(Received for publication, November 4, 1994; and in revised form, December 6, 1994)
From the
Affinity labeling studies and mutational analyses have implicated the involvement of a predicted domain of the insulin receptor (L1, amino acids 1-119) in ligand binding. In order to obtain a higher resolution localization of this ligand binding site, we have performed alanine scanning mutagenesis of this domain. Alanine mutant cDNAs encoding a secreted recombinant insulin receptor extracellular domain were expressed transiently in adenovirus transformed human embryonic kidney cells and the affinity of the expressed receptor for insulin was determined. Mutation of 14 amino acids located in four discontinuous peptide segments to alanine was disruptive of insulin binding: Segment 1, amino acids 12-15; Segment 2, amino acids 34-44; Segment 3, amino acids 64-67; and Segment 4, amino acids 89-91. The quantitative contribution of the four segments to the free energy of insulin binding was 1 > 3 > 2 > 4. Of the 14 amino acids whose mutation compromised insulin binding, 3 are charged, 3 hydrophobic, 5 aromatic, and 3 are amides.
Insulin initiates signal transduction in target cells by binding to a specific cell surface receptor(1) . This probably leads to conformational changes in the extracellular domains, which are transmitted across the cell membrane and result in activation of the receptor's tyrosine kinase activity. The molecular details of these events are obscure and will require a detailed understanding of the structure function relationships of the protein, in particular those of the extracellular domain.
The primary structure of this
region has been deduced from the predicted amino acid sequence of the
cloned human insulin receptor cDNA (2, 3) . It is
composed of two disulfide-linked heterodimers, each of which is
composed in turn of an M 135,000
subunit,
which is entirely extracellular, linked by a disulfide bond to an M
95,000
subunit, which has an extracellular
domain, a single
helical transmembrane domain, and an
intracellular domain containing the tyrosine kinase catalytic activity.
The
subunit contains a cysteine-rich domain homologous to that of
the epidermal growth factor receptor, and there are also possibly two
fibronectin type III repeats in the extracellular domain(4) .
Bajaj et al.(5) have proposed a hypothetical model
of the tertiary structure of the receptor extracellular domain based on
homologies between the primary structures of the epidermal growth
factor and insulin receptor families of tyrosine kinases(5) .
This model predicts that there are two homologous globular domains
flanking the cysteine domains: domain L1 containing amino acids
1-119 and domain L2 containing amino acids 311-428. Each
contains repeating structural motifs (Motifs I-V) composed of
helix,
turn
, and hypervariable structures. Since all
deletions and insertions occur in the hypervariable structures in the
sequence alignments obtained for these proteins with this model, it was
suggested that these may represent components of ligand binding
domains.
For the insulin receptor this proposal has received support
from recent experimental observations. Affinity labeling studies and
mutagenic analyses suggest the involvement of both the NH terminus of the molecule (6, 7) and also a
region COOH-terminal to the cysteine domain (8) in insulin
binding. Furthermore, studies of the ligand properties of chimeric
receptors produced from the insulin and the related IGF-1 receptors
indicate that residues 1-68 (9) and 325-524 (10) are involved in conferring ligand specificity, although
the role of the latter residues is probably minor. In addition point
mutations of amino acids located within the L1 domain, Asn-15 (11) and Phe-89(12, 13) , have been shown to
compromise high affinity insulin binding.
In view of this compelling evidence for the involvement of the L1 domain of the insulin receptor in ligand binding, we have undertaken, in the present study, a high resolution analysis of the residues involved by alanine scanning mutagenesis as a first step in elucidating the detailed mechanism of insulin receptor signal transduction. Since studies of insulin structure function relationships suggest a prominent role for aromatic residues in receptor interactions(14) , we performed both aromatic to alanine and charged to alanine mutagenesis. The results of these experiments indicate that the L1 domain of the insulin receptor contains an insulin binding site composed of four discontinuous polypeptide segments containing 14 amino acids, the mutation of which compromised insulin binding.
These constructs were expressed in 293EBNA cells (an adenovirus transformed human kidney cell line expressing EBV nuclear antigen) by transfection with 2 µg of Miniprep DNA using the commercially available lipofection reagent Lipofectamine (Life Technologies) according to the manufacturers' directions. For analysis of transient expression, media and cells were harvested 1 week after transfection. Conditioned medium was concentrated prior to assay using Centriprep 100 centrifugal concentrators (Amicon, Beverly, MA).
We chose to utilize the extracellular domain for these experiments, as it is expressed in large amounts by this expression system, and insulin only binds to a single homogeneous population of binding sites in this protein which have been shown to be contained within the L1 domain(9) , thus simplifying the analysis of binding data.
Insulin binding data were analyzed by the LIGAND program (20) in order to obtain the K of the
expressed protein. Trasfection and binding assays were repeated at
least once to confirm the K
of each mutant. Each
result is the mean of two experiments.
As described previously, insulin binding to recombinant
insulin receptor extracellular domain secreted by transiently
transfected 293EBNA cells displayed simple kinetics with a linear
Scatchard plot (data not shown)(19) . Analysis with the LIGAND
program (20) indicated a single population of binding sites
with a K of 1.41 ± 0.09
10
M (mean ± S.E., n = 6). Since previous studies utilizing alanine scanning
mutagenesis have demonstrated that meaningful changes in affinity
produced by a single alanine substitution range from 2- to 100-fold (21) , in the experiments described below we regarded any
mutant with a greater than 2-fold increase in K
, i.e.K
greater than 2.8
10
M, as causing a significant disruption
in insulin-receptor interactions.
The initial mutants analyzed were clustered charged/aromatic mutations. The results of these analyses are shown in Table 1, part A. None of these mutants displayed any abnormality except the the receptor with the simultaneous mutation of Phe-64 and Arg-65 (A64F/A65R), which did not appear to be secreted although Western blotting of lysates of the transfected cells revealed detectable levels of receptor precursor (data not shown), a pattern of disruption of post-translational processing indicative of impaired folding of the nascent proreceptor(11, 22) . Mutations of Phe-64 and Arg-65 were therefore analyzed individually (see below).
We then proceeded to perform alanine scanning mutagenesis of
individual charged amino acids (Table 1, part B). This identified
Arg-14 as a major determinant of insulin binding; mutation of this
residue to alanine led to the expression of a receptor whose affinity
for insulin was too low to be measured by the methodology used for this
study. In the same region mutation of D12 to alanine produced a 5-fold
reduction in affinity for insulin (K = 9.12
10
M). A further mutation of Glu-44
to alanine caused a 3-4 fold decrease in affinity (K
= 6.82
10
M). Mutation of Arg-65 to alanine had no effect on
expression or secretion of the resulting protein, and its affinity for
insulin was normal. Mutation of Glu97 resulted in impaired receptor
secretion, although the precursor of this mutant protein was detectable
by immunoblotting of lysates of transfected cells (data not shown),
again suggesting malfolding of the mutant.
We next undertook
aromatic to alanine mutagenesis (Table 1, part C). This revealed
4 additional residues whose mutation significantly compromised insulin
binding; Phe-39 (K = 35.70
10
M), Phe-64 (affinity too low to be
accurately measured), Phe-89 (K
= 5.17
10
M), and Tyr-91 (K
= 3.85
10
M). Phe-39
is located in a region that has previously been reported to be
important for conferring insulin specificity in studies of the binding
properties of chimeric insulin-IGF-1 receptors(9) . Phe-89 has
also previously been reported to be important for high affinity insulin
binding(12, 13) . Mutation of Tyr-60 and Phe-96 led to
a failure of secretion into the medium although the presence of their
precursors were visualized in cell lysates by Western blotting (data
not shown), suggesting that these mutations compromise the structure of
the receptor. It is of interest that Phe-96 is a neighbor of Glu-97
whose mutation to alanine also caused impaired expression in the
charged to alanine mutagenesis experiments, suggesting the importance
of this region for folding of the molecule into a native conformation.
These residues are conserved in the insulin receptor, the IGF-1
receptor, and the insulin receptor-related protein(23) ,
further emphasizing their critical roles in the maintenance of the
conformation of this family of proteins.
To further define the structures in which alanine mutations compromised binding, we mutated amino acids surrounding those that we had previously identified as being necessary for high affinity binding. To avoid potential structural perturbations, we did not mutate prolines, cysteines, or potential N-linked glycosylation sites. The results of these scans confirmed that the insulin binding epitope was composed of four discontinuous peptide segments; Segment 1 from Asp-12 to Asn-15, Segment 2 from Gln-34 to Glu-44, Segment 3 from Phe-64 to Tyr-67, and Segment 4 from Phe-89 to Tyr-91 (see Table 1, part D, and Fig. 1). In Segment 1, mutations produced decreases in affinity ranging from 6- to 7-fold (D12A) to too low to accurately determine (R14A).In the the second segment, mutations produced 3-fold (M38A) to 25-fold (F39A) reductions in affinity. In addition, mutations L33A, I35A, and L37A led to a failure of secretion of protein, although expression of precursor is detectable in cell lysates (data not shown). Mutations K40A, T41A, and R42A were without effect on insulin binding. In Segment 3 only mutations F64A and Y67A reduced affinity for insulin (too low to be determined and 2.2-fold, respectively). Mutations R65A and V66A produced receptors with normal affinity for insulin. In Segment 4 mutations reduced affinity from 2.5-fold (Y91A) to 6-fold (N90A).
Figure 1:
Alanine scanning mutagenesis of the
NH-terminal ligand binding site of the recombinant secreted
insulin receptor. Data from Table 1are expressed as a ratio of
the K
of the mutant to that of wild type
recombinant insulin receptor (K
(mut)/K
(wt)).
Results for amino acids 1-60 are shown in the upperpanel and those for amino acids 61-120 in the lowerpanel. Amino acids are designated by single-letter code. The K
(mut)/K
(wt)
could not be accurately determined for mutations of Arg-14 and Phe-64
(designated by *). K
(mut)/K
(wt) for the mutation of N15
is 250 as indicated on the figure.
In the present study we have identified 14 amino acids
organized into four discontinuous segments, which appear to be the
major functional determinants of the N-terminal ligand binding domain
of the insulin receptor - Segment 1 (Asp-12, Ile-13, Arg-14, and
Asn-15), Segment 2 (Gln-34, Leu-36, Met-38, Phe-39, and Glu-44),
Segment 3 (Phe-64 and Tyr-67), and Segment 4 (Phe-89, Asn-90, and
Tyr-91). Of the 14 amino acids, 3 are charged, 3 hydrophobic, 5
aromatic, and 3 are amides. The prominence of the aromatic residues
further emphasizes the role of aromatic interactions in insulin
receptor interactions. The predicted secondary structure of the four
segments according to the model of Bajaj et al.(5) is: Segment 1: strand,
helix (Motifs I and
II, respectively); Segment 2:
strand, loop (Motif II); Segment 3:
loop (Motif III); Segment 4: loop (Motif IV).
Thus, as suggested by
these authors, the predicted loop structures appear to play a prominent
role in ligand binding. However, in contrast to
immunoglobulins(24) , the ligand binding epitope is not
confined to these structures as both predicted strand and
helical structures are also involved. Precedents for this have been
reported for the growth hormone receptor(21) .
Several lines of evidence suggest that the decreases in affinity observed with these mutations are probably direct effects on ligand receptor interactions rather than the consequences of misfolding of the mutant proteins. Previous analyses of protein structure and function have shown that the effects of alanine mutants tend to be localized and nondisruptive of global protein structure(21) . In the case of the growth hormone-growth hormone receptor interactions, crystallographic studies have confirmed the involvement of determinants identified by scanning mutagenesis in hormone receptor interactions(25) . Second, in common with other membrane and secreted proteins (for review, see (26) ), studies of naturally occurring mutants of the insulin receptor associated with extreme insulin resistance (11) and of secreted COOH-terminal deletion mutants of the receptor (22) indicate that there is a strict requirement for folding into a native conformation prior to completion of post-translational processing and transport to the membrane or secretion. In the present study, all the mutants disruptive of insulin binding were secreted at levels comparable to that of the wild type protein and those of mutants that were without effect on insulin binding.
Of the four segments that we have identified, Segment 1 appears to be quantitatively the most important in its contribution to the free energy of binding, probably followed by Segment 3, and then 2, with the smallest contribution coming from Segment 4. Mutation of Arg-14 to alanine results in a receptor with an unmeasurably low affinity and mutation of Asn-15 to alanine producing a greater than 200-fold decrease. Interestingly a mutation at this position has been identified in a patient with extreme insulin resistance(11) . In this mutant the asparagine was mutated to lysine and resulted in impaired folding of the receptor and retention in the endoplasmic reticulum. However, those receptors that did reach the cell membrane exhibited a 5-fold decrease in affinity for insulin. In contrast the substitution of alanine results in the expression of a protein that appears to have native structure, since it is secreted in quantities comparable to that of the wild type receptor and post-translational processing does not appear to be impaired. The alanine substitution, however, produced a much more profound decrease in affinity than the lysine substitution, presumably because of the loss of the side chain. Phe-64 in segment 3 also appears to make a contribution to the free energy of binding comparable to that of Arg-14. However, accurate determination of the affinity of these residues will be required before we can definitively evaluate their relative contributions to the free energy of insulin binding.
Studies of insulin and IGF-1 specificity with chimeric receptors have identified amino acids 1-68 as being the major determinant of insulin specificity of the secreted soluble receptor(9) . Within this region it appears that amino acid differences in the region 38-68 are the most important. Thus, on the basis of the results of our scanning mutagenesis, one would predict Met-38, Phe-39, and Tyr-67 to be the major determinants of specificity with the major energetic contribution being provided by Phe-39. Certainly the combined effects of mutation of these residues to alanine are consistent with the observed differences in affinities between the secreted recombinant insulin and IGF-1 receptors for insulin (9) (assuming that resulting free energy changes of the combined mutations are additive; (27) ). The data acquired from scanning mutagenesis also allow us to predict the residues in the insulin receptor-related protein that contribute to the low affinity of this protein for insulin. There are significant sequence differences between the two proteins in Segments 1 and 4(23) . In Segment 1, Asn-15 of the insulin receptor is replaced by serine in the insulin receptor related protein; and in epitope 4, Phe-89 and Asn-90 of the insulin receptor are replaced by leucine and glycine, respectively. The combination of these substitutions is likely to cause a profound decrease in affinity for insulin. Experiments are currently in progress to test these predictions.
While it should be noted that these
results were obtained with a recombinant receptor that has an affinity
at least an order of magnitude lower than that of wild type
receptor(28) , there are good reasons to believe that the
results can be extrapolated to the native receptor. Recently two models
have been proposed to explain the complex kinetics of insulin
binding(28, 29) . Both propose that there are two
distinct receptor binding sites on the insulin molecule and that there
are two distinct insulin binding sites on each subunit, Site 1
and Site 2. Insulin first binds to the site with higher affinity, Site
1, on one
subunit and then cross-links the two heterodimers by
binding to Site 2 on the second
subunit, generating the high
affinity component of the receptor interaction. Binding of a second
insulin molecule in this manner disrupts the cross-linking of the first
and accelerates its dissociation (negative cooperativity). In the
recombinant secreted receptor and the isolated heterodimer, insulin
only binds to Site 1, and hence this interaction displays a lower
affinity than that of the native receptor and simple binding kinetics.
From the data presented by Shaffer, it is apparent that the affinity of
Site 1 is very much greater than that of Site 2. Thus it would be
expected mutations producing changes in the affinity of this binding
site for insulin, in the recombinant secreted receptor would also
produce comparable changes in affinity of the holoreceptor. This
conclusion is supported by experimental evidence for Segments
2-4. It has been reported that the relative increase in affinity
for insulin of chimeric IGF-1 receptors in which amino acids 1-62
have been substituted by the corresponding region of the insulin
receptor is comparable for both the secreted and full-length
recombinant proteins, suggesting that the contributions to the free
energy of binding of Met-38, Phe-39, and Tyr-67 that we have
demonstrated will be comparable in the both forms of the receptor.
Additionally, Schumacher et al.(13) have studied the
properties of the F89A mutation in the holoreceptor, and the changes in
affinity that they reported are very similar to those we have found for
the secreted form of this mutant.
This study thus confirms the utility of alanine scanning mutagenesis for the investigation of insulin-receptor interactions. This approach is has enabled us to map a discontinuous epitope of the insulin receptor involved in insulin binding, which contains polypeptide segments that would have escaped attention using previously employed systematic mutagenic approaches. However, while this analysis implies direct molecular interactions between this epitope and the insulin molecule, it does not provide proof of such interactions. Direct proof will require a high resolution structural analysis.