(Received for publication, August 2, 1996, and in revised form, October 28, 1996)
From the Departments of Medicine and Physiology and
Biophysics, State University of New York at Stony Brook,
Stony Brook, New York 11794 and ¶ Insulin Research, NOVO
NORDISK, 2880-Bagsvaerd, Denmark
Recent studies utilizing alanine scanning
mutagenesis have identified a major ligand binding domain of the
secreted recombinant insulin receptor composed of two subdomains, one
between amino acids 1 and 120 and the other between amino acids 704 and
716. In order to obtain a more detailed characterization of these
subdomains, we examined the binding of an insulin superanalog,
des-(B25-30)-[His-A8, Asp-B10, Tyr-B25 -carboxamide]insulin, to
alanine mutants of the ligand binding determinants of these subdomains.
cDNAs encoding mutant secreted recombinant receptors were
transiently expressed in 293 EBNA cells, and the binding properties for
this analog of the expressed receptors were evaluated. In general
des-(B25-30)-[His-A8, Asp-B10, Tyr-B25
-carboxamide]insulin
binding correlated with insulin binding, suggesting that both peptides
bound to the receptor in a similar manner. Alanine mutations of eight
amino acids (Asn15, Phe64, Phe705,
Glu706, Tyr708, Leu709,
Asn711, and Phe714) of the receptor produced
the most profound decreases in affinity for des-(B25-30)-[His-A8,
Asp-B10, Tyr-B25
-carboxamide]insulin, suggesting that interactions
with these amino acids contributed the major part of the free energy of
the ligand-receptor interaction. Mutation of Arg14 and
His710 to Ala produced receptors with undetectable insulin
binding but an affinity for des-(B25-30)-[His-A8, Asp-B10, Tyr-B25
-carboxamide]insulin only 8-23-fold less than for native receptor.
Further analog studies were performed to elucidate this paradox. The
receptor binding potencies of His-A8 and Asp-B10 insulins for these
receptor mutants appeared to parallel their relative potencies for
native receptor. In contrast the receptor binding potency of
des-(B25-30)-[Tyr-B25
-carboxamide]insulin was disproportionately
increased for these mutants when compared with its potency for native
receptor.
Insulin exerts its initial effects on target cells by binding to a
heterotetrameric cell surface receptor (for review see Ref. 1). The
receptor is composed of disulfide-linked heterodimers, which are
composed in turn of an extracellular subunit and a
subunit,
which is both extra- and intracellular, linked by disulfide bonds.
Insulin binding leads to the activation of the intrinsic tyrosine
kinase activity of the receptor
subunit cytoplasmic domain and
phosphorylation of intracellular substrates of the receptor, including
IRS-1 (2), IRS-2 (3), and SHC (4, 5), leading to the intracellular
events responsible for signal transduction (6). Structural and
mutational studies indicate that the receptor kinase is activated by
transphosphorylation of the receptor's cytoplasmic domain (7, 8), but
the molecular basis of the regulation of this process by ligand binding
still remains obscure. It is thought that by analogy to the
ligand-induced dimerization of the monomeric receptor tyrosine kinases,
insulin binding to the extracellular domain of the receptor either
produces an approximation of or a reorientation of the cytoplasmic
domains permissive for the transphosphorylation event (for review see Ref. 9).
The initial event in this pathway, the binding of insulin to its receptor, has been the subject of intensive study. The structure of insulin, as well as that of many analogs, has been solved at high resolution (for review see Ref. 10). However, it is only recently that a coherent model of its interaction with its receptor has been developed. A current consensus is that isoleucine A2, valine A3, valine B12, phenylalanines B24 and B25, tyrosine A19, asparagine A21, and the partially buried residues leucines A16 and B15 are the major determinants of the receptor binding site with threonine A8, serine B9, histidine B10, glutamate B13, and tyrosine B16 making minor contributions.1 The role of phenylalanines B24 and B25 in receptor binding have been subject to particular scrutiny (11-13), because mutations in these positions have been associated with diabetes (14). Systematic studies of insulin analogs modified in these positions (11-13) have lead to the suggestion that these residues are responsible for intial interaction with the receptor and that this interaction produces a conformational change in the COOH terminus of the B-chain, which is essential for high affinity receptor binding.
Considerably less is known about the ligand binding sites of the
insulin receptor. The results of affinity labeling experiments (15-18)
and studies with chimeric insulin receptors (19-21) point to the
involvement of at least three separate regions of the subunit in
the process of insulin binding; amino acids 1-120 (15, 16, 19, 20),
450-524 (17, 20, 21), and 714 -718 (18). More recently utilizing
alanine scanning mutagenesis, we have provided evidence for a major
ligand binding site composed of two subdomains widely separated in the
primary sequence. The amino-terminal subdomain is located between amino
acids 1 and 120 (22) and is composed of 14 amino acids arranged in four
discontinuous peptide segments. The carboxyl-terminal subdomain, amino
acids 704-716, consists of 12 amino acids (23) organized in three
discontinuous segments. Unfortunately, although these studies were
adequate for localization of this ligand binding domain, its detailed
functional characterization was not possible due to technical
limitations of the assay employed. Thus in the present study we have
further characterized this ligand binding site by evaluating the
binding of the des-(B25-30)[His-A8, Asp-B10, Tyr-B25
-carboxamide]insulin (X92)2 (24), an
insulin superanalog, to alanine mutants of the amino acids composing
this binding site. During this study, two alanine mutations were
identified in which the affinity of insulin was disproportionately
decreased compared with that of X92. Subsequent studies with other
insulin analogs showed that this compromise in affinity for insulin was
dependent on the presence of the amino acids of the COOH terminus of
the B-chain of the insulin molecule.
Mutant insulin receptor cDNAs were generated and expressed in transient transfection experiments as described previously (22, 23). The mutants are designated by the amino acids mutated to alanine using the single letter code followed by the number(s) indicating their position in the sequence of the insulin receptor, thus T704A is a mutant in which Thr704 of the receptor has been mutated to alanine. It should be noted that subsequent studies of the mutant V713A, which we previously reported to have a very low affinity for insulin (23), have shown that it has normal affinity for both insulin and X92 (data not shown). This mutant was therefore not utilized in this study. Human insulin and insulin analogs were synthesized as described previously (24). Carrier free 125I (IMS 30) was from Amersham Corp. Protease inhibitors were from Boehringer Mannheim. 293 EBNA cells were purchased from Invitrogen (San Diego, CA). Medium for tissue culture was from Mediatech (Herndon, VA), and serum was from Sigma. Anti-insulin receptor monoclonal antibody 18-44 (25) was generously provided by Dr. M. Soos and K. Siddle (University of Cambridge, Cambridge, UK). Affinity purified goat anti-mouse IgG was from Pierce.
Radioiodination of Insulin and AnalogsFor ligand binding studies insulin and X92 were iodinated with 125I by the lactoperoxidase method. Monoiodo-Tyr-A14 isomers were separated from the iodination reactions by reverse phase high pressure liquid chromatography using a C18 column (26).
Insulin and Analog Binding AssaysSoluble insulin receptor binding assays were performed using a microtiter plate antibody capture assay (22, 23). Microtiter plates (Immulon 4, Dynal Inc., Lake Success, NY) were incubated with affinity purified goat anti-mouse IgG (50 µl/well of 20 µg/ml solution in 0.2 M sodium carbonate, pH 9.4) for 2 h at room temperature. After washing and blocking for 15 min with 250 µl of SuperblockTM (Pierce), wells were then incubated overnight at 4 °C with a 1:100 dilution in SuperblockTM of crude ascites of anti-insulin receptor monoclonal antibody 18-44 (25). After washing with phosphate-buffered saline, wells were incubated for 4 h at 4 °C with soluble receptor diluted to give 10-20% [125I-Tyr-A14]insulin or X92 binding in the absence of added unlabeled insulin or analog under assay conditions. After washing with wash buffer (0.15 M sodium chloride, 20 mM Hepes, pH 7.8, 0.1% bovine serum albumin (w/v), 0.025% Triton X-100 (v/v), and 0.02% sodium azide (w/v)), wells were incubated for 48 h at 4 °C with [125I-Tyr-A14]insulin or X92 (12 pM) and varying concentrations of unlabeled insulin or analog in 100 µl of binding buffer (137.5 mM sodium chloride, 10 mM magnesium sulfate, 20 mM Hepes, pH 7.8, 0.5% bovine serum albumin (w/v), 0.025% Triton X-100 (v/v), and 0.02% sodium azide (w/v)). To terminate the assay, wells were aspirated and washed three times with 300 µl of ice-cold wash buffer and then counted.
Insulin binding data were analyzed by the LIGAND program (27) in order to obtain the Kd of the expressed protein. IC50 values were obtained from a four parameter logistic fit of competition curves using the program Kaleidagraph. Transfection and binding assays were repeated at least once to confirm the Kd of each mutant. Each result is the mean of at least two independent experiments.
Wild type and mutant insulin receptor cDNAs were transiently
expressed in 293 EBNA cells. Equilibrium binding studies were performed
on conditioned medium with labeled and unlabeled X92. As described
previously for insulin binding to this form of recombinant wild type
receptor (22, 23), binding of X92 displayed simple kinetics with a
linear Scatchard plot (data not shown). Analysis with the LIGAND
program indicated a single population of binding sites with a
Kd of 3.3 ± 0.01 × 1012
M (mean ± S.E., n = 6). This
represents an affinity for this analog, which is 180-fold greater than
that for insulin (Kd = 0.56 ± 0.02 × 10
9 M, mean ± S.E., n = 6).
The results of the analyses of alanine mutants of the insulin receptor
are shown in Table I; results are expressed as a ratio of the Kd of the alanine mutant to the
Kd of the wild type receptor, and the corresponding
values for insulin are shown for comparison. In general, for mutants in
which quantitation for both ligands was technically feasible, the
decrease in affinity was reasonably comparable for both ligands,
suggesting that the molecular mechanisms underlying binding of X92 and
insulin are similar. The exceptions to this finding were the mutants
D12A, R14A, N15A, Y91A, T704A, and H710A. The most extreme divergence was observed for R14A and H710A, which had an unmeasurable affinity for
insulin but only a 8- and 23-fold decrease in affinity for X92,
respectively (Tables I and II); IC50 values
for insulin displacement of labeled X92 from these mutant receptors,
shown in Table II, indicate that their affinity for insulin is
decreased approximately 500-1000-fold. These data also suggest that the COOH-terminal subdomain is the major contributor to the affinity of
this binding site for X92. Fig. 1 shows the free energy
contribution (G) of each mutated amino acid to the
ligand receptor interaction.3
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In order to determine which of the three modifications of the insulin
molecule present in X92 was responsible for the X92 binding properties
of the R14A and H710A mutants, we examined displacement of
[125I-Tyr-A14]X92 from wild type and the two mutant
receptors by unlabeled His-A8 insulin (X8), Asp-B10 insulin (X10), and
des-(B25-30)-[Thr-B25 -carboxamide] insulin (X137).
IC50 values for analog displacement of
[125I-Tyr-A14]X92 from wild type receptor of the analogs
are shown in Table II; the relative potencies for X92, X137, X10, X8,
and insulin in this assay were 205, 7.9, 4.1, 8.8, and 1, respectively. The IC50 values for the displacement of
[125I-Tyr-A14]X92 by these analogs from the mutant
receptors, R14A and H710A, are also shown in Table II. His-A8 insulin
exhibited increases in relative affinity for the R14A and H710A mutants (10- and 7-fold, respectively; Table II) that were comparable with the
9-fold increase in its affinity for wild type receptor (Table II). The
increased affinity of Asp-B10 insulin for the R14A and H710A mutants
(5.5- and 4-fold, respectively; Table II) was also comparable with the
4-fold increase in affinity observed for wild type receptor (Table II).
In contrast removal of the COOH-terminal pentapeptide of the insulin
B-chain and substitution of Phe B25 by Tyrosine
-carboxamide as seen
in X137 caused a disproportionate increase in affinity for the the R14A
and H710A mutant receptors (140- and 114-fold, respectively; Table II)
when compared with the 8-fold increase in affinity of this analog for wild type insulin receptor (Table II). Taken together these findings suggest that the compromised affinity of these mutations for insulin is
dependent in large part on the presence of the COOH-terminal pentapeptide of the insulin molecule.
In the present study we have used insulin analog binding to
perform a detailed characterization of a major ligand binding site of
the insulin receptor. In initial studies we examined the binding of the
insulin superanalog X92 to alanine mutants of the receptor with
compromised affinity for insulin. This analog has an affinity for the
receptor that is 180-200-fold that of insulin and is produced by a
triple modification of the native insulin molecule. Substitution of
threonine at position A8 by histidine, as is found in avian insulins,
increases the affinity of insulin for the insulin receptor 8-10-fold
(28); substitution of histidine at position B10 by aspartate produces a
4-5-fold increase in affinity, as originally described for a human
mutant insulin (29); and deletion of the COOH-terminal pentapeptide
combined with the replacement of phenylalanine B25 by tyrosine
-carboxamide also produces a 8-10-fold increase in affinity (24).
With the exception of the removal of the COOH-terminal pentapeptide of
the insulin B-chain, the precise structural mechanisms by which these
modifications affect affinity are unclear. However, it is probable that
the overall structure of this molecule and its conformation in the high
affinity receptor complex are therefore likely to be very similar to
that of insulin, and it is thus a valid tool for characterizing insulin-receptor interactions. This is confirmed by the overall similarity in the changes in affinity for the two peptides produced by
the mutations, with the exceptions Asp12,
Arg14, Asn15, Tyr91,
Thr704, and His710 to alanine (Table I). The
discrepancies between the effects of these mutations on the affinities
for insulin and X92 are presumably a reflection of the differences in
the molecular mechanisms underlying the interactions of the two
peptides with the insulin receptor; this is discussed further below for
the Arg14 and His710 to alanine mutations.
In a review of the additivity of the effects of mutations on proteins,
Wells concluded that for the majority of protein-protein interactions,
the free energy changes due to multiple mutations exhibited simple
additivity (30). However, it was noted that large deviations from
simple additivity could occur when the mutations produced large
structural perturbations or when the sites of mutation interacted with
one another. In the present study, the sum of the calculated
G values (33.8 kcal/mol) for all the mutations we have
evaluated (Fig. 1) is double that of the free energy of ligand binding
(14.9 kcal/mol) calculated from the dissociation constant of X92 for
the native receptor. It is unlikely that this is due to methodological
errors arising from the precision of the assay (see Table II). This
deviation from additivity has therefore probably arisen from major
structural perturbations or changes in intramolecular interactions
caused by the mutations. As we have discussed in previous studies of
these receptor mutants (21, 22), it is unlikely that there is any major
structural perturbation of the mutant proteins, and thus it is probable
that some of these mutations compromise insulin and X92 binding by
alteration of intramolecular interactions within the ligand binding
site of the receptor.
When the effects of the individual mutations on the free energy of the
interaction of X92 with the insulin receptor are considered individually, it is apparent that mutations in the COOH-terminal subdomain produce the largest effects. Alanine substitution of phenylalanine 705 and glutamate 706 had the greatest impact (>3 kcal/mol; Fig. 1), and with the exception of Val715 and
His710 (see below for more detailed discusssion) all other
substitutions in this subdomain (Tyr708,
Leu709, Asn711, and Phe714) also
produced profound decreases in free energy of ligand binding (2-3
kcal/mol); the perturbation of binding produced by mutation of
Thr704 to Ala appears to be an indirect effect (see Ref. 23
for detailed discussion). In the NH2-terminal subdomain
only two substitutions, Asn15 and Phe64,
produced comparable changes in free energy. Alanine mutation of all
other amino acids critical for high affinity insulin binding within
this subdomain only produced free energy changes ranging from 0.3 to
1.3 kcal/mol (Fig. 1). These findings are in marked contrast to the
findings of Clackson and Wells for growth hormone-receptor interactions
(31). This interaction has a dissociation constant (0.3 × 10
10 M) similar to that of the
insulin-receptor interaction. Mutational analysis of the ligand binding
site of the receptor indicates that the free energy of the interaction
is dominated by the contributions of two tryptophan residues that
contribute >4 kcal/mol each to the energy of ligand binding. With the
exception of one residue that contributes approximately 3.2 kcal/mol,
the contribution of each of the remaining 29 residues of the ligand
binding site, when considered individually is less than or equal to 2 kcal/mol. Thus, the molecular mechanisms underlying the ligand-receptor interactions may be somewhat different in the two systems. In the
growth hormone system the free energy of binding is generated from the
major contributions of two amino acids, whereas in the insulin system
it seems to be generated from more modest contributions of a larger
number of amino acids. This conclusion should, however, be interpreted
with caution because some of the insulin receptor mutations that we
have studied are probably disrupting intramolecular interactions within
the binding site rather than direct ligand receptor interactions, as
noted in the discussion in the preceding paragraph.
The striking discrepancies observed between the affinities of R14A and
H710A mutants for insulin and X92 are worthy of further discussion.
Studies with the individually substituted analogs suggest that this is
predominantly due to the removal of the COOH-terminal pentapeptide of
the insulin B-chain. This mirrors findings with certain insulin
analogs. Systematic studies of substitutions for phenylalanine B25 of
the insulin molecule indicate that the presence aromatic ring in
this position is essential for high affinity receptor insulin binding.
This appears only to apply to the full-length molecule because removal
of the COOH-terminal pentapeptide restores the receptor binding potency
of such substituted analogs (12, 13). These studies have lead to the
suggestion that an initial interaction with the receptor via B25
phenylalanine leads to a conformational change in the main chain of the
COOH terminus of the insulin molecule that is essential for the
completion of the interaction with the receptor (12, 13). This is
supported by the finding that insulin analogs that are cross-linked
between glycine A1 and lysine B29, in which movement of the COOH
terminus of the B-chain is constrained, exhibit varying decreases in
biological activity, depending on the length and flexibility of the
cross-link (32, 33), despite the lack of major perturbation of their structure. Solution NMR studies of the structure of Gly-B24 insulin provide further insights into possible structural mechanisms underlying these findings (34). In this analog the COOH terminus of the B-chain is
unfolded and disordered, exposing residues in the core of the molecule,
in particular valine A3, which has been shown to be essential for
receptor binding (35). It is therefore possible that interaction of
phenylalanine B25 with the receptor produces a similar change in
conformation of the insulin molecule, allowing completion of
interaction with the receptor.
Thus, in light of the finding that the interaction of the full-length
insulin molecule with the receptor mutants R14A and H710A resembles
that of insulin analogs substituted in the B25 position with wild type
insulin receptor, it is tempting to suggest that the mutated residues,
Arg14 and His710, interact with B25
phenylalanine of the insulin molecule to produce the conformational
changes in the main chain of the COOH terminus of the B-chain necessary
for high affinity insulin binding. Further studies of these mutants
with insulin analogs are in progress to test this hypothesis. It is,
however, unlikely that the effects of these mutations on the affinity
of the receptor for insulin are confined to disruption of interactions
with the aromatic ring of Phe-B25 of the insulin molecule because
they produce a 500-1000-fold decrease in affinity (Table II), whereas
substitution of Phe-B25 by alanine only results in a 100-fold decrease
in affinity (12). Further, the relative receptor binding potencies of
X137 for the mutants R14A and H710A are 36- and 64-fold less than that of this analog for native receptor, respectively (Table II). High resolution structural analysis of the hormone-receptor complex will be
essential to fully elucidate the molecular mechanisms underlying the
perturbation of the ligand-receptor interaction produced by these
mutations.
We are grateful to Drs. K. Siddle and M. Soos, University of Cambridge, Cambridge, UK for providing monoclonal antibodies to the insulin receptor.