0163-769X/92/1303-0566S03.00/0 Endocrine Reviews Copyright © 1992 by The Endocrine Society
Vol. 13, No. 3 Printed in U.S.A.
Mutations in the Insulin Receptor Gene SIMEON I. TAYLOR, ALESSANDRO CAMA*, DOMENICO ACCILI, FABRIZIO BARBETTIt, MICHAEL J. QUON, MARIA DE LA LUZ SIERRA, YOSHIFUMI SUZUKI, ELIZABETH ROLLER, RACHEL LEVY-TOLEDANO, EFRAT WERTHEIMER, VICTORIA Y. MONCADAJ, HIROKO KADOWAKI§, AND TAKASHI KADOWAKIH Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Disease, National Institutes of Health, Bethesda, Maryland 20892 C. Mutations that accelerate receptor degradation (class V) VII. Clinical Syndromes Due to Mutations in the Insulin Receptor Gene A. Leprechaunism B. Type A insulin resistance C. Rabson-Mendenhall syndrome D. Other syndromes E. Correlation of molecular defects with clinical syndromes F. Methods to screen for mutations in the insulin receptor gene G. Role of receptor defects in non-insulin-dependent diabetes mellitus VIII. Conclusion
I. Introduction II. Expression of the Insulin Receptor Gene A. Gene structure B. Transcription of insulin receptor mRNA C. Mutations decreasing levels of insulin receptor mRNA (class I) III. Insulin Receptor Structure and Biosynthesis A. Structural domains of the insulin receptor B. Posttranslational processing and assembly of oligomeric receptors C. Mutations that impair intracellular transport and post-translational processing (class II) IV. Insulin Binding A. Affinity and kinetics of insulin binding B. Mapping of the insulin binding domain C. Defects in insulin binding (class III) V. Insulin Receptor Tyrosine Kinase Activity A. Role of tyrosine kinase in mediating insulin action B. Mechanism whereby insulin stimulates receptor tyrosine kinase activity C. Tyrosine kinase domain: Structure-function relationships D. Mutations that impair receptor tyrosine kinase activity (class IV) VI. Endocytosis, Recycling, and Down-Regulation A. Receptor-mediated endocytosis B. Mutations in the tyrosine kinase domain impair receptor-mediated endocytosis
I. Introduction
T
Address requests for reprints to: Simeon I. Taylor, M.D., Ph.D., National Insitutes of Health, Building 10, Room 8S-239, Bethesda, Maryland 20892. * Present address: Istituto di Patologia Umana e Medicina Sociale, Facolta di Medicina e Chirurgia, Uiversita "G. D'Annunzio", Chieti, Italy. t Present address: Istituto San Raffaele, Rome, Italy. $ Present address: New England Deaconess Hospital, Boston, Massachusetts. § Present address: Institutes for Diabetes Care and Research, Asahi Life Foundation, Tokyo, Japan. || Present address: Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Tokyo, Japan.
HE insulin receptor is a cell surface glycoprotein that mediates the action of insulin upon target cells. The receptor was originally identified by its ability to bind the hormone (1-3). Over the past two decades, considerable progress has been made in defining the structure of the receptor molecule (4-7) as well as the biochemical mechanism by which it mediates insulin action (8-10). In addition, the insulin receptor has been identified as a target for pathological processes in human disease. In this review, we describe mutations in the insulin receptor gene that have been identified in patients with genetic forms of insulin resistance (6). Identification of these mutations has elucidated the molecular mechanisms that cause disease in these patients. In addition, the mutations have provided significant insights into the structure and function of the insulin receptor.
II. Expression of the Insulin Receptor Gene A. Gene structure The human insulin receptor gene contains 22 exons, and occupies in excess of 150 kilobase pairs of DNA on
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INSULIN RECEPTOR GENE MUTATIONS
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the short arm of chromosome 19 (bands pl3.2-*pl3.3) (11, 12). Typical of a "housekeeping" type of promoter, the promoter is GC-rich, lacks a TATA box, but contains several Spl binding sites (11, 13-16). In addition, an enhancer element has been identified 410-481 base pairs (bp) upstream from the initiator AUG codon (16). Several potential CAAT/enhancer binding protein (C/EBP) binding sites have been identified in the human insulin receptor gene, two in the 5'-flanking domain and one in the first intron (17). The precise importance of these various potential regulatory sites has not been completely elucidated. In addition, it seems likely there are other regulatory sites in the gene that remain to be identified. B. Transcription of insulin receptor messenger RNA Multiple different species of mRNA are transcribed from the insulin receptor gene. There are at least three causes of this heterogeneity of insulin receptor mRNA species. First, there are multiple distinct start sites for transcription located 350-550 bp upstream from the initiator AUG codon (14-16). Second, there are at least five alternative polyadenylation signals that are used, giving rise to mRNA species that range in length from approximately 5-11 kilobases (4,15,18). Third, exon 11, a short exon consisting of only 36 bp, undergoes variable splicing (19-21). Although these 36 nucleotides are present in virtually all of the insulin receptor mRNA molecules in liver, they are absent from insulin receptor mRNA in lymphocytes. Most other tissues contain a mixture of both types of transcripts. [N.B. In this review, we have used the numbering system of amino acids corresponding to the receptor encoded by the mRNA including the exon 11 sequence (5).] C. Mutations decreasing levels of insulin receptor mRNA (class I) Several different types of mutation have been identified that lead to a decrease in the level of insulin receptor mRNA (Fig. 1). As has been demonstrated in other genes, mutations that lead to premature termination of translation are frequently associated with a decrease in the level of mRNA (6). Several patients have had nonsense mutations (at codons 133, 897, and 1000) that had a cisdominant effect to decrease the levels of insulin receptor mRNA (Fig. 2) (22-24). Similarly, a point mutation has been described that altered the consensus sequence of the splice acceptor site at the 3'-end of intron 4 (25). As a result, the exon 4 sequence was spliced directly to the exon 6 sequence with skipping of exon 5. Because exon 5 contains 149 bp (i.e., not a multiple of 3), the skipping of exon 5 results in a frameshift and a premature chain termination codon; furthermore, this mutation in a splice
Class of Mutation
Synthesis
Transport to Plasma Membrane
Insulin Binding
Endocytosis, TransRecycling, membrane Signalling Degradation
-•X -•X -•X -•X
FIG. 1. Classification of mutations in insulin receptor gene. This cartoon summarizes the major steps in the life of an insulin receptor. First, the gene is transcribed, and the RNA is spliced. The mature mRNA is transported from the nucleus to the cytosol where it is translated by ribosomes on the rough endoplasmic reticulum. The receptor is transported through the endoplasmic reticulum and Golgi in which organelles it undergoes multiple posttranslational modifications. Eventually, the mature receptor is inserted in the plasma membrane. Insulin binds to the receptor on the cell surface. As a result, the receptor undergoes autophosphorylation and becomes activated as a tyrosine kinase. The interaction of insulin with its receptor initiates the various responses of the target cell to insulin. In addition, insulin binding triggers receptor-endocytosis. The acid pH in the endosome dissociates insulin from its receptor. Subsequent to receptor internalization, the receptor is either transported to lysosomes for degradation or recycled back to the plasma membrane for reutilization. The five major classes of genetic defects in receptor function are summarized in the table at the bottom of the figure. This classification scheme is an adaptation of the classification originally proposed by Brown and Goldstein (195) for genetic defects in the function of the LDL receptor.
site also had a cis-dominant effect to decrease the levels of mRNA. In contrast, at least one nonsense mutation (at codon 672) in the insulin receptor gene appeared not to decrease significantly the level of insulin receptor mRNA (26). Although some patients have been reported to have deletion mutations that result in frameshifts and premature chain termination (27-30), data have not been presented to address the question of whether these deletion mutations also decrease the level of the mutant mRNA. In several patients, evidence has been obtained that the level of insulin receptor mRNA is decreased despite
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Premature Termination Mutations in Insulin Receptor Gene a 1 a Phe-88,89 Trp Cys-rich
- » Stop
(Decreased mRNA levels)
Ex 3 A (Truncated receptor; fusion protein)
(155-312) 4
Cys-435
M ( A G —> GG (Defective splice acceptor; intron 4)
H ~
Cys-468 fl —
f E«5 A (Truncated receptor; luslon protein)
Cys-524 Gin
—> Stop
(Truncated receptor)
Exon 11 (718-729)
iArg Extracellular
"^ Stop (Decreased mRNA levels) (Truncated receptor; luslon protein)
Y//////////M(//X77, Tyr-972 ATP-Binding
(Codon 955; IrameshlH and premature stop) \nn 1000
(1003-1030)
Tyr-1158,1162,1163 •
~
-> Stop
(Decreased mRNA levels)
"A" (Codon 1109; frameshltt and premature stop) Tyr-1328,1334
P
(Truncated receptor; fusion protein)
FIG. 2. Map of premature termination mutations in insulin receptor gene. Key structural landmarks are identified at the left of the drawing of the receptor. Phe88, Phe89 (196), and the cysteine-rich domain (4) have all been implicated as playing a role in the insulin binding domain. Cys435, Cys468, and Cys624 are candidates to contribute sulfhydryl groups for formation of the disulfide bonds between adjacent a-subunits (44). Exon 11 is an exon that has been described to undergo variable splicing (19-21). As described in the text, the five (66,67) or six (61-65) tyrosine residues that are sites of autophosphorylation are indicated. The consensus sequence for an ATP binding domain is located between amino acid residues 1003-1030 (56). The locations of the 22 exons of the insulin receptor gene are indicated in the middle of the receptor cartoon. In addition, all of the published mutations causing premature chain termination are indicated on the right side of the cartoon.
the fact that no mutations were identified in any of the 22 exons of the allele from which the mRNA was transcribed (22, 31, 32). Although it seems likely that the mutations in these alleles map to important regulatory domains of the gene, specific mutations have not yet been demonstrated directly in any of these patients.
III. Insulin Receptor Structure and Biosynthesis A. Structural domains of the insulin receptor The nucleotide sequence of cloned insulin receptor complementary DNA has provided the information to deduce the primary amino acid sequence of the insulin receptor. Inspection of this sequence has allowed for the definition of multiple domains (Fig. 3): 1. Hydrophobic signal peptide. This consists of 27 hydrophobic amino acids at the N terminus of the proreceptor. The signal peptide is removed by proteolytic cleavage early in receptor biosynthesis, leaving histidine as the N terminus of the mature receptor molecule (4).
Vol. 13, No. 3
2. N-terminal repeat domain (amino acids 1-154). As will be discussed later in the review, the N-terminal domain of the insulin receptor appears to play an important role in ligand binding. This domain contains four repeats of a loosely conserved motif that is also homologous to the amino acid sequence of the receptors in the epidermal growth factor (EGF) receptor family (Table 1) (33). The best conserved feature in the motif is a central glycine residue that has been predicted to allow for a "turn" in the polypeptide chain conformation. It is intriguing that at least two of these conserved glycine residues (Gly31 and Gly366) have been identified as the sites of missense mutations in the insulin receptor genes of insulin-resistant patients (34, 35). In addition, there are usually hydrophobic amino acids located at positions 2, 5, and 8 amino acid residues upstream from the glycine residue (Table 1). It has been suggested that these amino acids may fold into a short amphipathic a-helical structure with hydrophobic amino acids oriented on the same face of the helix (33). Hydrophobic signal peptide (-27 --1) N-terminal repeat domain (1 -154)
Exons (amino acids) 1
(-27-7)
2
(7-191)
Cysteine-rich (155-312)
3 (191 -298)
2nd repeat domain
~ 2 9 8 - 348) 5 (348 - 396)
(313-428)
C-terminal cc-subunit
(429-731)
Proteolytic cleavage site (732 • 735)
Extracellular p-subunit (736 • 929)
6 (396-468) 7 (468-510) 8 (510-594) 9 (594 - 650) 10 (650-717) -729) 12 (729-821) 13 (821-867) 14 (867-921)
Transmembrane domain(930 • 952)
15 (921 - 955)
Juxtamembrane domain-
JMSJ955 • 978)
(953-1001)
17 (978- 1059) ~(1059 • 1096)
Tyrosine kinase domain(1002-1257)
19 (1096 -1150) 20 (1150 -1193) 21
C-terminal p-subunit
(1193 -1238)
22 (1238• 1355)
(1258-1355)
FIG. 3. Map of structural domains in the human insulin receptor. The various structural domain of the proreceptor are depicted in this figure. For comparison, the corresponding exons of the insulin receptor gene are depicted at the right side of the drawing.
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TABLE 1. Repeat motifs in the a-subunit of the insulin receptor Sequence
Motif Motif 1 Consensus sequence hIR hIR Motif 2
X--X-XX-G
I 1 311
HLYPGEVCPGMDIR VCHLLEGEKTI
14 321
G
hIR hIR Motif 3
1 15 NNLTRLHELENCSVIEGHLQILLMFKTRPEDFR 322 DSVTSAQELRGCTVINGSLIINIRGGNN 349
47
G
hIR hIR Motif 4
I 48 LSFPKLIMITDYLLLFRVY 350 LAAELEANLGLIEEISGYLKIRRSY
67 374
G
hIR hIR
I 68 GLESLKDLFPNLTVIRGSRLFFNYALVIFEMV 99 375 ALVSLSFFRKLRLIRGETLEIGNYSFYALDNQN 407
The repeat motifs in the a-subunit are listed. As proposed by Bajaj et al. (33), the most conserved element of this motif is the central glycine residue that is present in seven of the eight motifs. The only exception is that amino acid residue 58 is an aspartic acid instead of a glycine; however, the homologous amino acid is in fact glycine in the EGF receptor so that the sequence corresponding to amino acid residues 48-67 of the insulin receptor is nevertheless considered to comprise one repeat motif. A less well conserved feature of the repeat motif is the presence of hydrophobic amino acid residues (denoted below as X in the consensus sequence) at positions -2, -5, and -8 relative to the central glycine. In some of the motifs, a hydrophobic amino acid residue (denoted as x in the consensus sequence) is also found at position -3. To emphasize the hydrophobic amino acid residues upstream from the central glycine, they are printed in boldface type.
3. Cysteine-rich domain (amino acids 155-312). There is evidence [at least in the case of the highly homologous insulin-like growth factor I (IGF-I) receptor] that the cysteine-rich domain may play an important role in determining the specificity of ligand binding (36-42). This domain of the receptor contains 26 cysteine residues, most of which are probably involved in intrasubunit disulfide bonds. In addition, there are at least two other highly conserved amino acid residues (His209 and Pro219) that are identically conserved in all known members of the insulin receptor family (insulin receptor, IGF receptor, insulin receptor-related receptor) and the EGF receptor family (c-erb B-l, c-erb B-2, and c-erb B-3) (23, 43). 4. Repeat domain in the middle of the a-subunit (amino acids 313-428). This domain is homologous to the Nterminal repeat domain (amino acids 1-154), and also contains four repeats of the same motif described above (Table 1) (33). 5. C-terminal a-subunit domain (amino acids 429-731). This domain includes the 12 amino acid residues (amino acids 720-731) that are encoded by exon 11, the exon that undergoes variable splicing. Therefore, the length of this domain is variable,' either 291 or 303 amino acid residues, depending on whether or not exon 11 was present in the mRNA from which the receptor molecule
was translated. The functions of this domain are not known, but it has been proposed that it may contain the region of the receptor that allows for the binding interaction between the two a-subunits within the heterotetrameric a^fo form of the receptor (44). In addition, this domain contains a major immunogenic region (amino acids 450-601) that includes a major epitope recognized by several monoclonal anti-receptor antibodies as well as most anti-receptor autoantibodies from patients with type B insulin resistance (41). Furthermore, the presence of the 12 amino acid residues encoded by exon 11 causes a 2-fold reduction in the affinity of the receptor to bind insulin (45-47). 6. Proteolytic cleavage site (amino acids 732-735). There is a sequence consisting of four basic amino acids (ArgLys-Arg-Arg) located at the junction between the a- and /3-subunits that is presumed to be the site for proteolytic cleavage of the proreceptor (4, 5, 48, 49). 7. Extracellular [i-subunit domain (amino acids 736-929). This contains the site of O-linked glycosylation (50). The function of this domain has not been clearly defined. 8. Transmembrane domain (amino acids 930-952). There is a stretch of 23 amino acid residues (rich in hydrophobic amino acids) that is thought to be the single transmembrane domain of the receptor molecule (4, 5).
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9. Juxtamembrane domain (amino acids 953-1001). The juxtamembrane domain is the portion of the /3-subunit that is near the plasma membrane. This contains the signals that allow for receptor endocytosis (51-54). In addition, the juxtamembrane region contains Tyr972, an amino acid residue that has been proposed to play a role in binding some substrates for tyrosine-specific phosphorylation (55). 10. Tyrosine kinase domain (amino acids 1002-1257). This is the region with an amino acid sequence that most closely resembles that of other tyrosine kinases such as the EGF receptor (4, 56). This contains the consensus sequence for ATP binding (Gly-X-Gly-X-X-Gly Lys, amino acids 1003-1030) (56-60) as well as the three tyrosine residues (Tyr1158, Tyr1162, and Tyr1163) the phosphorylation of which appears to activate the ability of the receptor to phosphorylate other protein substrates (10, 61-67). 11. C-terminal fi-subunit domain (amino acids 12581355). This is the region of the intracellular domain where there is maximal divergence between the receptors for insulin and IGF-I (68). Therefore, it has been proposed to contribute to specificity in transmembrane signalling. Furthermore, this domain contains two tyrosine residues (Tyr1328 and Tyr1334) that undergo insulin-stimulated autophosphorylation (61-67). It also contains serine and threonine residues that undergo phosphorylation, including Thr1348 (the site of phosphorylation by protein kinase C) (69, 70) and Ser1305 and Ser1306 (sites of insulin-stimulated serine phosphorylation) (71). B. Posttranslational processing and assembly of oligomeric receptors
Although the mature insulin receptor contains two distinct subunits {a and /?), the proreceptor is a 1355 amino acid molecule that is encoded by a single gene (4, 5). The proreceptor undergoes cotranslational N-linked glycosylation (72-74). There are 15 potential sites for relinked glycosylation on the a-subunit, and four sites in the extracellular portion of the /3-subunit. The proreceptor has a mobility on NaDodSO4-polyacrylamide gel electrophoresis corresponding to Mr 190,000 (73, 75-76). Immediately after it is synthesized, the proreceptor does not have the ability to bind insulin (77). However, while still in the endoplasmic reticulum, the proreceptor folds into a conformation that is competent to bind insulin. It seems likely that the intrasubunit disulfide bonds are formed at this time. Subsequently, the receptor undergoes additional posttranslational processing steps within the endoplasmic reticulum and Golgi apparatus. 1. Assembly of receptor into an oligomeric form. Two 190
kilodalton (kDa) proreceptor molecules dimerize to form
Vol. 13, No. 3
a high molecular weight species linked by disulfide bonds (43, 74, 77, 78). At this point, the N-linked oligosaccharide chains remain in the high mannose form. 2. Proteolytic cleavage into subunits. The proreceptor undergoes proteolytic cleavage at a tetrabasic amino acid sequence (Arg-Lys-Arg-Arg) that separates the a- from the 0-subunit (4, 5, 43, 48, 49, 73-78). This gives rise to a disulfide-linked heterotetrameric a2^2 species. The immature pre-a- and pre-0-subunits have Mr values of 120,000 and 85,000, respectively. 3. Terminal processing of N-linked oligosaccharide. The N-linked oligosaccharide is processed with removal of glucose and mannose residues, and addition of other sugars (e.g. fucose and galactose) and sialic acid (72, 73). These processing steps, which occur in the Golgi apparatus, are associated with a decrease in the electrophoretic mobilities of the receptor subunits; the mature aand 0-subunits have Mr values of 135,000 and 95,000, respectively. 4. O-linked glycosylation. There are one or more oligosaccharides that are attached via O-linkage to the extracellular domain of the /?-subunit (50, 79), most likely in the cluster of threonine and serine residues in the extracellular domain of the /3-subunit (49): Asn Thr 756 Ser 7 5 7 Ser 758 Thr 759 Ser 7 6 0 Val Pro Thr 763 Ser 764 Pro
It is not established precisely at what point O-linked glycosylation occurs during receptor biosynthesis. 5. Acylation. There is evidence for acylation (both myristylation and palmitylation) of both subunits of the receptor. The fatty acids appear to be attached through both amide {a- and 0-subunits) and ester (/3-subunit) linkages (80). C. Mutations that impair intracellular transport and posttranslational processing (class II) Many mutations interfere with posttranslational processing of the insulin receptor (Figs. 1 and 4). In one case, the mutation specifically alters an amino acid sequence required for proteolytic cleavage, thereby directly impairing proteolytic processing (81-84). However, in several other cases, the mechanism is indirect. Presumbably, these mutations impair the normal folding of the receptor molecule (34, 43, 78, 85-88). As a consequence, there is a defect in transport of the receptor through the endoplasmic reticulum and Golgi. The defect in intracellular processing leads to a decrease in the number of receptors on the cell surface. Moreover, the defect in posttranslational processing occurs because most of the receptors do not reach the intracellular compartments where many
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August, 1992
INSULIN RECEPTOR GENE MUTATIONS
Missense Mutations in Insulin Receptor Gene
a Phe-88,89 Cys-rich (155-312)
Cys-435 Cys-468 Cys-524
Exon11 (718-729)
, . Asn
(Impaired transport to cell surface; -> Lys decreased binding affinity)
' ~ Val 2 8 - » Ala h-Gly31->Arg
(Impaired transport to cell surface)
] - His 209 -* Arg I* Leu 2 3 3 -» Pro
(Decreased binding)
(Impaired transport to cell surface)
' G l y 3 6 6 - * Arg 382 (Impaired transport to cell surface) 1 -Phe - > Val (Increased binding affinity; 460 decreased pH sensitivity) I * Asn - » Ser (Decreased pH sensitivity)
,-Arg
' _» e . . (Uncleaved receptor; a e r decreased binding affinity)
571
creases binding affinity, but because the defect is due to a specific conformational change introduced by the substitution of serine at position 735. Another site-directed mutation has been described that impairs proteolytic cleavage and decreases the affinity of insulin binding: that is, total deletion of the Arg-Lys-Arg-Arg sequence (49). Surprisingly, even when these four amino acids were deleted, approximately 20% of the mutant receptor molecules were still cleaved. This suggests that there must be a cryptic site that can be used for proteolytic cleavage. It is not known whether this cryptic site is sometimes used, even in the context of a proreceptor with a normal amino acid sequence. Nevertheless, the uncleaved A732~735-mutant receptor bound insulin with low affinity and was impaired in autophosphorylation and endocytosis (49).
Extracellular
2. Misfolding of receptor due to mutations in the Nterminal half of the a-subunit. At least five mutations Tyr-972 , . Arg" ->Gln ATP-Binding | (Fig. 4) have been identified in the a-subunit of the (1003-1030) '•-Gly -+Val receptor that impair intracellular transport through the fV>Lys1068->Glu Tyr-1158,1162,1163 endoplasmic reticulum and Golgi apparatus: Asn15—»Lys 1134 31 Decreased fe\>a ->Thr (78), Gly ^Arg (34), His20{UArg (43), Leu 233 ^Pro (86, tyrosine Tyr-1328,1334 *Ala - » Glu kinase 87), and Phe385WVal (83). Two other mutations have Met —> lie been identified in the same general location in the asubunit of the receptor, and these may also impair posttranslational processing of the receptor: Val28—*Ala (35), •_ 1200 . Car and Gly366—>Arg (35); however, the transfection studies Trp —* '*' have not yet been reported for these mutant receptors. FIG. 4. Missense mutations in the insulin receptor gene in insulinIn each case, impaired intracellular transport is associresistant patients. Key structural landmarks of the insulin receptor are identified at the left of the drawings (see Fig. 2). The locations of all of ated with a defect in posttranslational processing; howthe reported missense mutations are indicated in the right half of the ever, the completeness of the defect varies among the drawing of the receptor. different mutations. In those cases where it has been studied, the mutations inhibit the assembly of mutant of the enzymes required for posttranslational processing receptors into oligomers (43, 78). In addition, these muare located. tations inhibit terminal processing of the N-linked car1. Impaired proteolytic cleavage of proreceptor due to a bohydrate and proteolytic cleavage of the receptors into mutation in the proteolytic cleavage site. Two sisters with a- and /3-subunits (34, 43, 78, 85). This is demonstrated in experiments with NIH-3T3 cells transfected with type A insulin resistance have been identified who are 735 cDNA encoding either wild type or Arg209-mutant recephomozygous for a mutation substituting serine for Arg , tors (Fig. 5) (43). The transfected cells were pulse-labeled the last amino acid in the tetrabasic amino acid sequence by incubation for 45 min with [2-3H]mannose followed (Arg-Lys-Arg-Arg) located at the junction between the by a variable length chase in the presence of unlabeled a- and |8-subunits (Fig. 4) (81, 82). This mutation has mannose. The cells were solubilized in Triton X-100, been reported to impair proteolytic cleavage and to deand the insulin receptors were immunoprecipitated with crease the affinity of insulin binding to the uncleaved 735 anti-receptor antibody. The immunoprecipitates were Ser -mutant proreceptor (83, 84). Similar, although not first analyzed by NaDodSO4-polyacrylamide gel electroidentical, conclusions have been reached using alaninephoresis in the presence of reductant to study the postscanning mutagenesis (48). Substitution of alanine for 732 735 733 734 translational processing of the receptor. At the beginning either Arg or Arg (but not Lys or Arg ) impaired of the chase period, the major labeled band immunopreproteolytic cleavage of the proreceptor. The uncleaved cipitated by anti-receptor antibody corresponds to the mutant proreceptors were reported to bind insulin with receptor precursor (M 190,000). In cells expressing the r the same affinity as normally processed receptors in this 209 wild type (His ) receptor, the precursor is processed study. Taken at face value, these data suggest that it is rapidly, disappearing with a half-life of Ser) that causes a similar (although milder) defect in pH sensitivity and is also associated with a shortened receptor half-life (23, 168). Thus, although it is possible that some of the clinical differences among different patients may relate to selective insulin resistance in different branches of the insulin response pathway, this does not seem to be the major explanation of the observed clinical variation. 5. Genetic variation at different loci. According to this view, the clinical syndrome may be modulated by genetic factors at other genetic loci (187). For example, defects
585
have been reported in various growth factor receptors in patients with leprechaunism (187-189). It has been proposed that these may play a role in the growth retardation observed in patients with this syndrome. Although it would be possible to investigate genes encoding specific growth factor receptors that have been implicated (e.g., the EGF receptor), this type of study has not been done in these patients. If it were true that the difference between type A insulin resistance and leprechaunism were due to mutations at another genetic locus (e.g. the EGF receptor gene), then one would predict that mutations at the two genetic loci should segregate independently. In a given pedigree, one child might inherit the insulin receptor mutations in the absence of the mutations at the other locus and manifest type A insulin resistance according to this hypothesis. Another child in the same pedigree might inherit mutations in both the insulin receptor and the other locus and have the syndrome of leprechaunism. Thus, this theory would predict that patients with both syndromes should be found in the same family. However, to the best of our knowledge, this type of discordance has not been reported despite the fact that there have been multiple reported kindreds with two or more siblings having the same clinical syndrome, i.e., either leprechaunism (166, 172) or type A insulin resistance (81-85, 190, 191). Nevertheless, there is considerable evidence of subtle clinical variation between individuals within the same family who appear to have the same genotype at the insulin receptor locus (85, 191). Two sisters with type A insulin resistance (patients A-5 and A-8) provide one of the most striking examples of this phenomenon. They are both members of a consanguineous pedigree and are both homozygous for the Val382 mutation in the insulin receptor gene. Both patients are insulin resistant. However, patient A-5 has required treatment with several thousand units of insulin per day while her sister patient A-8 has not required insulin most of the time and has often been treated with dietary management. Furthermore, patient A-5 has such severe hyperandrogenism that she has never had spontaneous menses and did not experience withdrawal bleeding when she was treated with oral preparations of estrogen plus progestin. In contrast, despite some degree of hyperandrogenism, patient A-8 has ovulated spontaneously and has even achieved pregnancy on two occasions. It seems likely that the observed clinical differences are accounted for by polygenic factors that modulate the effect of the mutations at a major genetic locus (i.e. the insulin receptor gene). F. Methods to screen for mutations in the insulin receptor gene Many patients with mutations in the insulin receptor gene—especially those with two mutant alleles—are ex-
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TAYLOR ET AL.
tremely resistant to insulin, requiring therapy with as much as several thousand units of insulin per day (6). However, some patients—particularly those who are heterozygous for a single mutant allele—have more modest degrees of insulin resistance, comparable to that which is observed in patients with impaired glucose tolerance or non-insulin-dependent diabetes mellitus (Refs. 27, 28, 30, 31, 57, and 104-109 and unpublished observations of H. Kadowaki and T. Kadowaki and of H. Makino). Moreover, like most patients with non-insulin-dependent diabetes mellitus, some of these patients are obese (28, 104). It is possible that mutations in the insulin receptor gene provide a genetic predisposition that exacerbates the insulin resistance caused by obesity. Clearly, it would be of interest to determine the prevalence of these mutations in the population, and to determine whether these mutations could contribute to the pathogenesis of insulin resistance in a subpopulation of patients with non-insulin-dependent diabetes mellitus. The major obstacle to this type of study has been the amount of work required to obtain a definitive answer. Before considering specific studies of the insulin receptor gene in patients with non-insulin-dependent diabetes mellitus that have been reported, it is worthwhile to review the techniques that are required to detect various types of mutations: 1. Point mutations in the protein-coding sequence of the gene. Point mutations (i.e. either nonsense or missense mutations) can be detected by determining the nucleotide sequence of either genomic DNA or cDNA. Most of the mutations that have been identified to date fall into this category. In several respects, it is preferable to use genomic DNA for this purpose. The most important reason for this recommendation is that mutations causing premature chain termination usually reduce the level of mRNA transcribed from the allele with the mutation. Thus, there is a danger that most of the cDNA would be derived from the allele without the mutation. As a practical matter, most recent studies have utilized the polymerase chain reaction to amplify fragments of genomic DNA corresponding to each of the 22 exons of the insulin receptor gene together with the intron sequences immediately flanking each exon (22, 23, 25, 30, 31, 34, 35,104, 147-150, 186, 192). Accordingly, this strategy would detect mutations in the protein-coding sequence of the gene as well as mutations in the splice donor and acceptor sites in the introns. Furthermore, the same approach will detect insertions or short deletion mutations so long as they are entirely contained within a single exon. In most of the early studies, mutations were detected by determining the nucleotide sequence of either cloned DNA or DNA amplified by polymerase chain reaction (22-26, 31, 34, 57, 63, 85, 104, 106, 109, 184, 185).
Vol. 13, No. 3
However, more recently, several molecular scanning techniques have been used as a prelude to sequencing. These screening techniques allow for localization of variant sequences, which can then be analyzed definitively by sequencing the DNA. The major advantage of these techniques is to greatly reduce the work involved in studying large numbers of patients. The first such technique was heteroduplex mapping (193). However, because heteroduplex mapping has an unacceptably high rate of missing mutations, it has largely been supplanted by newer techniques: analysis of single-stranded conformational polymorphisms1 (SSCP) (147, 149) and denaturing gradient gel electrophoresis2 (DGGE) (Fig. 18) (35). Both of these techniques are estimated to have «90% sensitivity to detect point mutations. These techniques will detect any variation in the nucleotide sequence—both mutations and normal polymorphisms. Thus, in interpreting these data, it is extremely useful to have knowledge of the common polymorphisms in the nucleotide sequence (Table 3). 2. Deletion mutations. At least four deletion mutations 1 Double-stranded DNA assumes a double helical conformation under nondenaturing conditions. Thus, as a first order approximation, the conformation of double-stranded DNA does not depend on the nucleotide sequence. Consequently, the electrophoretic mobility of double-stranded DNA under nondenaturing conditions depends only on the length of the DNA. In contrast, the conformation of singlestranded DNA depends upon the nucleotide sequence. Because the shape of the single-stranded DNA determines its electrophoretic mobility, an alteration in the nucleotide sequence has the potential to alter the electrophoretic mobility of single-stranded DNA. Based on analysis of single-stranded conformational polymorphisms (SSCP), there is ~90% probability of detecting a change in a single nucleotide in a «500 nucleotide sequence if electrophoresis is carried out under four experimental conditions: 1) 4 C; 2) 4 C in the presence of 10% glycerol; 3) 24 C; and 4) 24 C in the presence of 10% glycerol (147, 149). 2 The nucleotide sequence determines the strength of the forces that stabilize the double helical conformation of double-stranded DNA. For example, it takes more energy to melt a G:C base-pair than an A:T base pair. Accordingly, if there is a change in the nucleotide sequence, this can alter the melting point of double-stranded DNA. For example, when genomic DNA is amplified by the polymerase chain reaction, both alleles will be amplified. If there is difference between the sequence of the two alleles, both sequences will be amplified to yield the two homoduplex molecules in which all of the nucleotides are matched perfectly. However, when the duplex DNA is denatured during the course of the polymerase chain reaction, there is a possibility that the sense strand derived from one allele will hybridize to the antisense strand derived from the other allele. This will yield heteroduplex molecules in which there are mismatched base pairs. In some cases, the two different homoduplex molecules will have different melting profiles. However, the heteroduplex molecules will almost always melt at lower temperature than the homoduplex molecules. In denaturing gradient gel electrophoresis (DGGE), duplex DNA is electrophoresed through a gradient of increasing concentrations of denaturants (usually, urea and formamide). As the duplex DNA is denatured, its electrophoretic mobility decreases. Accordingly, the nucleotide sequence determines the stability of the double-stranded conformation. This determines the concentration of denaturants required to melt the double-stranded conformation which, in turn, determines the electrophoretic mobility. It has been shown that the probability of detecting a change in a single base pair in a 500 bp sequence is «90% (35).
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INSULIN RECEPTOR GENE MUTATIONS
TABLE 3. Sequence polymorphisms in coding region of insulin receptor gene Amino Acid #
Amino Acid
Codon
A. Confirmed polymorphisms that do not alter amino acid sequence -20 234 276 519 523 642 1058
65%
GRADIENT OF DENATURANTS
25%
FIG. 18. Perpendicular DGGE of exon 2 of insulin receptor gene from patient's parents and from leprechaun/Verona-1. The amplified fragment of DNA corresponding to the 5'-half of exon 2 from the father (gel A) and mother (gel B) of a patient with leprechaunism was electrophoresed through a polyacrylamide gel with a 25-65% gradient of denaturants (urea and formamide). The gradient was linear, with the concentration of denaturants increasing from right to left. The direction of electrophoresis was from the top to the bottom of the gel (35). With the mother's amplified genomic DNA, a single melting curve is observed indicating that both alleles of her insulin receptor gene have the same sequence. In contrast, with amplified DNA from her father, multiple melting curves were observed, indicating that more than one species is present in the amplified DNA of the proband leprechaun/Verona-1. Thus, both the patient and her father are heterozygous for a nucleotide substitution in exon 2 encoding the Val28—* Ala mutation.
have been identified in the insulin receptor gene (27-30). These mutations were recognized initially because of the presence of abnormal bands on Southern blots. If the same DNA were analyzed by carrying out polymerase chain reaction with genomic DNA as template, the mutations would not have been detected. Consider, for example, the deletion of exon 14 (28). If one amplified exon 14 from the patient's genomic DNA, then one would get the normal size DNA fragment produced as a result of amplification of the normal allele. Because the oligonucleotide primers would not hybridize to the allele with the deletion, the experiment would not provide any evidence of the existence of the mutation. Accordingly, molecular scanning techniques such as SSCP and DGGE would fail to detect this type of mutation. Some deletions might be detected by determining the sequence of cDNA. However, most deletions will result in a frameshift so that they will introduce a premature chain termination codon. Accordingly, the mutation may selectively reduce the level of the mRNA transcribed from the mutant allele. Thus, analyzing the cDNA is an insensitive strat-
1062
Gly Gly Asp Asp Gin Gin Asp Asp Ala Ala Phe Phe His His Leu Leu
GGG (>90%) GGA (90%) GAT (90%) CTT (90%) ATG (
Vol. 13, No. 3 Printed in U.S.A.
Mutations in the Insulin Receptor Gene SIMEON I. TAYLOR, ALESSANDRO CAMA*, DOMENICO ACCILI, FABRIZIO BARBETTIt, MICHAEL J. QUON, MARIA DE LA LUZ SIERRA, YOSHIFUMI SUZUKI, ELIZABETH ROLLER, RACHEL LEVY-TOLEDANO, EFRAT WERTHEIMER, VICTORIA Y. MONCADAJ, HIROKO KADOWAKI§, AND TAKASHI KADOWAKIH Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Disease, National Institutes of Health, Bethesda, Maryland 20892 C. Mutations that accelerate receptor degradation (class V) VII. Clinical Syndromes Due to Mutations in the Insulin Receptor Gene A. Leprechaunism B. Type A insulin resistance C. Rabson-Mendenhall syndrome D. Other syndromes E. Correlation of molecular defects with clinical syndromes F. Methods to screen for mutations in the insulin receptor gene G. Role of receptor defects in non-insulin-dependent diabetes mellitus VIII. Conclusion
I. Introduction II. Expression of the Insulin Receptor Gene A. Gene structure B. Transcription of insulin receptor mRNA C. Mutations decreasing levels of insulin receptor mRNA (class I) III. Insulin Receptor Structure and Biosynthesis A. Structural domains of the insulin receptor B. Posttranslational processing and assembly of oligomeric receptors C. Mutations that impair intracellular transport and post-translational processing (class II) IV. Insulin Binding A. Affinity and kinetics of insulin binding B. Mapping of the insulin binding domain C. Defects in insulin binding (class III) V. Insulin Receptor Tyrosine Kinase Activity A. Role of tyrosine kinase in mediating insulin action B. Mechanism whereby insulin stimulates receptor tyrosine kinase activity C. Tyrosine kinase domain: Structure-function relationships D. Mutations that impair receptor tyrosine kinase activity (class IV) VI. Endocytosis, Recycling, and Down-Regulation A. Receptor-mediated endocytosis B. Mutations in the tyrosine kinase domain impair receptor-mediated endocytosis
I. Introduction
T
Address requests for reprints to: Simeon I. Taylor, M.D., Ph.D., National Insitutes of Health, Building 10, Room 8S-239, Bethesda, Maryland 20892. * Present address: Istituto di Patologia Umana e Medicina Sociale, Facolta di Medicina e Chirurgia, Uiversita "G. D'Annunzio", Chieti, Italy. t Present address: Istituto San Raffaele, Rome, Italy. $ Present address: New England Deaconess Hospital, Boston, Massachusetts. § Present address: Institutes for Diabetes Care and Research, Asahi Life Foundation, Tokyo, Japan. || Present address: Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Tokyo, Japan.
HE insulin receptor is a cell surface glycoprotein that mediates the action of insulin upon target cells. The receptor was originally identified by its ability to bind the hormone (1-3). Over the past two decades, considerable progress has been made in defining the structure of the receptor molecule (4-7) as well as the biochemical mechanism by which it mediates insulin action (8-10). In addition, the insulin receptor has been identified as a target for pathological processes in human disease. In this review, we describe mutations in the insulin receptor gene that have been identified in patients with genetic forms of insulin resistance (6). Identification of these mutations has elucidated the molecular mechanisms that cause disease in these patients. In addition, the mutations have provided significant insights into the structure and function of the insulin receptor.
II. Expression of the Insulin Receptor Gene A. Gene structure The human insulin receptor gene contains 22 exons, and occupies in excess of 150 kilobase pairs of DNA on
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INSULIN RECEPTOR GENE MUTATIONS
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the short arm of chromosome 19 (bands pl3.2-*pl3.3) (11, 12). Typical of a "housekeeping" type of promoter, the promoter is GC-rich, lacks a TATA box, but contains several Spl binding sites (11, 13-16). In addition, an enhancer element has been identified 410-481 base pairs (bp) upstream from the initiator AUG codon (16). Several potential CAAT/enhancer binding protein (C/EBP) binding sites have been identified in the human insulin receptor gene, two in the 5'-flanking domain and one in the first intron (17). The precise importance of these various potential regulatory sites has not been completely elucidated. In addition, it seems likely there are other regulatory sites in the gene that remain to be identified. B. Transcription of insulin receptor messenger RNA Multiple different species of mRNA are transcribed from the insulin receptor gene. There are at least three causes of this heterogeneity of insulin receptor mRNA species. First, there are multiple distinct start sites for transcription located 350-550 bp upstream from the initiator AUG codon (14-16). Second, there are at least five alternative polyadenylation signals that are used, giving rise to mRNA species that range in length from approximately 5-11 kilobases (4,15,18). Third, exon 11, a short exon consisting of only 36 bp, undergoes variable splicing (19-21). Although these 36 nucleotides are present in virtually all of the insulin receptor mRNA molecules in liver, they are absent from insulin receptor mRNA in lymphocytes. Most other tissues contain a mixture of both types of transcripts. [N.B. In this review, we have used the numbering system of amino acids corresponding to the receptor encoded by the mRNA including the exon 11 sequence (5).] C. Mutations decreasing levels of insulin receptor mRNA (class I) Several different types of mutation have been identified that lead to a decrease in the level of insulin receptor mRNA (Fig. 1). As has been demonstrated in other genes, mutations that lead to premature termination of translation are frequently associated with a decrease in the level of mRNA (6). Several patients have had nonsense mutations (at codons 133, 897, and 1000) that had a cisdominant effect to decrease the levels of insulin receptor mRNA (Fig. 2) (22-24). Similarly, a point mutation has been described that altered the consensus sequence of the splice acceptor site at the 3'-end of intron 4 (25). As a result, the exon 4 sequence was spliced directly to the exon 6 sequence with skipping of exon 5. Because exon 5 contains 149 bp (i.e., not a multiple of 3), the skipping of exon 5 results in a frameshift and a premature chain termination codon; furthermore, this mutation in a splice
Class of Mutation
Synthesis
Transport to Plasma Membrane
Insulin Binding
Endocytosis, TransRecycling, membrane Signalling Degradation
-•X -•X -•X -•X
FIG. 1. Classification of mutations in insulin receptor gene. This cartoon summarizes the major steps in the life of an insulin receptor. First, the gene is transcribed, and the RNA is spliced. The mature mRNA is transported from the nucleus to the cytosol where it is translated by ribosomes on the rough endoplasmic reticulum. The receptor is transported through the endoplasmic reticulum and Golgi in which organelles it undergoes multiple posttranslational modifications. Eventually, the mature receptor is inserted in the plasma membrane. Insulin binds to the receptor on the cell surface. As a result, the receptor undergoes autophosphorylation and becomes activated as a tyrosine kinase. The interaction of insulin with its receptor initiates the various responses of the target cell to insulin. In addition, insulin binding triggers receptor-endocytosis. The acid pH in the endosome dissociates insulin from its receptor. Subsequent to receptor internalization, the receptor is either transported to lysosomes for degradation or recycled back to the plasma membrane for reutilization. The five major classes of genetic defects in receptor function are summarized in the table at the bottom of the figure. This classification scheme is an adaptation of the classification originally proposed by Brown and Goldstein (195) for genetic defects in the function of the LDL receptor.
site also had a cis-dominant effect to decrease the levels of mRNA. In contrast, at least one nonsense mutation (at codon 672) in the insulin receptor gene appeared not to decrease significantly the level of insulin receptor mRNA (26). Although some patients have been reported to have deletion mutations that result in frameshifts and premature chain termination (27-30), data have not been presented to address the question of whether these deletion mutations also decrease the level of the mutant mRNA. In several patients, evidence has been obtained that the level of insulin receptor mRNA is decreased despite
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568
Premature Termination Mutations in Insulin Receptor Gene a 1 a Phe-88,89 Trp Cys-rich
- » Stop
(Decreased mRNA levels)
Ex 3 A (Truncated receptor; fusion protein)
(155-312) 4
Cys-435
M ( A G —> GG (Defective splice acceptor; intron 4)
H ~
Cys-468 fl —
f E«5 A (Truncated receptor; luslon protein)
Cys-524 Gin
—> Stop
(Truncated receptor)
Exon 11 (718-729)
iArg Extracellular
"^ Stop (Decreased mRNA levels) (Truncated receptor; luslon protein)
Y//////////M(//X77, Tyr-972 ATP-Binding
(Codon 955; IrameshlH and premature stop) \nn 1000
(1003-1030)
Tyr-1158,1162,1163 •
~
-> Stop
(Decreased mRNA levels)
"A" (Codon 1109; frameshltt and premature stop) Tyr-1328,1334
P
(Truncated receptor; fusion protein)
FIG. 2. Map of premature termination mutations in insulin receptor gene. Key structural landmarks are identified at the left of the drawing of the receptor. Phe88, Phe89 (196), and the cysteine-rich domain (4) have all been implicated as playing a role in the insulin binding domain. Cys435, Cys468, and Cys624 are candidates to contribute sulfhydryl groups for formation of the disulfide bonds between adjacent a-subunits (44). Exon 11 is an exon that has been described to undergo variable splicing (19-21). As described in the text, the five (66,67) or six (61-65) tyrosine residues that are sites of autophosphorylation are indicated. The consensus sequence for an ATP binding domain is located between amino acid residues 1003-1030 (56). The locations of the 22 exons of the insulin receptor gene are indicated in the middle of the receptor cartoon. In addition, all of the published mutations causing premature chain termination are indicated on the right side of the cartoon.
the fact that no mutations were identified in any of the 22 exons of the allele from which the mRNA was transcribed (22, 31, 32). Although it seems likely that the mutations in these alleles map to important regulatory domains of the gene, specific mutations have not yet been demonstrated directly in any of these patients.
III. Insulin Receptor Structure and Biosynthesis A. Structural domains of the insulin receptor The nucleotide sequence of cloned insulin receptor complementary DNA has provided the information to deduce the primary amino acid sequence of the insulin receptor. Inspection of this sequence has allowed for the definition of multiple domains (Fig. 3): 1. Hydrophobic signal peptide. This consists of 27 hydrophobic amino acids at the N terminus of the proreceptor. The signal peptide is removed by proteolytic cleavage early in receptor biosynthesis, leaving histidine as the N terminus of the mature receptor molecule (4).
Vol. 13, No. 3
2. N-terminal repeat domain (amino acids 1-154). As will be discussed later in the review, the N-terminal domain of the insulin receptor appears to play an important role in ligand binding. This domain contains four repeats of a loosely conserved motif that is also homologous to the amino acid sequence of the receptors in the epidermal growth factor (EGF) receptor family (Table 1) (33). The best conserved feature in the motif is a central glycine residue that has been predicted to allow for a "turn" in the polypeptide chain conformation. It is intriguing that at least two of these conserved glycine residues (Gly31 and Gly366) have been identified as the sites of missense mutations in the insulin receptor genes of insulin-resistant patients (34, 35). In addition, there are usually hydrophobic amino acids located at positions 2, 5, and 8 amino acid residues upstream from the glycine residue (Table 1). It has been suggested that these amino acids may fold into a short amphipathic a-helical structure with hydrophobic amino acids oriented on the same face of the helix (33). Hydrophobic signal peptide (-27 --1) N-terminal repeat domain (1 -154)
Exons (amino acids) 1
(-27-7)
2
(7-191)
Cysteine-rich (155-312)
3 (191 -298)
2nd repeat domain
~ 2 9 8 - 348) 5 (348 - 396)
(313-428)
C-terminal cc-subunit
(429-731)
Proteolytic cleavage site (732 • 735)
Extracellular p-subunit (736 • 929)
6 (396-468) 7 (468-510) 8 (510-594) 9 (594 - 650) 10 (650-717) -729) 12 (729-821) 13 (821-867) 14 (867-921)
Transmembrane domain(930 • 952)
15 (921 - 955)
Juxtamembrane domain-
JMSJ955 • 978)
(953-1001)
17 (978- 1059) ~(1059 • 1096)
Tyrosine kinase domain(1002-1257)
19 (1096 -1150) 20 (1150 -1193) 21
C-terminal p-subunit
(1193 -1238)
22 (1238• 1355)
(1258-1355)
FIG. 3. Map of structural domains in the human insulin receptor. The various structural domain of the proreceptor are depicted in this figure. For comparison, the corresponding exons of the insulin receptor gene are depicted at the right side of the drawing.
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INSULIN RECEPTOR GENE MUTATIONS
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TABLE 1. Repeat motifs in the a-subunit of the insulin receptor Sequence
Motif Motif 1 Consensus sequence hIR hIR Motif 2
X--X-XX-G
I 1 311
HLYPGEVCPGMDIR VCHLLEGEKTI
14 321
G
hIR hIR Motif 3
1 15 NNLTRLHELENCSVIEGHLQILLMFKTRPEDFR 322 DSVTSAQELRGCTVINGSLIINIRGGNN 349
47
G
hIR hIR Motif 4
I 48 LSFPKLIMITDYLLLFRVY 350 LAAELEANLGLIEEISGYLKIRRSY
67 374
G
hIR hIR
I 68 GLESLKDLFPNLTVIRGSRLFFNYALVIFEMV 99 375 ALVSLSFFRKLRLIRGETLEIGNYSFYALDNQN 407
The repeat motifs in the a-subunit are listed. As proposed by Bajaj et al. (33), the most conserved element of this motif is the central glycine residue that is present in seven of the eight motifs. The only exception is that amino acid residue 58 is an aspartic acid instead of a glycine; however, the homologous amino acid is in fact glycine in the EGF receptor so that the sequence corresponding to amino acid residues 48-67 of the insulin receptor is nevertheless considered to comprise one repeat motif. A less well conserved feature of the repeat motif is the presence of hydrophobic amino acid residues (denoted below as X in the consensus sequence) at positions -2, -5, and -8 relative to the central glycine. In some of the motifs, a hydrophobic amino acid residue (denoted as x in the consensus sequence) is also found at position -3. To emphasize the hydrophobic amino acid residues upstream from the central glycine, they are printed in boldface type.
3. Cysteine-rich domain (amino acids 155-312). There is evidence [at least in the case of the highly homologous insulin-like growth factor I (IGF-I) receptor] that the cysteine-rich domain may play an important role in determining the specificity of ligand binding (36-42). This domain of the receptor contains 26 cysteine residues, most of which are probably involved in intrasubunit disulfide bonds. In addition, there are at least two other highly conserved amino acid residues (His209 and Pro219) that are identically conserved in all known members of the insulin receptor family (insulin receptor, IGF receptor, insulin receptor-related receptor) and the EGF receptor family (c-erb B-l, c-erb B-2, and c-erb B-3) (23, 43). 4. Repeat domain in the middle of the a-subunit (amino acids 313-428). This domain is homologous to the Nterminal repeat domain (amino acids 1-154), and also contains four repeats of the same motif described above (Table 1) (33). 5. C-terminal a-subunit domain (amino acids 429-731). This domain includes the 12 amino acid residues (amino acids 720-731) that are encoded by exon 11, the exon that undergoes variable splicing. Therefore, the length of this domain is variable,' either 291 or 303 amino acid residues, depending on whether or not exon 11 was present in the mRNA from which the receptor molecule
was translated. The functions of this domain are not known, but it has been proposed that it may contain the region of the receptor that allows for the binding interaction between the two a-subunits within the heterotetrameric a^fo form of the receptor (44). In addition, this domain contains a major immunogenic region (amino acids 450-601) that includes a major epitope recognized by several monoclonal anti-receptor antibodies as well as most anti-receptor autoantibodies from patients with type B insulin resistance (41). Furthermore, the presence of the 12 amino acid residues encoded by exon 11 causes a 2-fold reduction in the affinity of the receptor to bind insulin (45-47). 6. Proteolytic cleavage site (amino acids 732-735). There is a sequence consisting of four basic amino acids (ArgLys-Arg-Arg) located at the junction between the a- and /3-subunits that is presumed to be the site for proteolytic cleavage of the proreceptor (4, 5, 48, 49). 7. Extracellular [i-subunit domain (amino acids 736-929). This contains the site of O-linked glycosylation (50). The function of this domain has not been clearly defined. 8. Transmembrane domain (amino acids 930-952). There is a stretch of 23 amino acid residues (rich in hydrophobic amino acids) that is thought to be the single transmembrane domain of the receptor molecule (4, 5).
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9. Juxtamembrane domain (amino acids 953-1001). The juxtamembrane domain is the portion of the /3-subunit that is near the plasma membrane. This contains the signals that allow for receptor endocytosis (51-54). In addition, the juxtamembrane region contains Tyr972, an amino acid residue that has been proposed to play a role in binding some substrates for tyrosine-specific phosphorylation (55). 10. Tyrosine kinase domain (amino acids 1002-1257). This is the region with an amino acid sequence that most closely resembles that of other tyrosine kinases such as the EGF receptor (4, 56). This contains the consensus sequence for ATP binding (Gly-X-Gly-X-X-Gly Lys, amino acids 1003-1030) (56-60) as well as the three tyrosine residues (Tyr1158, Tyr1162, and Tyr1163) the phosphorylation of which appears to activate the ability of the receptor to phosphorylate other protein substrates (10, 61-67). 11. C-terminal fi-subunit domain (amino acids 12581355). This is the region of the intracellular domain where there is maximal divergence between the receptors for insulin and IGF-I (68). Therefore, it has been proposed to contribute to specificity in transmembrane signalling. Furthermore, this domain contains two tyrosine residues (Tyr1328 and Tyr1334) that undergo insulin-stimulated autophosphorylation (61-67). It also contains serine and threonine residues that undergo phosphorylation, including Thr1348 (the site of phosphorylation by protein kinase C) (69, 70) and Ser1305 and Ser1306 (sites of insulin-stimulated serine phosphorylation) (71). B. Posttranslational processing and assembly of oligomeric receptors
Although the mature insulin receptor contains two distinct subunits {a and /?), the proreceptor is a 1355 amino acid molecule that is encoded by a single gene (4, 5). The proreceptor undergoes cotranslational N-linked glycosylation (72-74). There are 15 potential sites for relinked glycosylation on the a-subunit, and four sites in the extracellular portion of the /3-subunit. The proreceptor has a mobility on NaDodSO4-polyacrylamide gel electrophoresis corresponding to Mr 190,000 (73, 75-76). Immediately after it is synthesized, the proreceptor does not have the ability to bind insulin (77). However, while still in the endoplasmic reticulum, the proreceptor folds into a conformation that is competent to bind insulin. It seems likely that the intrasubunit disulfide bonds are formed at this time. Subsequently, the receptor undergoes additional posttranslational processing steps within the endoplasmic reticulum and Golgi apparatus. 1. Assembly of receptor into an oligomeric form. Two 190
kilodalton (kDa) proreceptor molecules dimerize to form
Vol. 13, No. 3
a high molecular weight species linked by disulfide bonds (43, 74, 77, 78). At this point, the N-linked oligosaccharide chains remain in the high mannose form. 2. Proteolytic cleavage into subunits. The proreceptor undergoes proteolytic cleavage at a tetrabasic amino acid sequence (Arg-Lys-Arg-Arg) that separates the a- from the 0-subunit (4, 5, 43, 48, 49, 73-78). This gives rise to a disulfide-linked heterotetrameric a2^2 species. The immature pre-a- and pre-0-subunits have Mr values of 120,000 and 85,000, respectively. 3. Terminal processing of N-linked oligosaccharide. The N-linked oligosaccharide is processed with removal of glucose and mannose residues, and addition of other sugars (e.g. fucose and galactose) and sialic acid (72, 73). These processing steps, which occur in the Golgi apparatus, are associated with a decrease in the electrophoretic mobilities of the receptor subunits; the mature aand 0-subunits have Mr values of 135,000 and 95,000, respectively. 4. O-linked glycosylation. There are one or more oligosaccharides that are attached via O-linkage to the extracellular domain of the /?-subunit (50, 79), most likely in the cluster of threonine and serine residues in the extracellular domain of the /3-subunit (49): Asn Thr 756 Ser 7 5 7 Ser 758 Thr 759 Ser 7 6 0 Val Pro Thr 763 Ser 764 Pro
It is not established precisely at what point O-linked glycosylation occurs during receptor biosynthesis. 5. Acylation. There is evidence for acylation (both myristylation and palmitylation) of both subunits of the receptor. The fatty acids appear to be attached through both amide {a- and 0-subunits) and ester (/3-subunit) linkages (80). C. Mutations that impair intracellular transport and posttranslational processing (class II) Many mutations interfere with posttranslational processing of the insulin receptor (Figs. 1 and 4). In one case, the mutation specifically alters an amino acid sequence required for proteolytic cleavage, thereby directly impairing proteolytic processing (81-84). However, in several other cases, the mechanism is indirect. Presumbably, these mutations impair the normal folding of the receptor molecule (34, 43, 78, 85-88). As a consequence, there is a defect in transport of the receptor through the endoplasmic reticulum and Golgi. The defect in intracellular processing leads to a decrease in the number of receptors on the cell surface. Moreover, the defect in posttranslational processing occurs because most of the receptors do not reach the intracellular compartments where many
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August, 1992
INSULIN RECEPTOR GENE MUTATIONS
Missense Mutations in Insulin Receptor Gene
a Phe-88,89 Cys-rich (155-312)
Cys-435 Cys-468 Cys-524
Exon11 (718-729)
, . Asn
(Impaired transport to cell surface; -> Lys decreased binding affinity)
' ~ Val 2 8 - » Ala h-Gly31->Arg
(Impaired transport to cell surface)
] - His 209 -* Arg I* Leu 2 3 3 -» Pro
(Decreased binding)
(Impaired transport to cell surface)
' G l y 3 6 6 - * Arg 382 (Impaired transport to cell surface) 1 -Phe - > Val (Increased binding affinity; 460 decreased pH sensitivity) I * Asn - » Ser (Decreased pH sensitivity)
,-Arg
' _» e . . (Uncleaved receptor; a e r decreased binding affinity)
571
creases binding affinity, but because the defect is due to a specific conformational change introduced by the substitution of serine at position 735. Another site-directed mutation has been described that impairs proteolytic cleavage and decreases the affinity of insulin binding: that is, total deletion of the Arg-Lys-Arg-Arg sequence (49). Surprisingly, even when these four amino acids were deleted, approximately 20% of the mutant receptor molecules were still cleaved. This suggests that there must be a cryptic site that can be used for proteolytic cleavage. It is not known whether this cryptic site is sometimes used, even in the context of a proreceptor with a normal amino acid sequence. Nevertheless, the uncleaved A732~735-mutant receptor bound insulin with low affinity and was impaired in autophosphorylation and endocytosis (49).
Extracellular
2. Misfolding of receptor due to mutations in the Nterminal half of the a-subunit. At least five mutations Tyr-972 , . Arg" ->Gln ATP-Binding | (Fig. 4) have been identified in the a-subunit of the (1003-1030) '•-Gly -+Val receptor that impair intracellular transport through the fV>Lys1068->Glu Tyr-1158,1162,1163 endoplasmic reticulum and Golgi apparatus: Asn15—»Lys 1134 31 Decreased fe\>a ->Thr (78), Gly ^Arg (34), His20{UArg (43), Leu 233 ^Pro (86, tyrosine Tyr-1328,1334 *Ala - » Glu kinase 87), and Phe385WVal (83). Two other mutations have Met —> lie been identified in the same general location in the asubunit of the receptor, and these may also impair posttranslational processing of the receptor: Val28—*Ala (35), •_ 1200 . Car and Gly366—>Arg (35); however, the transfection studies Trp —* '*' have not yet been reported for these mutant receptors. FIG. 4. Missense mutations in the insulin receptor gene in insulinIn each case, impaired intracellular transport is associresistant patients. Key structural landmarks of the insulin receptor are identified at the left of the drawings (see Fig. 2). The locations of all of ated with a defect in posttranslational processing; howthe reported missense mutations are indicated in the right half of the ever, the completeness of the defect varies among the drawing of the receptor. different mutations. In those cases where it has been studied, the mutations inhibit the assembly of mutant of the enzymes required for posttranslational processing receptors into oligomers (43, 78). In addition, these muare located. tations inhibit terminal processing of the N-linked car1. Impaired proteolytic cleavage of proreceptor due to a bohydrate and proteolytic cleavage of the receptors into mutation in the proteolytic cleavage site. Two sisters with a- and /3-subunits (34, 43, 78, 85). This is demonstrated in experiments with NIH-3T3 cells transfected with type A insulin resistance have been identified who are 735 cDNA encoding either wild type or Arg209-mutant recephomozygous for a mutation substituting serine for Arg , tors (Fig. 5) (43). The transfected cells were pulse-labeled the last amino acid in the tetrabasic amino acid sequence by incubation for 45 min with [2-3H]mannose followed (Arg-Lys-Arg-Arg) located at the junction between the by a variable length chase in the presence of unlabeled a- and |8-subunits (Fig. 4) (81, 82). This mutation has mannose. The cells were solubilized in Triton X-100, been reported to impair proteolytic cleavage and to deand the insulin receptors were immunoprecipitated with crease the affinity of insulin binding to the uncleaved 735 anti-receptor antibody. The immunoprecipitates were Ser -mutant proreceptor (83, 84). Similar, although not first analyzed by NaDodSO4-polyacrylamide gel electroidentical, conclusions have been reached using alaninephoresis in the presence of reductant to study the postscanning mutagenesis (48). Substitution of alanine for 732 735 733 734 translational processing of the receptor. At the beginning either Arg or Arg (but not Lys or Arg ) impaired of the chase period, the major labeled band immunopreproteolytic cleavage of the proreceptor. The uncleaved cipitated by anti-receptor antibody corresponds to the mutant proreceptors were reported to bind insulin with receptor precursor (M 190,000). In cells expressing the r the same affinity as normally processed receptors in this 209 wild type (His ) receptor, the precursor is processed study. Taken at face value, these data suggest that it is rapidly, disappearing with a half-life of Ser) that causes a similar (although milder) defect in pH sensitivity and is also associated with a shortened receptor half-life (23, 168). Thus, although it is possible that some of the clinical differences among different patients may relate to selective insulin resistance in different branches of the insulin response pathway, this does not seem to be the major explanation of the observed clinical variation. 5. Genetic variation at different loci. According to this view, the clinical syndrome may be modulated by genetic factors at other genetic loci (187). For example, defects
585
have been reported in various growth factor receptors in patients with leprechaunism (187-189). It has been proposed that these may play a role in the growth retardation observed in patients with this syndrome. Although it would be possible to investigate genes encoding specific growth factor receptors that have been implicated (e.g., the EGF receptor), this type of study has not been done in these patients. If it were true that the difference between type A insulin resistance and leprechaunism were due to mutations at another genetic locus (e.g. the EGF receptor gene), then one would predict that mutations at the two genetic loci should segregate independently. In a given pedigree, one child might inherit the insulin receptor mutations in the absence of the mutations at the other locus and manifest type A insulin resistance according to this hypothesis. Another child in the same pedigree might inherit mutations in both the insulin receptor and the other locus and have the syndrome of leprechaunism. Thus, this theory would predict that patients with both syndromes should be found in the same family. However, to the best of our knowledge, this type of discordance has not been reported despite the fact that there have been multiple reported kindreds with two or more siblings having the same clinical syndrome, i.e., either leprechaunism (166, 172) or type A insulin resistance (81-85, 190, 191). Nevertheless, there is considerable evidence of subtle clinical variation between individuals within the same family who appear to have the same genotype at the insulin receptor locus (85, 191). Two sisters with type A insulin resistance (patients A-5 and A-8) provide one of the most striking examples of this phenomenon. They are both members of a consanguineous pedigree and are both homozygous for the Val382 mutation in the insulin receptor gene. Both patients are insulin resistant. However, patient A-5 has required treatment with several thousand units of insulin per day while her sister patient A-8 has not required insulin most of the time and has often been treated with dietary management. Furthermore, patient A-5 has such severe hyperandrogenism that she has never had spontaneous menses and did not experience withdrawal bleeding when she was treated with oral preparations of estrogen plus progestin. In contrast, despite some degree of hyperandrogenism, patient A-8 has ovulated spontaneously and has even achieved pregnancy on two occasions. It seems likely that the observed clinical differences are accounted for by polygenic factors that modulate the effect of the mutations at a major genetic locus (i.e. the insulin receptor gene). F. Methods to screen for mutations in the insulin receptor gene Many patients with mutations in the insulin receptor gene—especially those with two mutant alleles—are ex-
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586
TAYLOR ET AL.
tremely resistant to insulin, requiring therapy with as much as several thousand units of insulin per day (6). However, some patients—particularly those who are heterozygous for a single mutant allele—have more modest degrees of insulin resistance, comparable to that which is observed in patients with impaired glucose tolerance or non-insulin-dependent diabetes mellitus (Refs. 27, 28, 30, 31, 57, and 104-109 and unpublished observations of H. Kadowaki and T. Kadowaki and of H. Makino). Moreover, like most patients with non-insulin-dependent diabetes mellitus, some of these patients are obese (28, 104). It is possible that mutations in the insulin receptor gene provide a genetic predisposition that exacerbates the insulin resistance caused by obesity. Clearly, it would be of interest to determine the prevalence of these mutations in the population, and to determine whether these mutations could contribute to the pathogenesis of insulin resistance in a subpopulation of patients with non-insulin-dependent diabetes mellitus. The major obstacle to this type of study has been the amount of work required to obtain a definitive answer. Before considering specific studies of the insulin receptor gene in patients with non-insulin-dependent diabetes mellitus that have been reported, it is worthwhile to review the techniques that are required to detect various types of mutations: 1. Point mutations in the protein-coding sequence of the gene. Point mutations (i.e. either nonsense or missense mutations) can be detected by determining the nucleotide sequence of either genomic DNA or cDNA. Most of the mutations that have been identified to date fall into this category. In several respects, it is preferable to use genomic DNA for this purpose. The most important reason for this recommendation is that mutations causing premature chain termination usually reduce the level of mRNA transcribed from the allele with the mutation. Thus, there is a danger that most of the cDNA would be derived from the allele without the mutation. As a practical matter, most recent studies have utilized the polymerase chain reaction to amplify fragments of genomic DNA corresponding to each of the 22 exons of the insulin receptor gene together with the intron sequences immediately flanking each exon (22, 23, 25, 30, 31, 34, 35,104, 147-150, 186, 192). Accordingly, this strategy would detect mutations in the protein-coding sequence of the gene as well as mutations in the splice donor and acceptor sites in the introns. Furthermore, the same approach will detect insertions or short deletion mutations so long as they are entirely contained within a single exon. In most of the early studies, mutations were detected by determining the nucleotide sequence of either cloned DNA or DNA amplified by polymerase chain reaction (22-26, 31, 34, 57, 63, 85, 104, 106, 109, 184, 185).
Vol. 13, No. 3
However, more recently, several molecular scanning techniques have been used as a prelude to sequencing. These screening techniques allow for localization of variant sequences, which can then be analyzed definitively by sequencing the DNA. The major advantage of these techniques is to greatly reduce the work involved in studying large numbers of patients. The first such technique was heteroduplex mapping (193). However, because heteroduplex mapping has an unacceptably high rate of missing mutations, it has largely been supplanted by newer techniques: analysis of single-stranded conformational polymorphisms1 (SSCP) (147, 149) and denaturing gradient gel electrophoresis2 (DGGE) (Fig. 18) (35). Both of these techniques are estimated to have «90% sensitivity to detect point mutations. These techniques will detect any variation in the nucleotide sequence—both mutations and normal polymorphisms. Thus, in interpreting these data, it is extremely useful to have knowledge of the common polymorphisms in the nucleotide sequence (Table 3). 2. Deletion mutations. At least four deletion mutations 1 Double-stranded DNA assumes a double helical conformation under nondenaturing conditions. Thus, as a first order approximation, the conformation of double-stranded DNA does not depend on the nucleotide sequence. Consequently, the electrophoretic mobility of double-stranded DNA under nondenaturing conditions depends only on the length of the DNA. In contrast, the conformation of singlestranded DNA depends upon the nucleotide sequence. Because the shape of the single-stranded DNA determines its electrophoretic mobility, an alteration in the nucleotide sequence has the potential to alter the electrophoretic mobility of single-stranded DNA. Based on analysis of single-stranded conformational polymorphisms (SSCP), there is ~90% probability of detecting a change in a single nucleotide in a «500 nucleotide sequence if electrophoresis is carried out under four experimental conditions: 1) 4 C; 2) 4 C in the presence of 10% glycerol; 3) 24 C; and 4) 24 C in the presence of 10% glycerol (147, 149). 2 The nucleotide sequence determines the strength of the forces that stabilize the double helical conformation of double-stranded DNA. For example, it takes more energy to melt a G:C base-pair than an A:T base pair. Accordingly, if there is a change in the nucleotide sequence, this can alter the melting point of double-stranded DNA. For example, when genomic DNA is amplified by the polymerase chain reaction, both alleles will be amplified. If there is difference between the sequence of the two alleles, both sequences will be amplified to yield the two homoduplex molecules in which all of the nucleotides are matched perfectly. However, when the duplex DNA is denatured during the course of the polymerase chain reaction, there is a possibility that the sense strand derived from one allele will hybridize to the antisense strand derived from the other allele. This will yield heteroduplex molecules in which there are mismatched base pairs. In some cases, the two different homoduplex molecules will have different melting profiles. However, the heteroduplex molecules will almost always melt at lower temperature than the homoduplex molecules. In denaturing gradient gel electrophoresis (DGGE), duplex DNA is electrophoresed through a gradient of increasing concentrations of denaturants (usually, urea and formamide). As the duplex DNA is denatured, its electrophoretic mobility decreases. Accordingly, the nucleotide sequence determines the stability of the double-stranded conformation. This determines the concentration of denaturants required to melt the double-stranded conformation which, in turn, determines the electrophoretic mobility. It has been shown that the probability of detecting a change in a single base pair in a 500 bp sequence is «90% (35).
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August, 1992
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INSULIN RECEPTOR GENE MUTATIONS
TABLE 3. Sequence polymorphisms in coding region of insulin receptor gene Amino Acid #
Amino Acid
Codon
A. Confirmed polymorphisms that do not alter amino acid sequence -20 234 276 519 523 642 1058
65%
GRADIENT OF DENATURANTS
25%
FIG. 18. Perpendicular DGGE of exon 2 of insulin receptor gene from patient's parents and from leprechaun/Verona-1. The amplified fragment of DNA corresponding to the 5'-half of exon 2 from the father (gel A) and mother (gel B) of a patient with leprechaunism was electrophoresed through a polyacrylamide gel with a 25-65% gradient of denaturants (urea and formamide). The gradient was linear, with the concentration of denaturants increasing from right to left. The direction of electrophoresis was from the top to the bottom of the gel (35). With the mother's amplified genomic DNA, a single melting curve is observed indicating that both alleles of her insulin receptor gene have the same sequence. In contrast, with amplified DNA from her father, multiple melting curves were observed, indicating that more than one species is present in the amplified DNA of the proband leprechaun/Verona-1. Thus, both the patient and her father are heterozygous for a nucleotide substitution in exon 2 encoding the Val28—* Ala mutation.
have been identified in the insulin receptor gene (27-30). These mutations were recognized initially because of the presence of abnormal bands on Southern blots. If the same DNA were analyzed by carrying out polymerase chain reaction with genomic DNA as template, the mutations would not have been detected. Consider, for example, the deletion of exon 14 (28). If one amplified exon 14 from the patient's genomic DNA, then one would get the normal size DNA fragment produced as a result of amplification of the normal allele. Because the oligonucleotide primers would not hybridize to the allele with the deletion, the experiment would not provide any evidence of the existence of the mutation. Accordingly, molecular scanning techniques such as SSCP and DGGE would fail to detect this type of mutation. Some deletions might be detected by determining the sequence of cDNA. However, most deletions will result in a frameshift so that they will introduce a premature chain termination codon. Accordingly, the mutation may selectively reduce the level of the mRNA transcribed from the mutant allele. Thus, analyzing the cDNA is an insensitive strat-
1062
Gly Gly Asp Asp Gin Gin Asp Asp Ala Ala Phe Phe His His Leu Leu
GGG (>90%) GGA (90%) GAT (90%) CTT (90%) ATG (
