(Received for publication, August 5, 1994; and in revised form, October 25, 1994)
From the
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.
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
-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
-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 ()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.
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.
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.
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 -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 -subunit before and after
incubation at 37 °C. The amount of
-subunit did not change
during the incubation of either receptor type. The experiment described
in Fig. 4B investigated whether or not the
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 -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
-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
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.
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.
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.
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.
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.''
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.
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 -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 -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.