A Comparative Structural Bioinformatics Analysis of the Insulin Receptor Famiy Ectodomain Based on Phylogenetic Information

A Comparative Structural Bioinformatics Analysis of the
Insulin Receptor Family Ectodomain Based on
Phylogenetic Information
Miguel E. Renterıá1¤
, Neha S. Gandhi1
, Pablo Vinuesa3
, Erik Helmerhorst1
, Ricardo L. Mancera1,2
*
1 Western Australian Biomedical Research Institute and School of Biomedical Sciences, Curtin University of Technology, Perth, Western Austrailia, Australia, 2 School of
Pharmacy, Curtin University of Technology, Perth, Western Austrailia, Australia, 3 Centro de Ciencias Geno´micas, Universidad Nacional Autońoma de Me´xico, Cuernavaca,
Morelos, Mexico
Abstract
The insulin receptor (IR), the insulin-like growth factor 1 receptor (IGF1R) and the insulin receptor-related receptor (IRR) are
covalently-linked homodimers made up of several structural domains. The molecular mechanism of ligand binding to the
ectodomain of these receptors and the resulting activation of their tyrosine kinase domain is still not well understood. We
have carried out an amino acid residue conservation analysis in order to reconstruct the phylogeny of the IR Family. We
have confirmed the location of ligand binding site 1 of the IGF1R and IR. Importantly, we have also predicted the likely
location of the insulin binding site 2 on the surface of the fibronectin type III domains of the IR. An evolutionary conserved
surface on the second leucine-rich domain that may interact with the ligand could not be detected. We suggest a possible
mechanical trigger of the activation of the IR that involves a slight ‘twist’ rotation of the last two fibronectin type III domains
in order to face the likely location of insulin. Finally, a strong selective pressure was found amongst the IRR orthologous
sequences, suggesting that this orphan receptor has a yet unknown physiological role which may be conserved from
amphibians to mammals.
Citation: Renterıá ME, Gandhi NS, Vinuesa P, Helmerhorst E, Mancera RL (2008) A Comparative Structural Bioinformatics Analysis of the Insulin Receptor Family
Ectodomain Based on Phylogenetic Information. PLoS ONE 3(11): e3667. doi:10.1371/journal.pone.0003667
Editor: Mark Isalan, Center for Genomic Regulation, Spain
Received May 8, 2008; Accepted October 20, 2008; Published November 7, 2008
Copyright: ß 2008 Renterıá et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Western Australian Biomedical Research Institute and School of Biomedical Sciences and School of Pharmacy, Curtin University of Technology
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: R.Mancera@curtin.edu.au
¤ Current address: Centro de Ciencias Geno´micas, Universidad Nacional Autońoma de Me´xico, Cuernavaca, Morelos, Mexico
Introduction
Insulin and the insulin-like growth factors (IGFs) are homologous
protein hormones that play distinct physiological roles in mammals
and other animals. Whilst the former is the primary regulator of
carbohydrate homeostasis and has effects on lipid and protein
metabolism [1,2] the latter stimulate cell growth, replication and
differentiation [3,4]. The mechanism of action of these hormones is
mediated by their specific binding to the Insulin Receptor (IR) or
the type 1 Insulin-like Growth Factor Receptor (IGF1R) [5].
The IR and IGF1R, along with the IR-Related Receptor (IRR)
[6,7,8] form subclass II of the Receptor Tyrosine Kinase (RTK)
superfamily [9], and unlike the other members which dimerise or
oligomerise upon ligand binding, the IR family members are pre-
formed covalently-linked homodimers (a2b2) consisting of several
structural domains [10]. It is possible that these receptors also
function as heterodimers, since IR/IGF1R hybrids have been
found in all tissues expressing both receptors [11,12] but their
physiological role remains unknown.
The IR is expressed in two isoforms IR-A (exon 112) and IR-B
(exon 11+) [13] that display differential kinase activity [14]. Both
isoforms have similar affinity for insulin [15]. However, IR-A
shows considerably higher affinity for IGF-1 and particularly for
IGF-2 than IR-B [16], and has been implicated together with the
IGF1R in malignant transformation [17,18].
Although no ligand has yet been associated to the IRR, its
expression in a variety of tissues including kidney, heart, liver and
pancreas has been reported [19]. Likewise, single and combined
IR family knockout models in mice were recently established [20],
suggesting that the IRR could function as an auxiliary member of
the IR family, a role that may extend to other co-expressed
recognition molecules, such as the TrkA receptor [21,22].
IR family members are synthesised as single-chain pre-
proreceptors, which are then glycosylated, folded, dimerised and
processed to produce the mature a2b2 receptors [23]. Each
receptor consists of an ectodomain, a transmembrane segment and
an intracellular tyrosine kinase. The ectodomain comprises two
leucine-rich repeat structural domains (usually referred to as L1
and L2) separated by a cysteine-rich (CR) region [24], followed by
three fibronectin type III domains (FnIII-1, FnIII-2 and FnIII-3)
[25,26], the second of which features an insert domain (ID) that
contains the site of cleavage between the a and b subunits and the
alternatively spliced exon 11.
The structural determination of the first three domains of the
IGF1R was reported in 1998 [27], facilitating the subsequent
mapping of functional regions to the L1 and CR domains that
contribute to ligand binding and affinity through alanine scanning
mutagenesis [28,29,30,31], chimeric receptor constructs
[32,33,34] and cross-linking [35,36,37] studies involving both IR
and IGF1R.
PLoS ONE | www.plosone.org 1 November 2008 | Volume 3 | Issue 11 | e3667

Attempts to obtain insights into the ectodomain arrangement
and ligand binding of the IR have included a three-dimensional
reconstruction based on images obtained by electron cryomicro-
scopy [38]. The three-dimensional structure of the intact IR dimer
ectodomain was recently determined by X-ray crystallography,
revealing an ‘‘inverted V’’ arrangement, wherein the first three
domains (L1-CR-L2) form one leg and the three FnIII domains
make up the other leg in each monomer [39–40]. The two
monomers are located in an anti-parallel orientation and are
linked by a disulphide bond at Cys 524. Similarly, there is a second
inter-a-chain disulphide bond at the Cys 682, Cys 683 and Cys
685 triplet in the insert domain, and the a and b chains are linked
within the monomer by a single disulphide bond at Cys647–
Cys860. The dimer crystal structure features two potential ligand
binding sites and helps rationalise many characteristics of ligand-
receptor binding, such as the existence of both low- (site 1) and
high-affinity (site 2) binding sites and negative cooperativity, as
inferred from Scatchard plots [41,42]. The domain arrangement
in the ectodomain crystal structure also suggests that receptor
binding site 2 involves one or more of the FnIII domains, as
opposed to a previously proposed model that suggested that the
first three domains of each monomer jointly participate in insulin
binding [38].
A crystal structure of only the first three domains of the IR was
also recently obtained, and a possible model of insulin binding to the
L1 domain was proposed [43]. There is evidence that the B chain
C-terminal of insulin contacts the insert domain of the IR
[37,44,45], presumably upon a conformational change of insulin
[37,46,47,48]. However, the insert domain could not be crystallised,
presumably due to its disordered conformation [39]. Hence, the
proposed model of the binding of insulin omitted the C-terminal
portion of its B chain. In the absence of a crystal structure of the
complex between insulin and its receptor, further investigation is
needed to determine the contribution of L2 and the three FnIII
domains to insulin binding and receptor ligand specificity.
Recently, studies involving the construction of chimeric
receptors have shown that there is a significant contribution of
L2 and particularly of FnIII-1 to insulin binding [49], but it was
not possible to determine the specific residues on these domains
that may be involved in contacting insulin. In order to map those
and other possible regions in the IR contributing to insulin
binding, we have performed a comparative structural bioinfor-
matics analysis of the insulin receptor family ectodomain based on
phylogenetic information.
Biological evolution has recorded vast and highly precise
information in genetic sequences. For this reason, amino acid
sequences are a powerful source of information for predicting
functional regions of proteins by analysing conservation patterns.
It is known that regions directly involved in biochemical functions,
such as binding surfaces, experience different selection pressure
from other regions on the surface of proteins [50]. In the same
way, non-polar amino acids in the interior of a protein may be
conserved due to structural and stability constraints as hydropho-
bic interactions are considered to be the driving force of protein
folding [51,52]. Although mutation rates and conservation scores
can be estimated for each amino acid position of a protein
sequence from a multiple sequence alignment, it is necessary to
correlate these data with their corresponding location in the three-
dimensional structure, since residues that are distant in sequence,
can be found in close proximity in the folded protein.
In view of the evidence that associates the FnIII domains of the
IR to insulin binding, we have attempted to map the evolutionarily
conserved regions of these domains in order to predict those
specific residues that might contact insulin. Homologous amino
acid sequences of the IR family ectodomain in mammals, birds,
amphibians and fish were retrieved from public databases through
a BLAST search, and were then classified into three different
orthologous sets corresponding to the IR, IGF1R and IRR. Each
set was subsequently aligned and evolutionary conservation scores
at each amino acid position were calculated for the IR and IGF1R
using the Rate4Site algorithm [53]. The resulting scores were
categorised into different conservation grades and projected onto
the three-dimensional X-ray structures of the IR family, as
available from the Protein Data Bank (PDB) [54]. We have aimed
to obtain a precise and detailed model of the ligand-binding
interactions and to identify the residues that are responsible for
ligand recognition specificity amongst paralogous receptors. This
knowledge may be used in future for the rational design of drugs to
treat diseases such as diabetes and cancer.
Through this amino acid conservation analysis we reconstructed
the phylogeny of the IR family and predicted with significant
accuracy the location of the well studied binding site 1 of the
IGF1R and IR. We have also predicted the potential location of
insulin binding site 2 on the FnIII-1 and FnIII-2 surface of the IR.
At the same time, we could not identify a conserved surface on the
L2 domain that may contact the ligand. We have also suggested a
possible mechanical trigger of the activation of the IR on the basis
of normal modes analysis of the low-frequency vibrations of this
receptor. Finally, a strong selective pressure was found amongst
the IRR orthologous sequences, suggesting that this orphan
receptor has a yet unknown physiological role which may be
conserved from amphibians to mammals.
Results and Discussion
Phylogenetic Analysis of the IR family
Invertebrates possess only a single homologous receptor of the
IR family [55]. In addition to its function in the regulation of
metabolism, insulin signalling in Drosophila Melanogaster (fruit fly)
and Caenorhabditis elegans (roundworm) has a role in lifespan and
reproduction control [56,57], whilst in Apis Mellifera (honeybee) it is
involved in caste determination and differentiation [58].
A significant step in the evolution of the IR family has been the
transition from a single invertebrate IR that regulates both growth
and metabolism to two different and specialised receptors that are
able to recognise and discriminate their specific ligands: the IR
and the IGF1R in vertebrates [57,59]. Studies in primitive
vertebrates suggest that gene duplication would have occurred
early in vertebrate evolution [60,61].
In order to study the phylogenetic relationships between the
distinct members of the IR family, a multiple sequence alignment
(MSA) including the 55 vertebrate ectodomain sequences listed in
Table 1, plus three IR family invertebrate homologous ectodo-
main sequences, was constructed using MUSCLE [62]. The first
53 residues of the Bos taurus 1GF1R protein (XP_606794.3) were
excluded because they were not homologous to the N-termini of
the other mammalian orthologues, as seen in the MSA and
confirmed by BLASTP searches. Bayesian and maximum
likelihood tree searches under the best-fitting model were
performed as described in the Methods section. Figure 1 shows
the best ML tree found under the substitution model with highest
posterior probability (JTT+G). Great model selection confidence
was also found for each of three individual sets (IR, IGF1R and
IRR) of orthologous sequences when evaluated with ProtTest [63].
The model selected was in all cases JTT+G, with an Akaike weight
ranging between 0.74 and 0.75 (the analysis is provided in
Supplementary File S1). All the deeper bipartitions of the
phylogeny were significantly supported ($0.95 SH-like P values
IR Family Analysis

or Bayesian posterior probabilities) by both types of tree searches,
as indicated on Figure 1. The two independent Bayesian MC3
runs yielded identical topologies (Robinson-Fould distance = 0)
which, when compared with that of the best-scoring ML tree
(Figure 1), differed only in a few bipartitions. These corresponded
to poorly resolved terminal clades which formed polytomies on the
Bayesian tree (shown in Supplementary File S2). Therefore, both
optimality criteria consistently and significantly support the
hypothesis that the IRR has a closer relationship to the IGF1R
as compared with the IR, suggesting that the IGF1R and IRR
share a common ancestor and that they are the product of a
second gene duplication event in the IR family evolution history
(Figure 1). This is consistent with the fact that no orthologous IRR
sequence could be found in fish genomes in this study. However,
the possibility of a loss of the IRR paralogous in fish remains to be
proved as it is not possible to establish with certainty that this
occurred with current available data. Nevertheless, a posterior
duplication of the IR and the IGF1R genes occurred in the zebra
fish lineage, presumably as a product of whole-genome duplica-
tion, therefore giving origin to two different functional versions (a
and b) of both the IR and the IGF1R [64]. Whilst both versions of
the IGF1R are required for proper zebra fish embryonic growth,
development and survival, IGF1Rb plays a considerably higher
role in spontaneous muscle contractility and motoneuron
development [65].
Both the IR and IRR contain an additional exon (exon 11) with
respect to IGF1R. In the IR, it gives rise to the two different IR-A
and IR-B isoforms, whereas in the IRR this exon is constitutively
expressed as part of the receptor. A recent study traced the
presence of the alternatively spliced exon 11 of the IR, showing
that it is a novel acquisition of mammals [66]. Furthermore, given
the highly divergent sequences and the phylogenetic relationship
of this exon amongst the IR family members, it was also proposed
that both exons were independently acquired by each paralogous
gene [66].
A possible selective advantage conferred by the evolutionary
acquisition of exon 11 by the IR is explained by the fact that
isoform B is predominantly expressed in insulin target tissues that
are involved in glucose homeostasis [13,67], which may be the
consequence of a more specialised function as a metabolic
receptor.
IR and IGF1R Conservation
With the increasing amounts of DNA and amino acid sequences
available in public databases, performing comparative multi-
species phylogenomics studies is now feasible. In the case of
families of genes, it is possible to study the evolution and
divergence of paralogous and orthologous proteins. This informa-
tion, along with protein structures, when available, can be used to
computationally predict functional regions of proteins.
Table 1. Orthologous sequences and their accession numbers obtained from the GenBank and ENSEMBL databases.
Species Common Name IR IGF1R IRR
Bos taurus Cow XP_590552 XP_606794.3 XP_001254386
Canis familiaris Dog XP_542108.2 XP_545828.2 XP_547526
Cavia porcellus Guinea Pig ENSCPOG00000011692 ENSCPOP00000004859 AAA37044
Coturnix japonica Japanese Quail BAF73401
Danio rerio Zebra Fish XP_690534.1, NP_001116701 NP_694500, NP_694501
Echinops telfairi Lesser Hedgehog Tenrec ENSETEP00000014592 ENSETEP00000009987
Equus caballus Horse XP_001496634 XP_001489815.1
Erinaceus europeus Western European Hedgehog ENSEEUP00000012248
Felis catus Cat ENSFCAP00000002790
Gallus gallus Chicken XP_418250.2 NP_990363.1
Gasterosteus aculeatus Threespine Stickleback Fish ENSGACP00000013853
Homo sapiens Human AAA59174.1 AAB22215 NP_055030
Macaca mulatta Rhesus Monkey XP_001100407.1 XP_014528
Microcebus murinus Mouse Lemur ENSMICP00000010917 ENSMICP00000009223 ENSMICP00000008186
Monodelphis domestica Gray Short-Tailed Opossum XP_001377572.1 XP_001372725.1 ENSMODP00000020864
Mus musculus Mouse NP_034698.2 NP_034643.2 NP_035962
Myotis lucifugus Bat ENSMLUP00000012576 ENSMLUP00000009532
Ochotonas princeps American Pika ENSOPRP00000004065 ENSOPRP00000014561
Oryctolagus cuniculus Rabbitt AAR04440
Pan troglodytes Chimpanzee XP_512324.2 & XP_512323.2 XP_001136377 XR_025504
Rattus norvegicus Rat EDL74923 NP_434694.1 XP_001068054
Sorex araneus Common Shrew ENSSARP00000011012
Scophthalmus maximus Turbot fish CAA12278
Sus scrofa Pig NP_999337.1
Tupaia belangeri Northern Treeshrew ENSTBEP00000014243
Xenopus laevis African Clawed Frog NP_001081702 NP_001081734.1 MP_001083465
doi:10.1371/journal.pone.0003667.t001
IR Family Analysis

In this study, the MSAs corresponding to the IR and IGF1R
ectodomain orthologous sets were used to estimate conservation
scores with the Rate4Site algorithm under the maximum-
likelihood model. The conservation scores were projected onto
the crystal structures of the IR family, as available from the Protein
Data Bank (PDB IDs: 2DTG, 1IGR, 2HR7), by using the ConSurf
server [68]. The conservation score at a particular position
corresponds to its evolutionary rate. Whilst some positions evolve
rapidly and are commonly referred to as ‘‘variable’’, other
positions evolve slowly and are referred to as ‘‘conserved’’. The
variations in the rate of conservation correspond to different levels
of purifying selection acting on each site. Purifying selection is
expected to be higher at structurally and functionally critical
positions, such as protein-protein binding surfaces.
In the IR dimer structure, the first three domains of each
monomer are packed against the three FnIII domains of the other
monomer, in such a way that the L1 and L2 domains from each
monomer correspondingly interact with the FnIII-3 and the FnIII-
1 domains of the other monomer. Figure 2 shows the degree of
residue-specific conservation in the structure of an IR monomer
ectodomain. The ‘‘inner’’ surface of the monomer exhibits a
considerable higher conservation than the ‘‘outer’’ surface. This is,
to a certain extent, due to the interactions between the L2 and
FnIII-1 domains from the same monomer, which might be an
indispensable requirement for the monomer to adopt the inverted
‘‘V’’ conformation. Furthermore, the conserved surfaces also
correspond to the regions involved in monomer-monomer
interactions.
A comparison of the overall conservation of the first three
domains of both the IGF1R and IR is shown in Figures 3A and B.
The ‘‘inner’’ surface of the L1 domain of the IR features a
conserved surface (shown in Figure 3D) formed by residues Asp12,
Arg14, Asn15, Gln34, Leu36, Leu37, Phe39, Tyr60, Leu62,
Phe64, Arg65, Tyr67, Leu87, Phe88, Phe89, Asn90, Tyr91,
Val94, Phe96, Glu97, Arg114, Arg118, Glu120 and Lys121,
which, on the basis of mutagenesis data, is likely to be involved in
ligand binding. Mutagenesis data was extracted from the
Receptors for Insulin and Insulin-like Molecules (RILM) online
database [69] and is listed in Table 2. The only residues that are
not strictly conserved are Asp12, Asn15, Asp59 and Phe96.
Figure 1. Evolution of the IR family. Maximum likelihood phylogeny of the IR family ectodomain inferred from amino acid sequences. The tree
shown was the best one found (lnL = 230836.65814) amongst 41 independent tree searches (see Materials and Methods) for 55 vertebrate and 3
invertebrate IR family ectodomain homologues. The numbers on the bipartitions indicate the ML SH-like P values/Bayesian posterior probability
support values. Only significant (P$0.95) values are shown. An asterisk (*) indicates that the bipartition was not significantly supported (P,0.95)
either by the highly conservative SH-like branch significance test [103] used to compute bipartition robustness under the ML criterion, or by the more
liberal Bayesian posterior probabilities. NCBI taxonomic ranks are provided for some clades. The scale indicates the number of expected substitutions
per site under the best fitting JTT+G model (shape parameter a = 0.865), which had a posterior probability of 1.
doi:10.1371/journal.pone.0003667.g001
IR Family Analysis

Likewise, the IGF1R features a similar conserved region
comprising residues Pro5, Asp8, Asn11, Tyr28, His30, Leu32,
Leu33, Tyr54, Leu56, Phe58, Arg59, Trp79, Leu81, Phe82,
Tyr83, Tyr85, Val85, Val88, Asn90, Arg112, Arg240, Phe241,
Glu242 and Phe251, that would serve as the IGF-1 binding site.
Figure 3C displays their conservation rates and the mutagenesis
data is listed in Table 3.
Ligand-Receptor Binding
The physiologically active form of insulin is a monomer
composed of two chains, an A chain of 21 amino acids and a B
chain of 30 residues, linked by two disulphide bonds at A7–B7 and
A20–B19. An additional intra-chain disulphide bridge is situated
between residues A6 and A11 [70,71]. IGF-1 and IGF-2 are
homologous peptides structurally related to insulin. The most
important structural difference of the IGFs with respect to insulin
is that they are single chain polypeptides that contain four
structural domains: A, B, C and D [4,72].
The most widely accepted model of insulin binding suggests that
the insulin molecule comprises two separate binding surfaces,
denominated as site 1 and site 2 [73]. These surfaces cross-link two
different binding sites on the ectodomain of the IR. The classical
binding surface of insulin (site 1) overlaps with the hexamer-
forming surface and involves residues A1–A3, A5, A19, A21 as
well as B12, B16, B23, B24 and B25 [73,74], whereas site 2
overlaps with the dimer-forming surface and comprises residues
A12, A13, A17, B10, B13 and B17 [42]. Moreover, it has been
demonstrated that high-affinity IGF1 binding to the IGF1R
involves the interaction between the IGF1 C-domain and the Cys-
rich region of the IGF1R [75]. The lack of the C-domain greatly
explains the low affinity of insulin for the IGF1R [75].
Nevertheless, an IGF1 analogue that binds with similar affinity
to insulin for the IR was produced recently by introducing four
insulin residues [76], which indicates that the IGF1 C-domain can
be accommodated in the insulin binding site. This evidence is in
agreement with previous experiments that showed that a single
chain insulin analogue, wherein the A and B chains are connected
by the C domain of IGF1, can bind to IR with the same affinity as
wild-type insulin [77].
There is considerable evidence that a conformational change
involving the C-terminal of insulin B chain occurs upon binding
[37,46,47,48]. The portion corresponding to residues B21–B30 is
believed to move away from its contact with residues A1 and A2, in
order to expose the hydrophobic ‘‘classic binding site’’ of insulin.
Furthermore, it has been proposed that the N-terminal portion of
the B chain (residues B1–B8) also experiences a change in
conformation, from an extended and stable form, known as the T
state to a less stable but more active form, known as the R state [47].
The conserved surfaces on the L1 and CR domains of the
IGF1R and IR, when contrasted with the available mutagenesis
data, reveal the strong correlation between the degree of
evolutionary conservation of an amino acid position and its
functional role, such as, in this case, its participation in a protein-
protein binding interface. These binding site interfaces are in
agreement with previous models of ligand binding and mutagen-
esis data [43,78].
In order to identify those specific amino acid positions subjected
to positive selection that might confer ligand-specificity to each
paralogous receptor, we looked for divergent selection patterns at
residues involved in ligand-binding. Interestingly, we found that
residues Tyr28, His30, Trp79 and Arg240 of the IGF1R have
diverged from their corresponding residues in the IR and IRR:
His, Gln, Ser/Tre and His. This partly explains why these
positions are less conserved than the rest of residues that contribute
to IGF-1 binding on the surface of the L1 domain, as can be
appreciated from Figure 3C.
The insert domain (ID) has been shown to play a role in insulin
binding. Cross-linking studies revealed that two consecutive insulin
residues, PheB24 and PheB25, contact two different domains of
the IR: L1 and ID, respectively [37]. Consequently, it is believed
that the ID is in close juxtaposition to the L1 domain. Recently,
complementation analysis showed that these interactions occurs as
a result of a trans mechanism, in which the ID and the L1 domain
that simultaneously contact insulin belong each to different
monomers of the IR [79].
Alanine scanning mutagenesis of the ID have indicated that
residues Thr704, Phe705, Glu706, Tyr708, Leu709, His710,
Asn711 and Phe714 display a considerable loss in insulin binding
Figure 2. Amino acid conservation in the IR ectodomain monomer. Amino acid conservation scores were classified into nine levels. This
figure shows the general conservation of the two faces of a single IR ectodomain monomer: facing towards both (A) outside and (B) inside of the
dimer. The colour scale for residue conservation is indicated in the figure. The molecular coordinates were taken from PDB structure 2DTG.
IR Family Analysis

affinity upon mutation [31]. We have found that these residues
show a strict conservation pattern in all 20 sequences of the IR
used in this study, from fish to mammals, which correlates with
their critical contribution to ligand binding. The corresponding
region in the IGF1R is also involved in IGF1 binding. Individual
mutation to alanine of residues Phe692, Glu693, Asn694, Leu696,
His697, Asn698 and Ile700 resulted in a 10- to 30-fold loss in
ligand binding, whilst mutation of Phe701 resulted in no
detectable ligand binding. Figure 4 illustrates using a logo
representation the evolutionary conservation of this region in the
three orthologous receptors. It can be seen that most of the IR
residues involved in binding are also evolutionary conserved in the
IGF1R and IRR, suggesting that whilst this region contributes to
ligand affinity, its contribution to ligand selectivity may be small.
A shortened IR, consisting of residues 1–601 and 650–719,
displays the same insulin binding properties as the holoreceptor,
suggesting that all residues needed for high affinity binding are
located within these regions [80]. What is not clear yet, though, is
whether this is also enough for triggering a conformational change
of the receptor that ultimately leads to signal transduction.
In this work, a region of conserved residues on the FnIII surface
that face the proposed L1 domain binding site in the IR was
identified. This region comprises residues Tyr507, Asn527,
Trp529, Lys557, Pro558, Trp559, Ser596, Val597, Pro598,
Leu599, Asp600 and Pro601. Based on the high level of purifying
selection acting on this region and its location and orientation
within the dimer structure, we suggest that it is a strong candidate
to act as the receptor binding site 2. Mutagenesis experiments
should be performed in order to investigate the magnitude of the
possible individual contribution of these residues. Moreover, the
contiguous residues Lys614, Trp615, Tp616, Pro617, Pro618, and
Pro621 display a strict conservation pattern, and their possible role
in binding or signal transduction should also be investigated
further. In an attempt to validate this prediction, a search for
Figure 3. Comparison of the overall amino acid conservation of the first three domains of the IR and IGF1R and their proposed
ligand binding sites. Amino acid conservation in the L1-CR-L2 domains of the (A) IGF1R and (B) IR. The rectangles indicate the regions that are
believed to be involved in ligand binding and the circled regions in B indicate the location of the major specificity regions between the IR and
IGF1R.[43] (C) A proposed binding site on the IGF1R surface is shown (available supporting mutagenesis data is listed in Table 3). (D) Insulin binding
surface on the L1 domain (mutagenesis data is listed in Table 2). Variations at non-strictly conserved positions on the binding surfaces are indicated.
Surfaces in C and D are in agreement with previous ligand binding models.[43,78]. The figures are based on the coordinates of the IGF1R (PDB code
1IGR) and IR (PDB code 2HR7).
IR Family Analysis

naturally occurring or engineered mutations on these residues was
carried out. However, no experimental evidence that could
provide insight into the functional role of this surface was found
reported in the RILM database [69] or in the NCBI SNPdb [81].
Furthermore, an evolutionary trace (ET) [82,83,84] run was
performed for the IR MSA, as described in the Materials and
Methods section. ET is an evolution-entropy hybrid method that
assigns a relative score of functional importance to each sequence
residue and subsequently ranks the residues by importance.
Consistent with the likely acting high selective pressure, ET
ranked first those hydrophobic residues located in the interior of
the protein as well as some residues at the monomer-monomer
and inter-domain interfaces (rho<1.00), whereas residues com-
prising both the receptor binding site in L1 and the proposed
binding site 2 scored moderately highly (rho<1.74–3.00 and
rho<1–2.59, respectively), forming two uniformly conserved
surfaces, as can be appreciated in Figure 5.
Figure 6 shows the conservation of a single dimer binding site,
formed by the three FnIII domains from one monomer (FnIII-1-
FnIII-2-FnIII-3) and the first three domains from the other
monomer (L1’-CR’-L2’) in the IR. It is evident once again that the
inner surfaces are more conserved than the outer ones. The
conserved residues that are likely to contact insulin upon binding
are listed in Table 4 and their location in the structure of the IR is
shown in Figure 7.
The IR is a highly glycosylated protein, comprising both N- and
O-linked glycosylation sites [85,86]. The functions of the N-linked
glycans attached to the IR include facilitating the correct folding of
the protein, processing of the proreceptor and dimer formation, as
Table 2. Alanine scanning and other mutagenesis data
available for conserved residues on the L1 binding interface of
IR (according to the RILM database).
IR Residue Mutation Reduction in affinity for insulin Reference
Asp12 D12A 5 to 6.2-fold [30,109]
Arg14 R14A +500-fold [109]
Asn15 N15A 134-fold [109]
Gln34 Q34A 3.2-fold [109]
Leu36 L36A 1.6-fold [109]
Leu37 L37A 20-fold (IR-A), 40-fold (IR-B) [31]
Phe39 F39A 10-fold [109]
Asp59 D59G1
4-fold [110]
Tyr60 Y60A Not secreted2
[30]
Leu62 L62P1
Insulin-resistant diabetes mellitus [110]
Phe64 F64A +500-fold [109]
Arg65 R65A No effect [30]
Tyr67 Y67A 3.2-fold [109]
Leu87 L87A 85% reduction [111]
Phe88 F88A No significant effect [112]
Phe89 F89A 4.8-fold [109]
Tyr91 Y91A 3.6-fold [109]
Val94 V94A 3 to 10-fold [31]
Phe96 F96A Not secreted2
[30]
Glu97 E97A 3 to 10-fold [31]
Arg114 ----- Data not available
Arg118 ----- Data not available
Glu120 E120A 3 to 10-fold [31]
Lys121 K121A 3 to 10-fold [31]
Thr704 T704A More than 500-fold [109]
Phe705 F705A More than 500-fold [109]
Glu706 E706A More than 500-fold [109]
Asp707 D707A 7 to 16-fold in the A isoform [109]
Tyr708 Y708A 218-fold [109]
Leu709 L709A 70-fold [109]
His710 H710A More than 500-fold [109]
Asn711 N711A 52-fold [109]
Val713 V713A 7 to 16 fold in the A isoform [109]
Phe714 F714A 182-fold [109]
Val715 V715A 8-fold [109]
1
Naturally occurring mutations.
2
When expressed transiently in adenovirus-transformed human embryonic
kidney cells.
Table 3. Alanine scanning and other mutagenesis data
available for conserved residues on the L1 and the CR binding
interfaces of the IGF1R (according to the RILM database).
IGF1R residue Mutation Reduction in affinity for IGF-1 Reference
Pro5 ----- Data not available
Asp8 D8A 9-fold [29]
Asn11 N11A 7.5-fold [29]
Tyr28 Y28A 4.5-fold [29]
His30 H30A 4.5-fold [29]
Leu32 L32A No significant effect [113]
Leu33 L33A 6-fold [29]
Tyr54 ----- Data not available
Leu56 L56A 5-fold [29]
Phe58 F58A 3-fold [29]
Arg59 R59A 5-fold [29]
Trp79 W79A 3-fold [29]
Leu81 L81A No significant effect [29]
Phe82 F82A No significant effect [29]
Tyr83 Y83A No significant effect [29]
Tyr85 Y85A No significant effect [29]
Val88 V88A No significant effect [29]
Asn90 N90A No significant effect [29]
Arg112 R112A No significant effect [29]
Arg240 R240A 2-fold [29]
Phe241 F241A 6-fold [29]
Glu242 E242A 4-fold [29]
Phe251 F251A 2-fold [29]
Phe692 F692A 30-fold [29]
Glu693 E693A 10-fold [29]
Asn694 N694A 10-fold [29]
Leu696 L696A 20-fold [29]
His697 H697A 10-fold [29]
Asn698 N698A 10-fold [29]
Ile700 I700A 20-fold [29]
Phe701 F701A Abolishes IGF1 binding [29]
IR Family Analysis

Figure 4. Conservation of the Insert Domain region involved in binding. Residues within the 700–715 fragment of the IR have been
implicated in ligand binding. This logo representation also shows the corresponding residues in the IGF1R and IRR. Residues with a gray box result in
considerable loss of binding when mutated to alanine, according to previous studies. Data is listed in Tables 2 and 3. Residues with a red box result in
a 200- to 500-fold loss of binding.
Figure 5. Functionally important residues in the IR predicted by Evolutionary Trace. Evolutionary Trace (ET) was performed on the 20 IR
sequences in order to compare with ConSurf predictions. ET assigns a relative score of functional importance to each sequence residue. Residues
predicted to be significantly important (rho#2.8) are shown in the figure. Residues comprising both the known L1 ligand-binding surface and the
proposed surface on FnIII display homogeneous functional scores, thus forming potential functional clusters. Likewise, residues implicated in
structural stability were assigned high scores. The figure was generated with ET Viewer 2.0.
IR Family Analysis

well as the transport of the functional receptor to the membrane
[4,87,88]. Studies aimed at investigating the effect of the removal
of N-linked glycosylation sites suggest that there are redundancies
in IR glycosylation, since many sites can be mutated individually
without compromising cell surface expression, receptor processing
or ligand binding [87]. On the other hand, when combinations of
sites are mutated, folding cannot be carried out properly [87]. We
looked at the conservation of the N-glycosylation sites and found
that 9 out of 19 sites showed strict evolutionary conservation:
Asn78, Asn111, Asn418, Asn514, Asn606, Asn624, Asn742,
Asn881 and Asn894, while Asn295, Asn337, Asn671 and
Asn743 showed a nearly strict pattern. Figure 8 shows the location
of the N-glycosylation sites within the dimer structure. Surpris-
ingly, several of these glycosylation sites were lost in IRR, such as
Asn16, Asn111, Asn215, Asn255, Asn282, Asn337 and Asn418,
which indicates that IRR has a different glycosylation pattern in
comparison to IR, supporting the evidence of redundancy in the
IR. Although the IR also contains mucin-type O-linked glycans
attached to six Ser/Tre residues located in the N-terminal portion
of the b chain, a recent study suggested that O-linked glycosylation
is unlikely to be functionally significant in the IR family [89]. In
the IGF1R there are only three O-linked glycosylations, whereas
in the IRR there are only two serine residues in the corresponding
portion and a single O-glycosylation site predicted [89].
Interestingly, we found a considerable number of conserved Gly
and Pro residues located within the FnIII-1 and FnIII-2 domains.
These residues may provide structural flexibility to this region, and
may also play a specific role in preventing aggregation, as
suggested by previous studies [90,91].
From our results, it is clear that the L2 domain of the IR plays
an important structural role in dimer formation through its
interaction with the FnIII-1 domain from the other monomer, and
that it contributes to the adoption of the ‘‘inverted V’’
conformation of the monomer through its interaction with the
FnIII-1 domain from the same monomer. However, we did not
find any conserved surface that could be involved in ligand
binding. Furthermore, the contribution of the L2 domain to ligand
binding in the IGF1R and IRR is still unclear.
Interestingly, in an attempt to develop insulin mimetic peptides,
the use of phage display methodologies led to the discovery of
three groups of peptides unrelated in sequence to insulin that
recognise three different sites on the IR surface [92]. The synthetic
combination of two of these sites resulted in a very potent, 36-
residue single chain peptide with insulin mimetic activity that had
an affinity for the IR comparable to that of insulin [93]. Further
studies found that activation of the IR-A by this peptide, named
S597, displays metabolic equipotency but low mitogenicity as
compared to activation by insulin, supporting the idea that insulin
Figure 6. Conservation of a single insulin receptor binding site. Each IR features two binding sites. Each one of them is formed by two
components: binding site 1 is contained in the first three domains of one monomer, and comprises the conserved surface on L1 and the carboxy-
terminal of the ID, which could not be crystallised in the IR ectodomain structure. In IGF-1, binding additionally involves the CR region. Binding site 2
is contained in the other monomer and it is thought to involve one or more of the FnIII domains. This figure shows the conservation of the (A) inner
and (B) outer surfaces of a single binding site. It is evident that the inner surface is considerably more conserved, due in part to its role in dimer
formation. The figure is based on the coordinates of PDB structure 2DTG and residue conservation is indicated in the same colour scale as in previous
figures.
IR Family Analysis

and S597 elicit different signaling and biological responses through
acting on the same IR isoform [94], later confirmed by gene
expression profile analysis [95]. It is thus believed that there is
more than one way to activate the IR.
Likewise, it has been reported that the soluble ectodomain of the
IR shows only low-affinity ligand binding, unless it is tethered by
transmembrane anchors, leucine zippers or Fc domains [32,40]. It
is also known that high affinity binding of insulin is accompanied
by a structural compaction of its receptor [96]. It may therefore be
necessary to consider that the conformation displayed by the IR in
PDB structure 2DTG may not be the actual conformation of the
receptor when insulin is bound to it.
Our residue conservation analysis reveals that, although a
portion of the conserved surface on the FnIII domains points
directly towards the conserved L1 surface, a slight rotation of the
FnIII-1/FnIII-2 domains would be needed in order for the
conserved surface to adopt an orientation that allows it to fully
contact the insulin binding site 2. To test the likelihood of this
conformational change, we performed a normal modes analysis
(NMA) of a single IR monomer in order to predict its low
frequency, high amplitude intrinsic vibrations. The results suggest
that the receptor is prone to rotate the FnIII-2/FnIII-3 conserved
surface towards the conserved surface on L1. On the other hand,
this movement was not observed in the dimer structure. This is
mainly explained by the restrictions imposed by inter-monomer
interactions in the crystal structure of the ectodomain dimer,
which may be different to the conformation that the receptor
adopts when it is anchored to the cell membrane. A possible full
rotation of the FnIII-2/FnIII-3 domains upon ligand binding
Table 4. Residues potentially involved in the second insulin
binding site (numbering according to PDB structure 2DTG).
IR IGF1R IRR
Tyr507 Tyr Tyr
Asn527 Asn Gln
Trp529 Trp Trp
Lys557 Lys Lys
Pro558 Pro Pro
Trp559 Trp Trp
Ser596 Ser Tre
Val597 Ile Val
Pro598 Pro Pro
Leu599 Leu Gln
Asp600 Asp Asp
Pro601 Val Val
Lys614 Lys Arg
Trp615 Trp Trp
Lys616 Asn Lys
Pro617 Pro Pro
Pro618 Pro Pro
Pro621 Pro Arg
Figure 7. Proposed receptor second ligand binding site. (A) A region of conserved residues on the FnIII domains was identified and is
proposed to act as the receptor binding site 2. Insulin is believed to cross-link both monomers in the high-affinity state of binding. Residues that are
predicted to be involved in forming the receptor second binding site, and the corresponding residues at the same positions in the IGF1R and IRR, are
listed in Table 4. (B) Representation of the location of the proposed binding site 2 within the dimer structure.
IR Family Analysis

could also act as a mechanical trigger for signal transduction in the
tyrosine kinase domain of the IR. Further experiments and
molecular dynamics simulations are needed to validate these
predictions.
Conclusions
Whilst the recent determination of the intact ectodomain
structure of the IR by X-ray crystallography has provided new
insights into the 3D arrangement of the receptor domains, a full
understanding of its interactions with insulin and its functional
activation remains elusive. The IR family thus remains a complex
but interesting system of study, particularly as the physiological
functions of heterodimeric receptors and the IRR are yet to be
discovered.
The physiological role of the IR/IGF1R hetero-dimers is unknown
and the physiological role of IRR is yet to be established. Our
sequence analysis indicates that IRR is highly conserved throughout
evolution, from Xenopus laevis to mammals, and that it differs from the
IGF1R and IR in some key residues for ligand specificity.
In this study, amino acid residue conservation scores have
revealed the different degrees of purifying selection acting on the
protein surface of the IR and IGF1R. We have used this
information to predict the location of the experimentally
characterised ligand binding sites on the surfaces of L1 and CR
as a control. These predictions were validated against the
mutagenesis data available from the RILM online database [69],
and were found to be in agreement with previous insulin binding
models. No conserved surface on L2 was found pointing towards
the receptor binding site. In addition, there does not appear to be
any evidence that directly relates L2 to ligand binding.
A region of conserved residues on the surface of the FnIII
domains was identified. Based on its location, this region is a
strong candidate to act as the receptor insulin binding site 2.
However, its location suggests the need for a slight ‘twist’ rotation
of the FnIII-2/FnIII-3 domains with respect to FnIII-1 in order to
face the likely location of insulin. This conformational change may
act as a mechanical trigger for receptor activation and signal
transduction. Further experiments and computer simulations are
needed in order to validate these predictions.
The insulin binding model that proposes that insulin cross-links
both receptor monomers in the IR also suggests that this is not
needed for IGF-1 binding, which only requires binding site 1. This
idea is supported by chimeric construct experiments that have
shown that both IR-A/IGF1R and IR-B/IGF1R hybrids behave
like the IGF1R.
Further crystallographic structures of both the low- and high-
affinity ligand/receptor complexes for the IR and IGF1R are
required to establish unambiguously the specific interactions
involved in ligand binding and receptor structural components
involved in these interactions, as well as to understand the nature of
the structural transitions that lead to the activation of the receptor
kinase. Due to the difficulties associated with the crystallisation of
transmembrane receptors, mutagenesis data and molecular dynam-
ics simulations may provide the easiest approaches to characterise
the molecular basis of ligand binding and receptor activation.
Finally, this study demonstrates that methods that estimate
amino acid sequence evolutionary conservation rates can provide
valuable information about regions of functional importance upon
the correct categorisation of homologous sequences into ortholo-
gous sets when crystal structures are available.
Figure 8. Conservation and location of the N-glycosylation sites of the IR. The figure shows the IR dimer structure. One monomer is
displayed in gray and the other one in light purple. N-glycosylation sites are highlighted according to their conservation grade. Numbering is shown
according to the 2DTG structure. Asn671, Asn730 and Asn743 lie within the un-crystallized ID.
IR Family Analysis

Materials and Methods
Data Sets
A BLAST (tblastn) [97] search was performed against the
GenBank non-redundant nucleotide database [98] and the
ENSEMBL nucleotide database [99], using NCBI and EN-
SEMBL web site tools [99,100]. The query sequences corre-
sponded to those of the IR family in humans: AAA59174.1 (IR),
AAB22215 (IGF1R) and NP_055030 (IRR). Homologous se-
quences were subsequently classified into three different sets (IR,
IGF1R and IRR) of orthologous sequences. Orthology was
validated by a bi-directional best hit procedure [101]. The
sequences and their accession numbers in the final sets for the
IR, IGF1R and IRR are listed in Table 1.
Additional modifications were made to the following sequences:
Bos Taurus IR mRNA was found to be reported in three separate
but overlapping transcripts and was merged manually by
removing the overlapping regions. Similarly, two different but
complementary mRNAs were found for Pan Troglodytes, and were
merged into a single sequence. Masked residues were removed
from Echinops Telfairi and Myotis Lucifugus IR and from Erinaceus
Europeus IGF1R sequences. The original and edited sequences can
be provided upon request to the authors.
Phylogenetic Analysis of the IR family
The three sets (IR, IGF1R and IRR) of orthologous sequences
listed in Table 1, and that corresponding to the IR family receptor
ectodomain of Bombyx Mori (Silkworm) [NP_001037011], Drosoph-
ila Melanogaster (Fruit fly) [NP_524436.2] and Lymnaea stagnalis
(Great pond snail) [CAA59353], were aligned separately using
MUSCLE [62] with three refinement rounds. The four sets of
Multiple Sequence Alignments (MSA) were finally merged into a
single one by profile to profile alignment using MUSCLE. All the
alignments and their respective phylogenetic trees used in this
study are provided in Supplementary File S3.
IR Family Phylogenetic Analysis
Bayesian (By) and maximum likelihood (ML) tree searches were
conducted using the MSAs produced by MUSCLE of the IR family
ectodomain, residues N-terminal from His1 or C-terminal from
Leu909 (human IR numbering as in the crystal structure) were not
taken into account in the alignments. ML tree searches were
performed with PhyML 2.4.5 [102,103] for each of the alignment sets
(IR, IGF1R and IRR) under the best-approximating model selected
by ProtTest [63] using the Akaike information criterion [104]. A ML
tree search was also performed for the full data set (including the 3
invertebrate sequences) under the model with the highest posterior
probability found by MrBayes, as explained below. In order to make
a more thorough search of tree space for the full dataset, 40 random
step-wise addition parsimony trees were generated with PAUP*4b10
[105] and used to initiate a corresponding number of ML searches on
a cluster of 27 dual core Pentium IV processors under Linux Rocks
3.3.0. A default PhyML search using a BioNJ seed tree was also used.
The tree yielding the highest log-likelihood (lnL) value was selected
amongst the 41 independent searches. The robustness of the ML
topologies was evaluated using a recently developed Shimodaira-
Hasegawa-like test for branches [103] implemented in PhyML v2.4.5
[102]. In brief, the test assesses whether the branch being studied
provides a significant likelihood gain, in comparison with the null
hypothesis that involves collapsing that branch, but leaving the rest of
the tree topology identical. We chose the Shimodaira-Hasegawa-like
procedure for assessing bipartition significance because the test is
non-parametric and much less liberal than the diverse (parametric)
approximate likelihood ratio tests (aLRTs) that are also implemented
in that program [103]. The resulting SH-like P-values therefore
indicate the probability that the corresponding split is significant. A
Bayesian estimation of phylogeny was performed with MrBayes 3.1.2
[106] for the full dataset. Two independent Metropolis-coupled
Markov-chain Monte Carlo (MC3
) simulations were run for 56105
generations, sampling every 100th
, using three heated chains
(temperature parameter set to 0.2) and a reversible-jump model
prior to use the chain for model selection. Each independent MC3
run had two replicates and was requested to use gamma-distributed
rates. The first 1000 samples (20%) were discarded as burnin.
Convergence and proper mixing of the chains was evaluated by visual
inspection of generation plots, comparison of the arithmetic and
harmonic lnL means of the two replicate runs, and calculation of
symmetric Robinson-Fould tree distances within replicates of the
same run (using the MrBayes sump output) and Treedist of the Phylip
package [107] to compute these distances between the majority rule
consensus trees obtained from each independent MC3
run (all
provided in Supplementary File S1 and Supplementary File S2).
The overall resolution of Bayesian and ML trees was evaluated
by computing diverse descriptive statistics of the SH-like P values
or Bayesian posterior probabilities parsed from the corresponding
phylograms using ad hoc Perl scripts [8].
Calculation of Consurf Conservation Scores
The conservation scores at each amino acid position were
calculated with the Rate4Site algorithm [53], under the maximum
likelihood (ML) principle providing both the IGF1R and the IR
MSAs and their corresponding ML trees calculated as explained
above. The conservation scores were projected onto the crystal
structures of the IR ectodomain (PDB code 2DTG), the IR first
three domains (PDB code 2HR7) and the IGF1R first three
domains (PDB code 1IGR), after submitting the data to the
ConSurf server [68,104]. Consurf results for both the IR and the
IGF1R sets are provided in Supplementary File S4.
Evolutionary Trace Calculation
Evolutionary Trace calculations were performed by running the
ET Wizard module coupled into the Evolutionary Trace Viewer
2.0 remotely, providing the IR MSA and the 2PDB E chain as
input. Complete ET results are provided in Supplementary File
S5.
Normal Modes Analysis
Normal modes analysis (NMA) was used to predict the
equilibrium low frequency, high amplitude inter-domain move-
ments of an IR monomer, using the Elastic Network Model (ENM)
as available through the ElNe´mo web server [108]. PDB structure
2DTG was used as input for these calculations. The five lowest
frequency normal modes were computed and the minimum and
maximum perturbations were set to 2150 and 150 DQ,
respectively. The output in PDB format corresponding to the
fourth model is provided in Supplementary File S6.
Supporting Information
Supplementary File S1 Supplemental Information for the
Maximum-likelihood Analyses
Found at: doi:10.1371/journal.pone.0003667.s001 (0.10 MB
PDF)
Supplementary File S2 Supplemental Information for the
Bayesian Analyses
Found at: doi:10.1371/journal.pone.0003667.s002 (0.65 MB
PDF)
IR Family Analysis

Supplementary File S3 Zip file containing all multiple
sequence alignments and phylogenetic trees used in this study
Found at: doi:10.1371/journal.pone.0003667.s003 (0.03 MB ZIP)
Supplementary File S4 Zip file containing Consurf scores for
both the IR and the IGF1R ectodomains
Supplementary File S5 Zip file containing Evolutionary Trace
results for the IR
Supplementary File S6 Zip file containing PDB structures
obtained through normal modes calculations.
Acknowledgments
We are grateful to Eleanor Morgan and Dr. Steve Bottomley for their kind
advice and valuable discussions. Moreover, MER gratefully acknowledges
the Peace Scholarship Program and the Secretariat of Public Education of
Mexico for making possible his stay in Australia as a visiting scholar.
Author Contributions
Conceived and designed the experiments: MER EH RLM. Performed the
experiments: MER PV. Analyzed the data: MER NSG. Wrote the paper:
MER NSG PV RLM.
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