(Received for publication, October 17, 1995)
From the
A recent affinity labeling study has suggested that amino acids
704-717 of the C terminus of the insulin receptor represent a
contact site for insulin. To determine whether these amino acids are
part of a ligand binding site, we have performed alanine-scanning
mutagenesis of this region. Mutant cDNAs encoding recombinant secreted
receptors were transiently expressed in 293 EBNA cells, and their
insulin binding properties were evaluated. Of the 14 residues in this
region only 4 amino acids, Asp-707, Val-712, Pro-716, and Arg-717,
could be mutated to alanine without compromising insulin binding. The
reduction in affinity resulting from the individual mutation of the
remaining amino acids varied from an increase in K to 3.69
10
M (Asn-711)
to greater than 10
M (Thr-704, Phe-705,
Glu-706, and His-710); the K
of native
secreted recombinant receptor is 0.56
10
M.
Insulin initiates signal transduction in target cells by binding to a specific cell surface receptor, which is a member of the growth factor receptor tyrosine kinase superfamily of proteins(1) . Ligand binding leads to the activation of the receptor's tyrosine kinase activity and the initiation of intracellular signaling. Mutational studies of receptor signaling and the elucidation of the structure of the receptor's tyrosine kinase catalytic domain suggest that kinase activation is effected by intramolecular transphosphorylation of the constituent tyrosine kinase catalytic domains of the receptor heterotetramer(2, 3) . The molecular details of the mechanism by which insulin binding initiates the transphosphorylation event remain obscure and will require an understanding of the molecular organization of the extracellular domain of the receptor and the molecular basis of insulin binding.
The
insulin receptor is composed of two disulfide-linked heterodimers, each
of which is composed in turn of a 135-kDa subunit (entirely
extracellular) linked by a disulfide bond to a 95-kDa
subunit,
which has an extracellular domain, a single
helical transmembrane
domain, and an intracellular domain containing the tyrosine kinase
catalytic activity(1) . While the tertiary structure of the
extracellular domain has not been elucidated, the presence of several
characteristic structural motifs can be predicted from inspection of
the deduced amino acid sequence(4, 5) . The
subunit contains a cysteine-rich domain homologous to that of the
epidermal growth factor receptor(4, 5) , and there are
also two fibronectin type III repeats; the first is composed of the C
terminus of the
subunit and the N terminus of the
subunit,
and the second is composed of the C terminus of the extracellular
region of the
subunit(6) .
Bajaj et al.(7) 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. This model predicts that
there are two homologous globular domains flanking the cysteine-rich
domains: domain L1 containing amino acids 1-119 and domain L2
containing amino acids 311-428. Each contains repeating
structural 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.
This proposal has received support from recent experimental
observations(8, 9, 10, 11, 12) .
We have recently performed alanine-scanning mutagenesis of the L1
domain of the insulin receptor and have shown it it to contain a ligand
binding domain composed of 14 amino acids organized in four
discontinuous peptide segments(13) . However, it is unlikely
that this is the complete insulin binding site, as secreted recombinant
receptors with deletions N-terminal to the C terminus of the
subunit fail to bind insulin(14) . Further, mutant receptors
which are not proteolytically cleaved into mature
and
subunits only bind insulin with low affinity(15) . Recently
Kurose et al.(16) , using affinity labeling
techniques, identified a novel insulin contact site just proximal to
the C terminus of the
subunit between threonine 704 and lysine
718. In the present study we have performed alanine-scanning
mutagenesis of this region of the receptor and demonstrated that it is
a major determinant of insulin binding.
These constructs were expressed in 293 EBNA cells (an adenovirus-transformed human kidney cell line expressing Epstein-Barr virus nuclear antigen) by transfection with 2 µg of miniprep DNA using the commercially available lipofection reagent Lipofectamine (Life Technologies, Inc.) according to the manufacturer's directions. For analysis of transient expression, medium and cells were harvested 1 week after transfection. Conditioned medium was concentrated prior to assay using Centriprep 100 centrifugal concentrators (Amicon, Inc., 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(8, 13) , thus simplifying the analysis of binding data. Further, it has been demonstrated that affinity changes produced by mutations of this form of receptor show good correlation with those observed in the membrane-bound receptor as a consequence of the same mutation(8, 9, 13, 22) .
Insulin binding data were
analyzed by the LIGAND program (24) in order to obtain the K of the expressed protein. Transfection and
binding assays were repeated at least once to confirm the K
of each mutant. Each result is the mean of two
experiments.
Mutant receptor cDNAs were transiently expressed in 293 EBNA
cells. To evaluate expression and post-translational processing of the
receptor, conditioned medium from transfected cells and detergent
lysates of the cells were analyzed by Western blotting with an antibody
directed toward the C terminus of the subunit of the insulin
receptor. In conditioned medium, insulin receptor
subunit, M
135,000, was detectable in all transfections (Fig. 1). In detergent lysates of cells, a protein of M
160,000, corresponding to the predicted mobility
of the secreted receptor
precursor(20, 26, 27) , was expressed in all
transfected cells (Fig. 1). The M
135,000
band observed in the cell lysates probably represents a nonspecifically
reacting protein as it is detectable in equivalent amounts in
transfected and nontransfected 293 EBNA cells (data not shown). For
each transfection the amount of detectable
subunit relative to
precursor was similar (Fig. 1), suggesting that the mutations
did not cause any major perturbation of post-translational processing
of the receptor.
Figure 1:
Immunoblotting of mutant receptors. 293
EBNA cells were transfected with cDNAs encoding alanine mutants of the
secreted recombinant insulin receptor extracellular domain. One-week
post-transfection-conditioned medium was harvested and concentrated and
cells were lysed in detergent as described under ``Materials and
Methods.'' For each transfection, equivalent amounts of medium (M) and lysate (L) were separated by
SDS-polyacrylamide gel electrophoresis on 7.5% reducing gels and
transferred to polyvinylidene difluoride membranes. Membranes were
probed with a polyclonal antibody directed toward the insulin receptor
subunit and visualized by enhanced chemiluminescence (ECL).
Wild-type receptor is designated WT, and the mutants are
designated by the amino acids mutated to alanine, using the single
letter code, followed by the numbers indicating their positions in the
sequence of the insulin
receptor(4, 5) .
As in our previous study(13) , insulin
binding to recombinant insulin receptor extracellular domain secreted
by transiently transfected 293 EBNA cells displayed simple kinetics
with a linear Scatchard plot (data not shown). However, analysis with
the LIGAND program (24) indicated a single population of
binding sites with a K of 0.56 ± 0.02
10
M (mean ± S.E., n = 6) in contrast to our earlier study (13) in which
we obtained a K
of 1.41 ± 0.09
10
M (mean ± S.E., n = 6) due to the modification of the assay
conditions(28) . Since studies utilizing alanine-scanning
mutagenesis have demonstrated that meaningful changes in affinity
produced by a single alanine substitution range from 2- to 100-fold (29) , in the experiments described below we regarded any
mutant with a greater than 2-fold increase in K
(i.e. K
greater than 1.2
10
M) as demonstrating a significant
disruption of insulin-receptor interactions.
The effects of alanine
mutations of amino acids in the region between threonine 704 and lysine
718 on the dissociation constant (K) of the
secreted receptor for insulin are shown in Table 1. Mutation of
Asp-707, Val-712, Pro-716, and Arg-717 appeared to be without
deleterious effect on insulin binding; in fact mutation of Asp-707,
Val-712, and Pro-716 produced small increases in affinity for insulin (K
= 0.28, 0.31, and 0.37
10
M, respectively). Mutation of all other
amino acids in this region produced marked perturbations of insulin
binding, with K
values of the mutants varying from
3.69
10
M for mutation of Asn-711
to levels too high to be determined accurately by our assay method (K
> 10
M) for
Thr-704, Phe-705, Glu-706, His-710, and Val-713 (Table 1). These
data are represented graphically in Fig. 2as ratios of the K
of the mutant to that of wild type in order to
demonstrate the relative contributions of the individual amino acids to
the free energy of insulin binding.
Figure 2:
Alanine-scanning mutagenesis of the
C-terminal subunit 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).
Mutant receptors are designated by the amino acids mutated to alanine,
using the single letter code, followed by the numbers indicating their
positions in the sequence of the insulin
receptor(4, 5) . The K
Mut/K
WT could not be accurately determined for mutations of T704A, F705A, E706A, H710A, and V713A (designated by *).
Our results demonstrate that the region between amino acids
704 and 717 of the insulin receptor subunit appears to play a
major role in high affinity insulin binding by the secreted recombinant
receptor. Of the 14 residues in this region only 4 amino acids,
Asp-707, Val-712, Pro-716, and Arg-717, can be mutated to alanine
without severely compromising insulin binding. The reduction in
affinity resulting from the individual mutation of the remaining amino
acids varied from an increase in K
of 3.69
10
M (Asn-711) to greater than
10
M (Thr-704, Phe-705, Glu-706, and
His-710). As with our previous findings for the L1 domain(13) ,
it appears that the aromatic amino acids of this region of the receptor
(Phe-705, Tyr-708, and Phe-715) play a prominent role in insulin
binding.
Several lines of evidence suggest that the decreases in affinity observed as a consequence of these mutations, with the possible exception of Thr-704 (see below), 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(29) . 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(30) . Second, in common with other membrane and secreted proteins (for review, see (31) ) studies of naturally occurring mutants of the insulin receptor associated with extreme insulin resistance (32) and of secreted C-terminal deletion mutants of the receptor (6) 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 similar to both those of the wild-type protein and those of mutants that were without effect on insulin binding, and their post-translational processing was normal.
While we have not been able to accurately determine the affinity of all mutants in either this study or our previous study because of technical limitations of our assay procedure, it appears that the amino acids in this region make as great if not greater contribution to the free energy of insulin binding than do those in the L1 domain(13) . In the L1 domain mutation of only 2 amino acids, Arg-14 and Phe-64 to alanine, produced a decrease in affinity that was too great to be determined (13) . Mutation of Asn-15 to alanine produced a 250-fold decrease in affinity, and mutation of Phe-39 to alanine caused a 35-fold decrease (13) . Mutation of the remaining amino acids implicated in insulin binding only produced decreases in affinity ranging from 3- to 15-fold (13) . In contrast, in the scan of amino acids 704-717, mutation of 4 amino acids (Thr-704, Phe-705, Glu-706, and His-710) produced decreases in affinity that were too great to be accurately quantitated, and mutation of 3 amino acids (Tyr-708, Leu-709, and Phe-714) caused a greater than 120-fold decrease in affinity. Certainly the magnitude of these free energy contributions is sufficient to account for the finding that secreted recombinant proteins generated from receptor cDNAs encoding truncations N-terminal to this region fail to bind insulin, although they are secreted from transfected cells as stable proteins(6) . Also they are of sufficient magnitude to account for the low affinity of the uncleaved precursor of the alternately spliced isoform of the receptor lacking the amino acids encoded by exon 11 (15) . The proteolytic cleavage of this form of the receptor may, therefore, be a prerequisite for this region of the receptor to adopt its normal conformation. Also their role in insulin binding may account for the variation in the affinity for insulin of the two alternately spliced isoforms of the receptor(33) .
The
equivalent region of the insulin-like growth factor-1 receptor (amino
acids 690-704) is highly conserved (34) and thus may play
a similar role in ligand binding. There are only two nonconservative
substitutions, Val-690 for Thr-704 and Ser-699 for Val-712, and of
these substituted residues only Thr-704 appears to be critical for high
affinity insulin binding. However, its role may be indirect. Chimeric
receptors produced by replacement of the N terminus of the insulin-like
growth factor-1 receptor by the corresponding region of the insulin
receptor bind insulin with relatively high
affinity(8, 10) . This observation effectively
excludes the possibility of the participation of this side chain in a
direct interaction with insulin, unless compensatory substitutions are
present in the insulin-like growth factor-1 receptor. Thus there may be
a necessity for a -methyl group in this position for the
appropriate orientation of other side chains critical for insulin
binding. It has also been suggested that the C-terminal region of the
subunit is a determinant of ligand specificity of the insulin and
insulin-like growth factor-1 receptors (35) . The
conservatively substituted residues, Asp-707, Tyr-708, and Val-713,
could, therefore, play a role in mediating specificity for insulin.
In two recently proposed models of insulin-receptor
interactions(28, 36) , it has been suggested that
insulin has two receptor binding sites and that the receptor
heterodimer in turn has two ligand binding sites. High affinity insulin
binding is generated by the asymmetrical binding of one insulin
molecule to the two receptor heterodimers constituting the
holoreceptor, i.e. Site 1 of the insulin molecule binds to
Site 1 on the first heterodimer and Site 2 of the insulin molecule
binds to Site 2 on the second heterodimer according to Schaffer's
nomenclature(28) . In the isolated heterodimer and the secreted
receptor extracellular domain, only one receptor insulin binding site
on the insulin molecule (Site 1) binds to its corresponding site on the
receptor heterodimer (Site 1), producing low affinity interactions (28) . Since in both this and our previous study (13) we have examined the effects of alanine mutations on the
affinity of the secreted recombinant insulin receptor, the ligand
binding determinants we have identified are those of binding Site 1
according to this model of insulin-receptor interactions. More
quantitative analyses will be necessary to determine whether the free
energy contributions of the amino acids, which we have identified in
the N and C terminus of the subunit as being essential for high
affinity insulin binding, are sufficient to account for the free energy
of insulin binding of this form of the receptor. In addition, high
resolution structural analysis will be essential to fully characterize
the role of these amino acids in insulin binding.