I, which are similar to the value reported by Gammeltoft and Gliemann (8) for insulin binding to fat cells, but are 3 to 30 times less than that given by Cuatrecasas (17). These constants are of a reasonable order of magnitude (see LDiscussion ) for a reaction between FIG. 10. A, receptor degradation in buffer containing low -(60 mm) NaCl. Membranes (0.6 mg ner ml) were incubated from 0 to 226min ai 30 prior to addition of ~2SI-insulin 0.6 no. Incubation was then continued for 30 min at 30 and bound hormone measured. Data are the mean A S.E. for two experiments each done in triplicate. B, receptor degradation in buffer containing 2 M NaCl. Conditions as in legend to A. tion in maximal binding after 4 hours. In high ionic strength media (Fig. IOB), receptor degradation was more rapid with a half-time of about 40 min. Spontaneous degradation of receptor sites has not been reported by others studying this reaction, although Kono (11) studying insulin binding to fat cells and Gavin (13) studying insulin binding to lymphocytes have observed a fall off in binding after prolonged incubation. The term degradation is used here only to imply loss of ability to bind insulin to receptors on the membrane and does not exclude the possibility of solubilized receptors. With intact lymphocytes the receptor degradation is at least in part solubilization of the receptor (19), with liver membrane this does not appear to be the case.3 DISCUSSION The quantitative aspects of insulin interaction with its receptor have been measured in at least seven different laboratories by ten different investigators using a variety of preparations of fat, liver, and lymphocytes. The results are summarized in Table 111. Seven investigators report a heterogeneity of sites while three find homogeneity; affinity constants vary from as high as 2 X lolo M- to as low as 1.3 X 105 M-l. We have attempted to resolve some of these differences by studying insulin interaction with a highly 3 C. R. Kahn, unpublished observation.

7 TABLE Aflnity constants for insulin receptor iuteractiou Investigators Multiple orders of sites Kahn et al. 0 Liver membrane Freychet et al. 6 Fat cell membrane; Gavin et al. Goldfine House Hammond Marinetti et al. et al. el al. Single order of sites Gammeltoft and Gliemann Cuatrecasas Kono and Barham 5 Present study III Tissue Lymphocytes, cultured Lymphocytes, peripheral Thymic lymphocyte Liver membrane Fat cell membrane Liver membrane Isolated fat cells 37 Isolated fat cells Particulate fraction, liver Isolated fat cells TABLE IV TelIlperature Affmity constant 2.0 x x x lower affinity 1.2 x x x x x x x X x x x X x x x x x 108 Equilibrium constants for insulin irlteraction with purified live plasma membranes at 30 Constant I High affinity-low capacity site Low affinity-high capacity site Affinity constant. 2.0 X lo9 M-I 2.1 x 10 M-l Number of sites. 4 X 101o/mg 90 X 10IO/mg Binding capacity. 6.6 X lo-l4 moles/mg 1.5 X lo-i2 moles/mg 0 Mean of five determinations on three separate membrane preparations. S.E. is f0.2 for the high affinity receptor and +O.l for the low affinity receptor. purified and well characterized fraction of liver plasma membranes. We have also considered the competing reactions of insulin degradation and receptor degradation which have been, for the most part, neglected in other quantitative studies. Our results show that the population of insulin receptors in the purified liver membrane can indeed be considered heterogeneous with respect to affinity for insulin (Table IV). The displacement curve may be analyzed using a Scatchard plot composed of three orders of receptor sites: a high affinity-low capacity site with an equilibrium constant of 2 x lo9 M-l, a low affinity-high capacity site with an equilibrium constant of 2 x IOx M-I, and a site which we have designated as nonspecific (low affinity and very high capacity). At both 30 and 4, we were unable to detect sites of any higher affinity. We have discussed the limitations of analysis of a nonlinear Scatchard plot. Berson and Yalow (23) in their study of mixed antibody populations point out how analysis of limited data, especially if it is near one end or the other of a Scatchard plot, can result in a linear appearance of the curve. Thus, if the data analyzed were obtained only at very low insulin concentrations a high K (109) will result; if the center of the curve is analyzed an intermediate K (108) will result, and if orlly very high concentrations of insulin were used, a very low K (IO ) would be found. If graphical methods other than Scatchard analysis are used, it is important to consider the regions of the curve given the most weight in these analyses since a similar result may occur. Since the great majority of receptor sites are of the 2 X lo8 M-l affinity, it is important to analyze the entire displacement curve in detail if all orders of sites are to be detected. The data analyzed by Kono (Fig. 3 in Reference ll), for example, are restricted to the region of the displacement curve above M (the size of the tracer used), and thus it would be impossible to observe a site of higher affinity which might be significantly saturated at this hormone concentration. Cuatrecasas, on the other hand, has restricted his analysis to data obt,ained 0111~ at very low insulin concentrations (Fig. 1 in Reference 16) and neglected the data at higher insulin concentrations (Fig. 2 in Reference 16) ; thus, he reports ody a very high affinity constant. Also, since higher temperatures significantly decrease the number of sites of higher affinity, it is possible that at 37 the high affinity receptor would not be detected, while at lower temperatures it might be very obvious (Fig. 5). Although tliffereilces in the observed rquilibrium constants may be related to the tissue studied, existing data do not suggest this to be the case (Table III). Hammond et al. (7) and Gavin et al. (13) report affinity constants similar to ours in studies of fat cell membranes and lymphocytes, respectively. This model with a heterogeneous population of receptors is also not unique to the insulin-receptor interaction. A similar model has been proposed for the interaction of adrenocorticotropic hormone with its receptor (31), epinephrine with its receptor (33, 34), glucagon with its receptor (34), and osytocin with its receptor (35). The biological significance of a class of low affinity receptor sites has been questioned by Cuatrecasas (36) who reports ody a single class of high affinity receptors in both fat and liver. Similarly, Marinetti el al. (5) who report three classes of receptor sites for insulin in liver speculate that only the high affinity sites represent the specific sites associated with the physiological action of the hormone. Kono and Rarham (11) and Gammeltoft and Gliemann (8) both of whom have found a single class of receptors with an affinity constant of about 2 x 10R M- observed that in the fat cell maximal insulin stimulation of glucose oxidation was observed when ody a small fraction of the total binding sites were occupied. Kono (37) has pointed out, however, that certain effects are observed ody with higher concentrations of insulin. Thus, he suggests that the individual low affinity receptor is important, and cells are equipped with a large number of receptors so that they are able to bind the necessary amount of insulin even at low hormone concentrations. Several other factors also suggest that biological effects may be coupled to the low affinity receptors. We have observed that insulin analogues displace Ii&Iinsulin from the low affinity-high capacity receptor with the same relative order as their bioactivity (2). Even at very low concentrations of insulin, 50 to 70 y. of the bound hormone is on the low affinity receptor (Figs. 5 and 7). Furthermore, if only the high affinity receptors in liver were important they would be almost continuously saturated due to the high portal blood concentrations of insulin (38). Finally, Goldfine et al. (15) studying insulin

8 2256 stimulated a-amino isobutyric acid influx in thymocytes demonstrated a close correlation with insulin binding, even though the concentrations involved were above the normal physiologic range. Thus, from the existing data, it appears that there is no reason to consider some of the receptors <active and others inactive, even though a maximal response may be achieved with only a minority of receptor sites occupied (i.e. reserve receptors ). Studies (30) of the glucagon receptor in liver have revealed a similar broad dose-response of both binding and activation of adenylate cyclase with an approximate K of 2.5 X lo* M-l. Although exact kinetic constants cannot be obtained, our estimated values are similar to those reported by Gammeltoft and Gliemann (8) for insulin binding to fat cells and are remarkably similar to those obtained by Person and Yalow (23) in their study of insulin interaction with antibodies. This seems a reasonable comparison in terms of the size of the reacting species. It is possible to calculate a theoretical maximal forward reaction rate from the Smoluchowski equation (39). This assumes a diffusion limited process where each collision of the reacting species results in a reaction. For insulin interaction with its receptor, this equation gives the value of 6 X lo* M-l s-l (assuming the receptor has a molecular weight of 150,000 or more). It would not be surprising if the actual value for the forward reaction rate is much lower than the theoretical maximum since the reacting species almost certainly would have to be preferentially aligned to react, since most collisions would not result in a reaction, and since insulin may have to overcome an activation energy to react with its receptor. From the data obtained at 4 and 30, it is possible to calculate approximate values for the thermodynamic constants of the reaction of insulin with its receptor (Table V). These calculations must be regarded as preliminary because of the possibility of a temperature-dependent phase transition in the membrane, but are valuable because they provide some insight into the nature of the reaction. Although the AHo is negative, it is interesting that most of the free energy comes from the entropic term. The same is true for the reaction of insulin with its antibody where remarkably similar values for AFO, AHo, and AS0 have been reported (23). In addition to the complex interaction of insulin with its receptor, two other simultaneous reactions are occurring which we have considered in evaluation of the data: insulin degradation and receptor degradation. Insulin degradation appears to be a proteolytic process (22). The K, for degradation is 1.7 x lo- M, i.e. the enzyme has a much lower affinity for insulin than either the high or low affinity receptor discussed here. The degrada- degradation and the fate of the insulin receptor are currently under study. In summary, we have studied the quantitative aspects of insulin interaction with its receptor. This complex reaction presents several interesting aspects in analysis of data. We find a receptor population which can be described by a model with a minimum of three receptor sites. The estimated kinetic constants observed are similar to those observed in the insulin-antibody reaction. It is important to point out again, however, that this analysis is not a unique solution of the data. All of the data presented here could be fit with a model in which there is negative cooperativity between sites. Such possibilities are currently being investigated. On the other hand, the proposed model is useful for the comparison of results in situations in which the insulin-receptor interaction is altered (40) and because it points out some of the complexities and difficulties in doing quantitative studies in these systems. Note Added in Proof-Since submission of this manuscript, De Meyts et al. (41) have presented experimental evidence that there are interactions between insulin receptor sites of a type consistent with negative cooperativity. If these are present, the apparent values of K and Ro do not have their usual precise physicochemical meaning. Such site-site interaction, however, does not esclude the possibility of coexistent site heterogeneity. Acknowledgments-The authors would like to thank Drs. P. Gorden, J. R. Gavin, III, I. D. Goldfine, and J. E. Rall for their helpful advice during this study; J. Boone for his excellent technical assistance; Ms. L. Perry and D. Watkins for secretarial assistance; and S. Gammeltoft for making available unpublished data. The authors would also like to acknowledge the referee for the Journal of Biological Chemistry whose rigorous review lead to a better understanding of the complexity of these studies. REFERENCES 1. ROTH, J. (1973) Metabolism 22, FREYCHET, P., ROTH, J., AND NEVILLE, D. M., JR. (1971) Proc. Nat. Acad. Sci. U. S. 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10 Quantitative Aspects of the Insulin-Receptor Interaction in Liver Plasma Membranes C. Ronald Kahn, Pierre Freychet, Jesse Roth and David M. Neville, Jr. J. Biol. Chem. 1974, 249: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at