©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Deletion of Lysine 121 Creates a Temperature-sensitive Alteration in Insulin Binding by the Insulin Receptor (*)

(Received for publication, August 5, 1994; and in revised form, October 25, 1994)

Ruichun Liu (1) Jian Zhu (3) Nicholas Jospe (2) Richard W. Furlanetto (2) William Bastian (3) James N. Livingston (3)(§)

From the  (1)Department of Biochemistry and the (2)Division of Pediatric Endocrinology, Department of Pediatrics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 and (3)Miles Pharmaceutical Division, West Haven, Connecticut 06515

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Recently we reported the deletion of Lys-121 in one allele of the insulin receptor gene from a child with severe insulin resistance. In the present work, this mutant receptor (M121) was shown to have an abnormal sensitivity to temperature and an alteration in ``negative cooperativity.'' In contrast to the wild-type receptor (HIRC), insulin binding by the M121 receptor was rapidly and irreversibly lost at temperatures above 30 °C with the phosphorylated form of the receptor being more temperature-sensitive than the nonphosphorylated form. Although insulin binding activity was lost, Western analysis and other studies showed that the mutant receptor remained intact. Measurements of I-insulin dissociation at 21 °C in the presence of native insulin (an estimate of negative cooperativity) demonstrated a difference between the mutant and wild-type receptor. Insulin dissociation from the mutant receptor was not as pronounced as that found with the wild-type receptor. Thus, an abnormality in insulin binding by the mutation was evident at lower ``permissive'' temperatures. The results of these and other studies argue that Lys-121 occupies an important position for the regulation of insulin receptor conformation. This regulation apparently influences negative cooperative interactions with insulin and modulates signal transduction.


INTRODUCTION

A large amount of work has been conducted on mapping the insulin binding site(s) in the insulin receptor in attempts to understand how the ligand binding process stimulates the receptor's tyrosine kinase activity and regulates other phenomena such as negative cooperativity and receptor internalization. Site-directed mutagenesis(1, 2, 3, 4, 5) , the generation of insulin receptor chimeras(6, 7, 8, 9, 10) , and affinity labeling procedures (11, 12, 13, 14) have provided new insights, including the identification of regions in the receptor important for high affinity insulin binding and for modulating receptor binding affinity. However, a number of unresolved issues remain. The relative contributions to insulin binding by the amino terminus of the alpha-subunit, the high cysteine region, and the regions toward the carboxyl terminus of this subunit need to be clarified(15) . Affinity labeling experiments have also provided some conflicting evidence, with one group identifying a region in the high cysteine domain and another group identifying the amino terminus as important for insulin binding(15) . Finally, more information is needed on regions that support changes in conformation which regulate beta-subunit tyrosine kinase activity and/or insulin binding affinity (i.e. negative cooperativity).

Recently we described the presence of a deletion of Lys-121 in one allele of the insulin receptor gene of a severely insulin-resistant child with leprechaunism(16) . When expressed in CHO (^1)cells, this mutation caused a marked decrease in the signaling activity of the receptor which was accompanied by an extremely poor biological response to the hormone. In attempts to evaluate further the impact of the mutation on other actions of the receptor, we undertook studies of insulin processing and internalization. This work uncovered the finding that the mutation caused an abnormal sensitivity of the purified receptor to physiologic temperatures and to an alteration in the insulin-mediated dissociation of radiolabeled insulin, i.e. a change in negative cooperativity. These and other findings argue that Lys-121 plays an important role in mediating conformational changes that accompany the activation of the tyrosine kinase activity and the insulin-stimulated reduction in ligand binding affinity.


EXPERIMENTAL PROCEDURES

Materials

The CHO cell lines CHO-HIRC and CHO-M121 have been described in detail previously(16) . In brief, both lines were derived from stable transfections with cytomegalovirus-driven expression plasmids that expressed wild-type (HIRC) insulin receptor or the Lys-121 deletion mutation (M121) prepared by site-directed mutagenesis. Following selection by G418, cells were selected by fluorescence-activated cell sorting using an antibody against the alpha-subunit of the human insulin receptor(2) . An enriched population of cells was obtained which expressed similar amounts of wild-type insulin receptor or mutant receptor as determined by fluorescence-activated cell sorting. I-Insulin (2,000 Ci/mmol), [P]ATP (3,000 Ci/mmol), and L[S]methionine (1,000 Ci/mmol) were obtained from Amersham Corp. Polyclonal antibody against the carboxyl-terminal portion of the insulin receptor (a-CT) was a gift from Dr. Jaime Flores-Riveros, Miles Inc. Monoclonal antibody against phosphotyrosine (a-PY) and rabbit polyclonal antibody against the alpha-subunit (residues 657-670) of the human insulin receptor were purchased from Upstate Biotechnology, Inc., Lake Placid, NY. Pansorbin was obtained from Calbiochem. Protein G-Sepharose was from Pharmacia Biotech Inc. Alkaline phosphatase-conjugated anti-rabbit and anti-mouse immunoglobulin were from Dako Corp., Carpinteria, CA. 5-Bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium phosphatase substrate was bought from Kirkegaard and Perry Laboratories, Inc. Wheat germ agglutinin (WGA) gel was obtained from EY Labs Inc., San Mateo, CA.

Flow Cytometry Studies

The concentration of wild-type and mutant insulin receptors on the surface of CHO cells was determined by flow cytometry studies exactly as reported previously(2) . In the present studies, a monoclonal antibody, A-IR(1) was used as described.

Measurement of I-Insulin Binding, Internalization, and Degradation by CHO Cells

Standard insulin binding studies were conducted at 4 and at 24 °C in 24-well plates as described previously(2, 17) . In studies conducted at 37 °C, I-insulin was added, and the incubation continued for the indicated times. The medium was removed, and the cells were washed with ice-cold Krebs buffer, pH 7.4. The cells were dissolved in 1 N NaOH and the radioactivity determined to assess the total amount of radiolabeled insulin associated with the cells. In parallel studies, the cells were washed with ice-cold ``acid'' Krebs, i.e. pH 3.5, to remove cell surface-bound insulin. Solubilization and determination of the radioactivity allowed the amount of insulin in internal compartments of the cell to be determined (18, 19) . To evaluate insulin degradation, the media from the cells were subjected to 10% trichloroacetic acid precipitation(18) . Nonspecific associations of radiolabeled insulin were determined by the inclusion of excess native insulin (5,000 ng/ml) in parallel incubations.

Measurement of the Temperature Sensitivity for Insulin Receptors

Mutant and wild-type insulin receptors were partially purified from CHO cells by WGA affinity chromatography(20) . The WGA-purified receptors were incubated for the indicated time and temperature and then chilled to 4 °C. A soluble insulin binding assay was conducted at 4 °C as described, and the receptor-insulin complexes were precipitated by polyethylene glycol(21) . In some studies, the receptors were purified further using a-CT, which was raised against the amino acid sequence CNGRVLTLPRSNPS present in the carboxyl terminus of the mouse beta-subunit. This antibody immunoprecipitates mouse, rat, and human insulin receptors and does not precipitate the IGF-I receptor (data not shown). The antibody was absorbed to Pansorbin and then incubated with WGA extracts. Following extensive washes with 50 mM Tris, 0.1% Triton X-100, and 0.15 M NaCl, pH 7.4, and once with this buffer plus 0.5 M LiCl, the preparation was used to conduct the various studies described.

To prepare autophosphorylated insulin receptor, 100 µg of WGA extract was autophosphorylated under conditions described previously(2, 16) and then incubated with a-PY absorbed to protein G-agarose. After 3 h at 4 °C, the beads were collected and washed extensively with a 1% Triton buffer (50 mM Tris, 1% Triton X-100, pH 6.5) at 21 °C to remove bound insulin. The agarose beads were then washed three times with 50 mM Tris, 0.1% Triton X-100, and the receptor eluted from a-PY with 50 mMp-nitrophenyl phosphate. The eluted receptor was used in the various studies of temperature inactivation.

Western Blot Analysis of Insulin Receptors

WGA extracts containing the mutant and wild-type receptors (5 µg of protein/sample) were placed in 1% SDS, 50 mM Tris, pH 6.8, and 50 mM beta-mercaptoethanol, heated at 100 °C for 5 min, and then subjected to SDS-polyacrylamide gel electrophoresis (7.5% acrylamide resolving gel). The protein was transferred to Immobilon-P membrane, and the membrane was blocked with 5% milk in 50 mM Tris, pH 7.4, 0.05% Tween 20, and 0.1 M NaCl solution. The membrane was probed with antibody against the alpha-subunit of the insulin receptor (1 µg/ml) and the blot developed with alkaline phosphatase-conjugated anti-rabbit immunoglobulin using standard methods.

Insulin Dissociation Experiments

Fifty µg of WGA protein was incubated overnight at 4 °C with 0.5 µCi of I-insulin and a-CT absorbed to Pansorbin. The Pansorbin samples were washed twice in ice-cold 50 mM Tris, 0.1% Triton X-100, 1% bovine serum albumin, and 0.1 M NaCl, pH 7.4, and resuspended in 200 µl of this buffer. Dissociation of I-insulin was initiated by adding 50 µl of the suspension to 9.95 ml of buffer maintained at 22 °C. Native insulin was present in or absent from the dilution buffer as indicated. At different times after the dilution, 0.5-ml aliquots were subjected to centrifugation at 10,000 times g for 1 min at 4 °C, and the radioactivity in the pellet was determined by -counting. The amount of I-insulin bound to the receptor at zero time was determined by diluting the 50-µl aliquot into 9.95 ml of buffer maintained at 4 °C, and 0.5-ml samples were subjected immediately to centrifugation and the radioactivity in the pellets determined.


RESULTS

Abnormal Processing of Insulin by CHO Cells That Express the Mutant Insulin Receptor

Initial studies of the M121 receptor examined its ability to internalize and process insulin during insulin treatment of CHO cells at 37 °C. Fig. 1shows that CHO-HIRC and CHO-M121 cell lines have equal numbers of cell surface insulin receptors as measured by flow cytometry using an antibody specific for the alpha-subunit of the human insulin receptor. The results also indicate that each cell line is generally homogeneous for the concentration of insulin receptors, i.e. no significant subpopulation of cells exists which express significantly more or less receptors than the general cell population. Insulin binding studies have been conducted previously with both cell lines at 21 °C, and the results showed similar amounts of insulin binding activity(16) .


Figure 1: Laser flow cytometry of CHO-HIRC and CHO-121 cells. The studies were conducted as described under ``Experimental Procedures.''



Fig. 2illustrates the ability of the two cell lines to specifically bind, internalize, and degrade insulin at 37 °C. In these studies, I-insulin was added to confluent CHO-HIRC and CHO-M121 cells. At the indicated times, the amounts of I-insulin associated with the cell surface, the amounts internalized by the cells, and the amount of degraded insulin in the medium were determined. Fig. 2A shows that the amount of I-insulin bound to CHO-M121 was approximately 50% that bound to CHO-HIRC. The alteration in M121 cell-associated insulin also correlated with the reduction in the amount of I-insulin internalized, which was 20-30% the amount found in CHO-HIRC (Fig. 2A). This reduction in insulin internalization was parallel to a reduction in the amount of degraded insulin in the medium (Fig. 2B).


Figure 2: Time course for insulin binding, insulin internalization, and insulin degradation by CHO cells. Panel A, confluent CHO-HIRC and CHO-M121 were incubated at 37 °C with I-insulin. At the indicated times, the total amount of hormone associated with the cells (squares) or the acid-resistant (intracellular) amount (triangles) were determined as described under ``Experimental Procedures.'' Panel B, degradation of I-insulin by CHO cells was measured at the indicated times. The data are representative of four separate experiments.



The marked discrepancy between the ability of CHO-M121 and CHO-HIRC cells to process insulin might result from a number of causes, including the kinase signaling defect noted previously for this mutation(16) . Alternatively, it could be related to an abnormal temperature sensitivity of binding, which had escaped detection in earlier work since our previous insulin binding measurements were made at temperatures of 22 °C or lower.

M121 Insulin Receptor Loses Its Ability to Bind Insulin at Elevated Temperatures

The insulin binding activity of WGA-purified receptors was determined after incubation for the indicated times at 37 °C (Fig. 3A). As shown, the incubation of wild-type receptor for periods up to 350 min did not alter its ability to bind insulin when the receptor was subsequently cooled to 4 °C and binding activity measured. In contrast, the mutant receptor's binding activity rapidly decreased (t of approximately 45 min) during the 37 °C incubation.


Figure 3: Effect of incubation at 37 °C on the insulin binding activity of the partially purified M121 and wild-type (HIRC) insulin receptors. Panel A, WGA-purified receptors were incubated for the indicated period of time at 37 °C and then chilled to 4 °C. Insulin binding activity was measured at 4 °C (see ``Experimental Procedures''). The B/F value for insulin binding at the 0 time point (100% binding) for both receptor types was approximately 0.35. The results are the mean of three separate experiments. Panel B, insulin receptors in the WGA extract were further purified by a-CT and immobilized on Pansorbin. Following this step they were incubated at 37 °C for the indicated periods of time. The samples were then cooled to 4 °C, and insulin binding studies were performed on the immobilized receptors. The B/F value for the 0 time point for the HIRC receptor was 0.42 and for M121 receptor was 0.45. Panel C, WGA extracts containing the receptors were subjected to insulin-mediated autophosphorylation as described under ``Experimental Procedures.'' The phosphorylated receptors were immunoprecipitated by a-PY and protein G-agarose. The immunoprecipitates were washed in a 1% Triton X-100, slightly acid (pH 6.5) buffer to remove insulin, and the receptors were eluted from the antibody with p-nitrophenyl phosphate. These eluates underwent incubation at 37 °C for the times indicated after which they were tested for insulin binding activity at 4 °C.



Similar studies were also conducted on receptors purified by precipitation with a-CT, an antibody specific for the carboxyl terminus of the beta-subunit. This immobilized preparation showed normal insulin binding characteristics at 4 and 21 °C for wild-type and M121 receptors (data not shown). In contrast, as illustrated in Fig. 3B, the mutant receptor again demonstrated a marked reduction in insulin binding activity after incubation at 37 °C. Such results with an immunopurified receptor preparation argue that non-receptor contaminants in the WGA extract do not contribute to the loss of insulin binding activity by the mutant receptor.

Fig. 3C provides additional data that support a change in the temperature sensitivity of the mutant receptor. In this study wild-type and mutant receptors underwent insulin-stimulated autophosphorylation. The autophosphorylated receptors were purified with an a-PY, a step that also allowed insulin to be removed in anticipation of conducting insulin binding measurements. As shown, the phosphorylated mutant receptor is much more sensitive to 37 °C treatment than the nonphosphorylated form, i.e. almost complete destruction of insulin binding activity by 10 min as compared with a 50% reduction of binding by the nonphosphorylated mutant receptor by 40-50 min. As in the other studies, the wild-type receptor was stable to incubation at 37 °C, even when in the phosphorylated form.

Experiments were conducted to evaluate whether or not the mutant receptor remained intact during the 37 °C incubation. Fig. 4A shows a Western blot of the wild-type and mutant receptor alpha-subunit before and after incubation at 37 °C. The amount of alpha-subunit did not change during the incubation of either receptor type. The experiment described in Fig. 4B investigated whether or not the alpha(2)beta(2) holoreceptor structure remains intact during the 37 °C incubation. In these studies the receptors were incubated with [P]ATP at 21 °C in the presence and absence of insulin. The receptor preparations were then incubated at 37 °C for 1 h, conditions that destroy 50% of the insulin binding activity of the mutant receptor. As the nonreducing SDS-polyacrylamide gel electrophoresis shows, the size of the mutant holoreceptor was similar to the wild-type receptor. No evidence was found of smaller receptor fragments that would indicate disruption of its tetrameric structure.


Figure 4: Structural examination of M121 and HIRC receptors after incubation at 37 °C. Panel A, Western blot of the alpha-subunit was performed on the WGA extracts after the indicated period of incubation of WGA extracts at 37 °C. The arrow indicates the position of the alpha-subunit. Panel B, SDS-polyacrylamide gel electrophoresis of the nonreduced receptors following autophosphorylation at 22 °C in the presence or absence of insulin as described under ``Experimental Procedures.'' The receptors were either placed at 4 °C or subjected to a 1-h incubation at 37 °C. The receptors were collected by polyethylene glycol precipitation and subjected to SDS-polyacrylamide gel electrophoresis using a 5% resolving gel as described above but in the absence of reductant.



These results clearly show that insulin binding by the isolated mutant receptor is abnormally sensitive to incubation at 37 °C. Moreover, the failure to retain insulin binding activity is not due to gross structural changes in its alpha(2)beta(2) architecture. A more detailed examination of the temperature sensitivity of mutant and wild-type receptors is shown in Fig. 5. In this study the receptors were immunopurified with a-CT and immobilized on Pansorbin. They were then subjected to the indicated temperature for 1 h and cooled to 4 °C for measurement of insulin binding activity. As shown, there is a sharp drop in insulin binding activity by the M121 receptor as the temperature is raised above 30 °C. In contrast, the insulin binding activity of the wild-type receptor is stable up to temperatures of 40 °C.


Figure 5: The effect of temperature on insulin binding activity by M121 and HIRC receptors. The receptors were immobilized with the a-CT antibody on Pansorbin. They were incubated for 1 h at the indicated temperatures and then chilled to 4 °C. Insulin binding activity was measured at 4 °C. The results are the mean ± S.E. of three separate experiments.



Fig. 6illustrates Scatchard plots of insulin binding data for the two receptor types before and after incubation at 37 °C. In agreement with previous findings(16) , the Scatchard plot obtained at 21 °C for the mutant receptor is very similar to that for the wild-type receptor (panel A). As expected, 37 °C treatment for 45 min caused a marked decrease in the amount of high affinity insulin binding activity by the mutant receptor preparation (panel B). This indicates that the loss in binding activity of the M121 receptor is due to an irreversible inactivation of the binding site rather than a decrease in binding affinity.


Figure 6: Scatchard plots of insulin binding by M121 and HIRC receptors before and after incubation at 37 °C. Panel A, insulin binding studies using WGA-purified receptors were conducted at 22 °C in the absence of a 37 °C treatment period. Panel B, the receptors were incubated for 45 min at 37 °C before conducting the insulin binding studies at 22 °C. The results are representative of three separate experiments.



Studies of Insulin Dissociation

Although Scatchard plots of the mutant and wild-type receptors were similar in the unheated preparations, which suggest normal insulin binding kinetics for the mutation, a more extensive analysis of the insulin binding kinetics was undertaken which included the measurements of insulin dissociation rates. The receptors immobilized on Pansorbin were first allowed to bind I-insulin at 4 °C, and dissociation of the hormone was induced by dilution with buffer maintained at 22 °C. Control studies show that the conditions used to bind I-insulin to the receptors resulted in the labeling of about 5% of the total insulin binding sites (data not shown). In addition the steps involved in the washing of the immobilized receptors and their dilution for dissociation measurements ensured that essentially no rebinding of the tracer insulin occurs once it has dissociated from the binding site.

As shown in Fig. 7A, the rate of dissociation in the absence of any native insulin in the dilution buffer was slow for both wild-type and mutant receptors. The addition of 100 nM insulin to the dilution buffer caused an acceleration in the rate of insulin dissociation from both receptor types. However, this insulin-mediated effect was much less with the mutant than with the wild-type receptor, i.e. at 2.5-min dissociation in the presence of 100 nM insulin, the mutant receptor retained approximately 60% of the insulin bound at zero time compared with the 33% retained by the wild-type receptor. These findings clearly demonstrate a difference in the insulin binding properties of the mutant receptor, even in the absence of heat treatment. The same qualitative differences between mutant and wild-type receptors were found when the receptor preparations underwent a 37 °C incubation for 45 min before the dissociation experiments (Fig. 7B).


Figure 7: Studies of negative cooperativity with M121 and HIRC receptors. Panel A, receptors from WGA extracts were immobilized with a-CT on Pansorbin. The immobilized receptor preparations were maintained at 4 °C and labeled with I-insulin. Studies of the rate of dissociation at 22 °C were conducted as described under ``Experimental Procedures.'' The triangles (also shown as +) indicate dissociation in the presence of 100 nM insulin. The squares represent dissociation in the absence of native insulin. Similar amounts of specifically bound I-insulin were used to initiate these experiments (at 0 time, HIRC = 47,500 cpm and M121 receptor = 38,700 cpm). Panel B, the receptor preparations were first treated at 37 °C for 45 min before being chilled to 4 °C and labeled with I-insulin. Cooperativity experiments were then conducted as described in panel A. The amount of labeled insulin bound to the receptor preparations at 0 time differed for HIRC receptors (41,400 cpm) and M121 receptors (20,300 cpm) even though the same amount of material was used as for the experiments shown in panel A. This difference is expected because of the loss of binding activity by the M121 receptor when incubated at 37 °C.



Fig. 8illustrates the dose-response characteristics for the insulin-mediated accelerated dissociation of the bound ligand. The reduced response of the mutant receptor is again evident, showing approximately one-half the response of the wild-type receptor. However, the shapes of the curves are not different between the two receptor types. Maximal effect is present at 100-300 nM insulin, and half-maximal effect is produced by 3-5 nM hormone. The similarity also includes the typical biphasic shape of the curve in which very high insulin concentrations actually impede the negative cooperative effects(22, 23) .


Figure 8: Insulin dose-response characteristics for the negative cooperativity effect. The rate of dissociation mediated by the indicated concentration of insulin in the dissociation buffer was measured using the immobilized receptor preparations. The results are plotted as the amount of I-insulin that dissociates from the receptors in 5 min at 22 °C.



Protection of the Insulin Binding Site with Insulin

Table 1provides the results from a study of the protective effect of insulin on temperature inactivation of insulin binding by the Lys-121 mutation. In these studies the receptor immobilized on a-CT and Pansorbin was incubated with the indicated concentration of insulin or IGF-I during a 37 °C treatment period for 2 h. In these studies this treatment reduced insulin binding activity of the receptor to 35%. However, in the presence of insulin concentrations as low as 0.3 nM, the loss of insulin binding activity was reduced. Thirty nM insulin provided a retention of 80% of the insulin binding activity.



Specificity for this protection effect was demonstrated by a parallel experiment in which IGF-I was used in place of insulin. Only at the highest concentration of 30 nM IGF-I was there a clear protection of the binding site. Thus, the presence of insulin in the binding pocket stabilizes this region and protects it from inactivation by heat treatment.

Sensitivity of the Mutant Receptor to Alkylating and Reducing Reagents

The effect of iodoacetamide on insulin binding was examined to determine if the 37 °C incubation produced abnormally positioned disulfide bridges (Table 2). However, as indicated, iodoacetamide did not protect the mutant receptor against temperature-induced inactivation of insulin binding. Instead, at 100 mM iodoacetamide, the mutant receptor showed a marked increase in the rate of inactivation. Thus, formation of new disulfide bridges during a 37 °C incubation does not appear to contribute to the loss of insulin binding.



A second group of experiments probed the receptor with the reducing reagent, DTT (Table 2). From these results, it is clear that even at 21 °C the mutant receptor is more sensitive to DTT than the wild-type receptor. Conducting the experiment at 37 °C increased the effect of DTT on insulin binding by both the wild-type and mutant receptors (data not shown). Here again, the mutant receptor was much more sensitive to DTT than the wild-type receptor.

Effects of Trypsin on Insulin Binding Activity

Studies were conducted to determine the effect of mild trypsin treatment on insulin binding activity of wild-type and mutant receptors (Fig. 9). The incubations were standardized for the same amount of protein and insulin binding activity and then treated with trypsin for the indicated period of time. Insulin binding activity at tracer I-insulin concentration by the wild-type receptor increased significantly following 5-60 min of trypsin treatment, which is similar to the effect of elastase treatment on adipocyte insulin binding reported previously(24) . In marked contrast, the M121 receptor rapidly lost insulin binding activity, with 50% of the binding lost at 1 h of protease treatment. These findings support further the presence of substantial alterations in the conformation of the M121 receptor.


Figure 9: Effect of mild proteolysis on insulin binding by M121 and wild-type receptors. WGA extracts (3 µg of protein in 15 µl) were treated for the indicated times with 1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (0.15 µg) at 22 °C. At the times shown, the reaction was terminated by the addition of trypsin inhibitor (0.5 µg). Insulin binding activity was measured by the soluble binding assay as described under ``Experimental Procedures.''



Mutant Insulin Receptors in Membranes from CHO Cells

The previous findings raise the question of how the M121 receptor with intact insulin binding activity can be isolated from CHO cells grown at 37 °C. This issue was examined by conducting insulin binding studies with membranes isolated from CHO-HIRC and CHO-M121 cells (Fig. 10). As shown, insulin binding by mutant and wild-type receptors in the CHO membranes demonstrated the same responses to 37 °C incubation. In both preparations, there is a slow and gradual lose of insulin binding activity. Therefore it is possible to retrieve active M121 receptor because the membrane environment protects its insulin binding activity.


Figure 10: Effect of 37 °C incubation on insulin binding by membranes from CHO-M121 and CHO-HIRC cells. The membrane preparations were incubated at 37 °C for the indicated periods of time and then cooled to 4 °C. Insulin binding activity by the membranes was determined during a 16-h incubation at 4 °C as described under ``Experimental Procedures.'' The B/F ratio at the 100% point for the membranes for both cell types was standardized at 0.56. The results are the mean ± S.E. of three experiments.



Although the mutant receptor is protected against temperature-induced inactivation when in the cell membrane, the receptor continues to show the same abnormal response to insulin-induced dissociation of bound tracer hormone as the detergent-solubilized receptor (Fig. 11). In fact the membrane data from the studies of insulin dissociation are very similar to those from the studies of the isolated and purified receptors.


Figure 11: Measurement of negative cooperativity in membranes from CHO-M121 and CHO-HIRC cells. The membranes were allowed to bind I-insulin during a 16-h incubation at 4 °C. The membrane preparations were diluted 100-fold in dilution buffer at 22 °C in the absence (squares) and presence (triangles) of 100 nM insulin. At the indicated times, the membranes were collected by rapid centrifugation and the radioactivity remaining in the membrane fraction determined. The values for the 100% point at 0 time were 2,800 cpm for CHO-HIRC and 2,300 cpm for the CHO-M121. The results are representative of three separate experiments.




DISCUSSION

Deletion of Lys-121 in the insulin receptor generates a mutation with unusual properties that include the alterations in its signaling activity as reported previously (16) and as shown by the present work, changes in its temperature sensitivity for insulin binding, and alterations in its negative cooperative interactions. Although a number of other insulin receptor mutations have been studied (for review see (25) ), this mutation is the first to demonstrate such a widespread combination of alterations and is the first to show abnormal sensitivity to physiologic and subphysiologic temperatures.

From these findings we hypothesize that Lys-121 is an important functional constituent in a region that mediates conformational changes. It is clear that Lys-121 is not part of the contact sites which binds insulin with high affinity since this function of the mutant receptor is normal at permissive temperatures under steady-state conditions. In addition, other data from site-directed mutations and from the construction of insulin receptor/IGF-I receptor chimeras have not placed Lys-121 directly in the binding pocket(15) . The previous work generally argues that the amino-terminal portion of the alpha-subunit and the cysteine-rich region are the main (direct) determinants of high affinity insulin binding activity.

The interpretation of the data which we favor is based on an important role for Lys-121 in ligand-mediated conformational changes. Such a role would explain the altered regulation of the tyrosine kinase activity of the mutant receptor (16) and the change in ligand-mediated insulin dissociation. It also highlights a possible relationship between conformational changes that activate the kinase domain and changes that contribute to negative cooperativity.

Several findings argue for changes in the conformation of the mutant receptor. Among these of course is the abnormal response to temperature, an effect that irreversibly destroys high affinity insulin binding activity. This destruction can be prevented by insulin, demonstrating that occupancy of the binding site helps maintain the receptor's structural integrity.

Although alterations are induced by temperatures above 30 °C, abnormalities can also be detected at temperatures below 30 °C. For example, mild trypsin treatment at 21 °C produces differential effects on insulin binding between wild-type and mutant receptors. In the wild-type receptor, mild proteolysis appears to relieve some constraints that allow insulin binding activity actually to increase. In contrast, insulin binding by the mutant receptor decreases rapidly during proteolysis. Since the mutation does not increase the number of trypsin-sensitive sites, an increase in trypsin sensitivity is likely due to conformational differences between the mutation and wild-type receptors.

Other evidence of conformational differences includes the altered sensitivity of the mutant receptor to chemical probes. We chose to probe the binding activity with reducing and alkylating reagents, since the mutation does not alter the number of potential disulfide bridges or the number of sulfhydryl groups. Interestingly, the insulin binding activity of the mutant receptor was more sensitive to either reagent than the wild-type receptor. Such changes argue that critical disulfides and sulfhydryl groups are more available to these reagents in the mutant receptor, which is again consistent with an altered conformation.

An especially interesting phenomenon that was altered in the mutation at permissive temperatures is negative cooperativity. The Lys-121 mutation apparently reduces ligand-induced acceleration of the ``off'' rate for I-insulin by about 50% when compared with the wild-type receptor. There remains, however, a remnant of the negative cooperative effect in the mutant receptor, a component that demonstrates a normal biphasic dose-response relationship(22, 23) . Thus, the mutant receptor has about a 50% reduction in insulin-mediated negative cooperativity which parallels the 50% reduction in insulin-stimulated tyrosine kinase activity(16) .

Negative cooperativity has a controversial history in which various explanations have been formulated to explain accelerated ligand dissociation from the receptor(23, 26) . Although the underlying reason for the alteration in cooperativity is not clear, especially since the molecular events required for the cooperative effect are not well understood, the present findings add support to the concept that negative cooperativity is an integral, functional part of the insulin receptor.

Among the questions that the new findings raise is the lack of a correlation between the steady-state insulin binding data (Scatchard plot) and the kinetic data that measure insulin dissociation. One would expect a reduction in negative cooperativity to diminish the curvilinear property of the Scatchard plot. However, very little if any difference exists between the plots for the mutant receptor, which displays a reduced negative cooperativity, and the wild-type receptor, which displays normal cooperativity. This lack of agreement may arise from a greater sensitivity provided by dissociation studies for determining receptor binding affinity or from an inability of negative cooperativity to contribute significantly to the curvilinearity of Scatchard plots.

The Lys-121 deletion is not the only mutation in the insulin receptor which disrupts negative cooperativity. Kadowaki et al.(4) have reported a Glu substitution for Lys-460 which has a reduction in negative cooperativity. Like the Lys-121 deletion, the Glu-460 substitution demonstrated a normal curvilinear Scatchard plot. However, the substitution of Arg at the 460 site reduced negative cooperative effects even more than the Glu substitution and further altered the receptor's binding affinity. The Scatchard plot for this mutant receptor was not curvilinear, indicating in this case a correspondence between a loss of cooperativity and a loss of curvilinearity of the Scatchard plot.

Neither the Arg or Glu substitution mutations for Lys-460 mimicked exactly the findings from the M121 mutation since the biphasic insulin dose-response relationship on the remaining cooperative effect was lost in the 460 mutations. Moreover, the Glu-460 mutation demonstrated an altered pH profile for insulin binding(27) , a change not evident in the Lys-121 mutation (data not shown). The Glu-460 mutation also had an increased resistance to temperature inactivation of insulin binding, and it differed further from the Lys-121 mutation by having a normal ability to signal, i.e. a normal insulin-mediated tyrosine kinase activity and ability to promote insulin action in fibroblasts. It was concluded that the reason for the marked insulin resistance associated with the Glu-460 mutation is not an alteration in signaling or insulin binding, but a faulty ability of the cell to synthesize the mutant receptor(4) .

In summary, studies of the Lys-121 mutation have demonstrated a new region in the insulin receptor which mediates conformational changes. These experiments also provide correlative findings that argue for a relationship between the insulin activation of the tyrosine kinase domain in the beta-subunit with the phenomenon of negative cooperativity. A more detailed understanding of the Lys-121 region may offer new insights into the structural changes required for the insulin receptor to activate its kinase domain and to alter its insulin binding affinity.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant RO1 DK40394. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Pharmaceutical Division, Miles Inc., 400 Morgan La., West Haven, CT 06516-4175. Tel.: 203-931-5308; Fax: 203-937-2686.

(^1)
The abbreviations used are: CHO, Chinese hamster ovary; M121 mutation, the deletion of Lys-121 in the human insulin receptor; CHO-M121, CHO cells that express the M121 mutation; CHO-HIRC, CHO cells that express the wild-type human insulin receptor; a-CT, antibody against the carboxyl-terminal portion of the insulin receptor; a-PY, antibody against phosphotyrosine; WGA, wheat germ agglutinin; IGF, insulin-like growth factor; DTT, dithiothreitol.


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