Download Human Insulin-Receptor Gene

Perspectives in Diabetes
Human Insulin-Receptor Gene
SUSUMU SEINO, MITSUKO SEINO, AND GRAEME I. BELL
The human insulin-receptor (hlNSR) gene spans a
region of >120,000 base pairs (bp) on the short arm of
chromosome 19. It is comprised of 22 exons or coding
regions that vary in size from 36 to >2500 bp. To a
large degree, the introns appear to divide the hlNSR
gene into segments that encode structural and/or
functional elements of the hlNSR protein. The exonintron organization of the hlNSR gene provides a clue
to the evolutionary history of this gene and suggests
that it is a mosaic constructed from protein-coding
regions recruited from other genes. Eight mutations in
the hlNSR gene that result in expression of structurally
abnormal proteins have been described. These
mutations are associated with insulin resistance and
provide insight into the role of the hlNSR gene in the
development of diabetes mellitus. Diabetes 39:129-33,
1990
nsulin exerts a wide variety of effects on responsive cells,
resulting in increased glycogen, lipid, and protein synthesis (1-6). These effects include stimulation of glucose,
amino acid and ion uptake, enhanced tyrosine and serine
phosphorylation of cellular proteins, modulation of enzymatic
activity of regulatory enzymes by dephosphorylation, and
stimulation or repression of transcription of specific genes.
In addition, insulin promotes cell growth. Although the cellular responses to insulin are diverse and in many instances
tissue or cell specific, all are mediated by the insulin receptor
(INSR).
The human lNSR (hlNSR) is an integral membrane protein
present on the surface of all cells. The surface concentration
From the Howard Hughes Medical Institute ana the Departments of Medicine.
Biochemistry, and Molecular Biology, The University of Chicago, Chicago,
llllnois
Address c:orrespondence and reprint requests to Susumu Seino, MD, DMS.
Howard Hughes Med~calInstitute, The University of Chicago, 5841 South
Maryland Adenue, Box 391, Chlcago, IL 60637
Received for publication 30 May 1989 ana accepted in revised form 9
August 1989.
DIABETES, VOL. 39, FEBRUARY 1990
of receptors varies widely from as few as 40 on circulating
erythrocytes to >200,000 on adipocytes and hepatocytes.
The mature hlNSR is a heterotetramer of two a-subunits of
719 or 731 amino acids* and two 620-amino acid p-subunits
(7,8). The insulin-binding a-subunit and the membranespanning protein tyrosine kinase p-subunit are generated by
proteolytic processing of a common single-chain precursor.
Cleavage of the proreceptor occurs during ~ntracellular
transfer from the endoplasmic reticulum to the plasma membrane. After cleavage of the proreceptor, the nascent a- and
p-subunits are glycosylated on asparagine (N linked) and
possibly serinelthreonine residues (0linked). Thus, the aand p-subunits have apparent M, of 135,000 and 95,000,
respectively, which are much greater than those predicted
from their sequences: the a- and p-subunits have predicted
M, of 82,000 or 84,000 and 70,000, respectively. In the absence of the a-subunit, the endogenous tyrosine kinase of
the p-subunit is constitutively active, suggesting that one
function of the a-subunit is to repress the activity of the
tyrosine kinase (9). Insulin binding to the a-subunit probably
induces a conformational change in the receptor that relieves
this repression.
The identification of genetic defects in the hlNSR that are
associated with extreme insulin resistance and glucose intolerance has provided new insight into the possible role of
the hlNSR in the development of non-insulin-dependent diabetes mellitus (NIDDM) (10-16). It is appropriate to consider the contribution of genetic variation in the hlNSR gene
in relation to the development of the common forms of
NIDDM. Is there a subpopulation of diabetic patients who
have mutations in the hlNSR that impair its function but less
severely than those associated with extreme insulin-resistant
syndromes? We are in a position to address this important
question
'The numbering of the amino acid res~duesof the hlNSR is for a-subunit and
precursor of 731 and 1355 amno acids, respectively The four basic amino
acids at the proreceptor cleavage site are presumed to be removed from the
COOH-terminal of the a-subunit during processing of the proreceptor
129
EXON-INTRON ORGANIZATION OF hlNSR GENE
The hlNSR gene is located on the distal short arm of human
chromosome 19 in the region of bands p l 3 . 3 4 p13.2 (17).
The low-density lipoprotein (LDL)-receptor gene is also in
this region of chromosome 19 and is located toward the
centromere and -10-15 x lo6 base pairs (bp) from the
hlNSR gene (18). Most of the hlNSR gene has been isolated
as a series of overlapping DNA fragments in the bacteriophage-A (19). These fragments span a region of >130,000
bp, which includes both the gene and flanking regions (Fig.
1). We have sequenced -13,000 bp of the hlNSR, including
the 5'-flanking promoter region, each exon, and part of each
intron (19, unpublished observations). The gene is composed of 22 exons and 21 introns. The exons range in size
from 36 bp (exon 11) to >2500 bp (exon 22, most of which
codes for the 3'-untranslated region of hlNSR mRNA),
whereas the 21 introns that separate the exons vary in size
from -500 to >25,000 bp (19). All of the introns interrupt
protein-coding regions of the gene. The actual size of the
hlNSR gene is uncertain because parts of introns 2. 3, 9,
and 10 were not isolated, but it must be >120,000 bp. MullerWieland et al. (20) isolated a fragment of the hlNSR gene
that contains both exons 1 and 2, and their data indicate
that intron 1 is -25,000 bp. The distribution of the exons is
rather striking. Exons 1-1 1, which encode the a-subunit of
the receptor, are dispersed over >90,000 bp. In contrast,
exons 12-22, which encode the p-subunit, are located together in a region of -30,000 bp. The promoter region of
the hlNSR gene has also been characterized, and its features
are reviewed in Seino et al. (19).
The exon organizationof the hlNSR gene appears to reflect
the structural organization of the protein, because many of
the exons code for structural or functional modules of the
protein (Fig. 1). Exons 1-3 code for the signal peptide, part
of the insulin-binding region (21), and a very cysteine-rich
domain of the protein that may also interact with insulin (22),
respectively. The region encoded by exons 2-5 is also homologous to the corresponding region of the epidermal
growth factor-receptor gene (19). Residues encoded by
exon 6 appear to be located at the interface between adjacent a-subunits in the heterotetrameric form of the hlNSR
Mutations
Exon
Protein
domains
and contribute to the cooperative site-site binding interactions of the hlNSR (23). Exon 11 is the smallest exon, only
36 bp in size, and alternative splicing of this exon results in
the synthesis of hlNSR proteins having a-subunits with different COOH-terminal sequences (7,8,24). Exon 12 codes
for the tetrabasic amino acid sequence Arg-Lys-Arg-Arg,
which is the site in the proreceptor molecule cleaved by the
proteolytic processing enzyme, thereby generating the aand p-subunits. Exon 12 also encodes the NH,-terminal of
the p-subunit; 20% of the amino acids in this region of the
p-subunit are either serine or threonine, suggesting that the
NH,-terminal of the p-subunit may represent a region of Olinked oligosaccharide modification. Exon 15 codes for the
membrane-spanning region of the p-subunit. Thus, exons
1-14 encode the extracellular region of the hlNSR, whereas
exons 16-22 encode the region that is localized within the
cell. Exon 16 codes for a 22-amino acid segment that separates the transmembrane and tyrosine kinase domains. Exons 17-21 code for the protein tyrosine kinase; the amino
acids involved in ATP binding by the kinase, residues 10031008 (Gly-Gln-Gly-Ser-Phe-Gly)and 1030 (Lys), are all encoded by exon 17. The tyrosine residues that are autophosphorylated by the kinaseon insulin binding are encoded
by exons 20 (Tyr 1158, 1162, and 1163) and 22 (Tyr 1328
and 1334). Exon 22 also codes for the highly charged
COOH-terminal of the p-subunit. The data suggest that exons encode functional/structuraI domains of the hlNSR protein and that the hlNSR gene, like the LDL-receptor gene
(25,26), is a mosaic constructed from exons recruited from
other sources.
ALTERNATIVE SPLICING GENERATES
AMINO ACID-SEQUENCE ISOFORMS OF hlNSR
The a- and (3-subunits are generated by proteolytic processing of a precursor of 1370 or 1382 amino acids, which
includes a 27-amino acid signal peptide in addition to the
1343 or 1355 proreceptor molecule (7,8). The difference in
size of the a-subunit and its precursors is due to tissuespecific, possibly developmentally regulated, alternative
splicing of exon 11, which codes for a 12-amino acid segment at the COOH-terminal of the a-subunit (24). Brain and
0
./I+
-B+wt+*~
I
2
Signal
peptide
Insulin
binding
00
/'
3
Cysteine
rich
4
5 6 7 8 9
10 11
a-subunit
association spliced exon
-
L
-
V
1
12 13
a -subunit
14 15 16 17 18192021 22
-
I
J
a- and @-subunit
association
EGF receptor homology
\
0
processing site
kinase
~ransmbmbrane
Receptor recycling (?)
\
0
0
\
-
@-subunit
FIG. 1. Map of human insulin-receptor (hlNSR) gene. Relative locations and sizes of exons (m) and introns are indicated. Oblique lines, regions of
nonsense; -, deletion.
introns 2, 3, 9, and 11 that have not been cloned. Location of mutatlons in hlNSR gene are indicated: 0, mlssense; 0,
Table 1 describes mutations in greater detail. Domains of hlNSR protein encoded by each exon or group of exons are below gene map. kb,
Kllobase.
DIABETES. VOL. 39. FEBRUARY 1990
-
spleen express almost exclusively the 719-amino acid asubunit, whereas other tissues, including placenta, liver, kidney, and adipose tissue, express a-subunits of both 719 and
731 amino acids. It is unknown whether both types of asubunit are expressed in an individual cell, thereby generating a mixed population of receptors, including proteins
having two a-subunits of either 719 or 731 amino acids and
hybrid molecules with one a-subunit of each type. However,
because all cultured cell lines that have been examined
express only a single type of a-subunit, we suspect that
individual cells express only one form of a-subunit, i.e., of
either 719 or 731 amino acids. The biological consequences
of this diversity of hlNSR isoforms is unknown. Transfection
of cultured cells with cloned genes encoding recombinant
hlNSR protein representing both hlNSR isoforms has indicated that each is biologically active and capable of binding
insulin, stimulating autophosphorylation, and mediating postreceptor responses (27-29). However, preliminary studies
suggest that the hlNSR isoform with an a-subunit of 731
amino acids may have reduced affinity for insulin (30). Additional studies of the splicing of hlNSR mRNA might account
for some of the observed heterogeneity in size of the protein
expressed in different tissues.
GENETIC VARIATION IN hlNSR GENE
Genetic disorders of extreme hormone resistance provide a
unique opportunity to examine the functional consequences
of expression of an abnormal receptor. Extreme insulin resistance is associated with three syndromes: the type A syndrome of insulin resistance and acanthosis n~gricans,leprechaun~srn,and the Rabson-Mendenhall syndrome (31).
Recent studies have identified eight different mutations in
one or both of the INSR alleles of individuals with these
syndromes (Fig. 1; Table 1). These mutations affect both the
biosynthesis of the receptor and its biochemical properties
and include four mutations in the a-subunit (1 patient is a
compound heterozygote and has different mutations in each
of the parental INSR alleles; 10-12), one at the proreceptor
processing site (13), and three in the p-subunit (14-16). Two
of the a-subunit mutations (Pro'" and
appear to alter
the structure of the protein such that posttranslational pro-
-
cessing and transport of the receptor to the plasma membrane is delayed, thereby resulting in reduced numbers of
receptors on the cell surface (1 1,12). Individuals expressing
both a normal allele and the Pro2" mutation have mild insulin
resistance.
Subject 3 is a compound heterozygote expressing two
different a-subunit mutations (10; Table 1). There is a nonsense mutation in the codon for amino acid 672 of the paternally derived allele, which results in the synthesis of a
truncated 671-amino acid fragment of the a-subunit; such
a molecule would be expected to be secreted from the cell
and to be biologically inactive. This patient's maternally derived hlNSR allele has a missense mutation (GIu4=O)that results in the expression of a protein with qualitative abnormalities in insulin binding, including increased stability of the
insulin-INSR complex. Such a mutation might impair the release of insulin in the acidic endosome after internalization
of the insulin-hlNSR complex and thereby affect hlNSR recycling. Cells from the patient's mother, which express both
normal and mutant (GIuJ60)INSRs, also exhibited qualitative
abnormalities in insulin binding similar to the patient; however, the mother was neither insulin resistant nor diabetic.
By contrast, the father, who expresses both the normal INSR
protein and the truncated molecule, was moderately insulin
resistant with impaired glucose tolerance, although not
allele in the paovertly diabetic. It is unclear why the GIu~~O
tient is less effective than a normal allele in complementing
the nonsense mutation.
A mutation at the proreceptor processing site (Ser735)results in the expression of fully glycosylated but uncleaved
proreceptors on the cell surface (13). This mutant INSR is
capable of binding insulin and undergoing insulin-stimulated
autophosphorylation, albeit weakly and at high insulin concentrations, and provides an elegant demonstration that the
hlNSR acquires normal insulin affinity and sensitivity only
after proteolytic separation of the a- and p-subunits.
Three mutations in the p-subunit that represent postbinding defects in insulin action have been described, including
two missense mutations (Va11°08and SerlZoo;14,15) and a
deletion involving exons 17-22 that results in the expression
of a receptor lacking the tyrosine kinase domain (16). All are
TABLE 1
Mutations at human insulin-receptor locus associated with insulin resistance in subjects 1-7
Ethnic origin
Biochemical properties of
mutant protein
Leu 233 -+ Pro
Phe 382 + Val
Lys 460 -, Glu
Gln 672 -t AM
Dutch
Venezuelan
American
Delayed transport to cell surface
Delayed transport to cell surface
Qualitative abnormalities in insulin binding
Truncated a-subunit
11
10
Arg 735
Japanese
Impaired proreceptor processing
13
Gly 1008 -, Val
Trp 1200 -, Ser
Japanese
American
15
14
> 10-kb deletion
Japanese
Decreased tyrosine kinase activity
Decreased receptor affinity and
autophosphorylation
Decreased tyrosine kinase activity
Subject
Mutation
Proreceptor processing site
4
P-Subunit
5
6
-
Ser
Exon
Ref.
12
16
Subjects 1 2, and 4 are homozygous for indicated mutation. Subject 3 is a compound heterozygote having inherited different mutations
from each parent. Subjects 5-7 are heterozygous and express both mutant and normal insulin-receptor proteins. AM, amber (TAG)
translation termination codon; kb, Kilobase.
DIABETES, 'JOL 39, FEBRUARY 1990
131
associated with decreased or defective tyrosine kinase activity or autophosphorylation. The mutation at residue 1008
is of a conserved glycine residue that is part of the ATP
binding site of all protein kinases. The codominant expression of normal lNSRs and receptors having mutations in the
tyrosine kinase domain appears to result in suppression of
the function of the normal protein. The dominant negative
character of these mutations may be due to the fact that the
hlNSR is a heterotetramer and that heterotetramers formed
from two mutant subunits and mixed heterotetramers having
one normal and one mutant subunit are inactive or have
reduced activity. As a consequence, only 25% of the receptors would possess full biological activity.
In addition to mutations in the hlNSR gene that are associated with insulin resistance, population-association studies suggest that there is also genetic variation in the region
of the hlNSR gene that increases (32) or decreases (33) the
risk for NIDDM. The increased risk could be due to the
expression of abnormal receptors that suppress the function
of the normal receptors as suggested above. The decreased
risk associated with some hlNSR haplotypes might be due
to genetic variation that increases expression of the hlNSR
and results in increased numbers of receptors per cell.
Along with nucleotide substitutions that result in the
expression of an abnormal protein, silent substitutions in the
coding region of the hlNSR gene that alter the sequence of
the gene and mRNA but rot the protein have been described
(8,10,19t). Several restriction-fragment-length polymorphisms have also been reported at the hlNSR locus (3342) and all appear to be located within introns.
NIDDM suggested that the INSR gene was not the major
susceptibility locus for NIDDM, at least in this population
(46), the INSR gene may still contribute to the development
of NlDDM in a small but measurable subpopulation of patients. The isolation and characterization of the hlNSR gene
and recent technical developments provide a strategy for
examining the contribution of this gene to the development
of NlDDM by amplifyingindividual exons of the hlNSR gene
with the polymerase chain reaction (PCR) and then sequencing the amplified DNA. Where should we look for hlNSR
mutations? Because mutations in the tyrosine kinase domain
of the hlNSR are associated with severe insulin res~stance
in the heterozygous state, whereas heterozygosity for mutations in the a-subunit appears to be associated with lesssevere insulin resistance, the NlDDM subjects should first
be classified as to the degree of insulin resistance. In the
most-resistant subjects, PCR amplification and sequencing
of exons 17-21, which code for the tyrosine kinase domain
of the hlNSR, should be the first priority. In the less-resistant
patients, the analysis should begin with the amplification and
sequencing of exons 2 and 3, which appear to be involved
in insulin binding. Examination of the sequences of selected
regions of the hlNSR gene in 50-100 well-characterized
NlDDM subjects could provide valuable insight into the contribution of this gene to the development of this disorder.
ACKNOWLEDGMENTS
The studies from our laboratory were supported by the Howard Hughes Medical Institute, the Diabetes Research and
Training Center of The University of Chicago (DK-20595),
and a gift from Sandoz Research Institute.
CONCLUSION
The causes of NlDDM are unclear. Although impaired p-cell
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