Perspectives in Diabetes Human Insulin-Receptor Gene SUSUMU SEINO, MITSUKO SEINO, AND GRAEME
The human insulin-receptor (hINSR) 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 hINSR gene into segments that encode structural and/or functional elements of the hINSR protein. The exonintron organization of the hINSR 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 hINSR 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 hINSR gene in the development of diabetes mellitus. Diabetes 39:129-33, 1990
I
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 INSR (hINSR) is an integral membrane protein present on the surface of all cells. The surface concentration
From the Howard Hughes Medical Institute and the Departments of Medicine, Biochemistry, and Molecular Biology, The University of Chicago, Chicago, Illinois. Address correspondence and reprint requests to Susumu Seino, MD, DMS, Howard Hughes Medical Institute, The University of Chicago, 5841 South Maryland Avenue, Box 391, Chicago, IL 60637. Received for publication 30 May 1989 and accepted in revised form 9 August 1989.
DIABETES, VOL. 39, FEBRUARY 1990
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of receptors varies widely from as few as 40 on circulating erythrocytes to >200,000 on adipocytes and hepatocytes. The mature hINSR is a heterotetramer of two a-subunits of 719 or 731 amino acids* and two 620-amino acid (3-subunits (7,8). The insulin-binding a-subunit and the membranespanning protein tyrosine kinase (3-subunit are generated by proteolytic processing of a common single-chain precursor. Cleavage of the proreceptor occurs during intracellular 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 serine/threonine residues (O linked). Thus, the aand (3-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 Mr of 82,000 or 84,000 and 70,000, respectively. In the absence of the a-subunit, the endogenous tyrosine kinase of the (3-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 hINSR that are associated with extreme insulin resistance and glucose intolerance has provided new insight into the possible role of the hINSR in the development of non-insulin-dependent diabetes mellitus (NIDDM) (10-16). It is appropriate to consider the contribution of genetic variation in the hINSR gene in relation to the development of the common forms of NIDDM. Is there a subpopulation of diabetic patients who have mutations in the hINSR 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 residues of the hINSR is for a-subunit and precursor of 731 and 1355 amino 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.
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and contribute to the cooperative site-site binding interactions of the hINSR (23). Exon 11 is the smallest exon, only 36 bp in size, and alternative splicing of this exon results in the synthesis of hINSR proteins having a-subunits with different COOH-temninal 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 NH2-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 NH2-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 hINSR, 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 kinase on 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/structural domains of the hINSR protein and that the hINSR gene, like the LDL-receptor gene (25,26), is a mosaic constructed from exons recruited from other sources.
EXON-INTRON ORGANIZATION OF hINSR GENE
The hINSR gene is located on the distal short arm of human chromosome 19 in the region of bands p13.3-> 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 - 1 0 - 1 5 x 106 base pairs (bp) from the hINSR gene (18). Most of the hINSR gene has been isolated as a series of overlapping DNA fragments in the bacteriophage-\ (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 bpof thehlNSR, 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 hINSR mRNA), whereas the 21 introns that separate the exons vary in size from - 5 0 0 to >25,000 bp (19). All of the introns interrupt protein-coding regions of the gene. The actual size of the hINSR 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 hINSR 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 1-11, 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 hINSR gene has also been characterized, and its features are reviewed in Seino et al. (19). The exon organization of the hINSR 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 hINSR
ALTERNATIVE SPLICING GENERATES AMINO ACID-SEQUENCE ISOFORMS OF hINSR
The a- and p-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 >10 kb
Mutations
4
Exon Protein domains
OO
D O
5 6 789
ion
• ••••• Signal peptide
Cysteine rich
Insulin binding
II
O
O
•• •••••••••
12 13
14 I5I6 I7I8I92O2I 22
Tyrosine a-subunit Alternatively Proreceptor Kinase association spliced exon processing site Transmembrane Receptor recycling (?)
a-and /3-subunit association
EGF receptor homology a-subunit _J L 10
20
30
40
50
J 60
70
80
90
100
/3-subunit I 110
L 120
130 kb
FIG. 1. Map of human insulin-receptor (hINSR) gene. Relative locations and sizes of exons ( • ) and introns are indicated. Oblique lines, regions of introns 2, 3, 9, and 11 that have not been cloned. Location of mutations in hINSR gene are indicated: 0 , missense; Q, nonsense; Pro Phe 382 -> Val Lys 460 -» Glu Gin 672 -» AM
3 5 6 10
Dutch Venezuelan American
Delayed transport to cell surface Delayed transport to cell surface Qualitative abnormalities in insulin binding Truncated a-subunit
12 11 10
Arg. 735 -> Ser
12
Japanese
Impaired proreceptor processing
13
Gly 1 0 0 8 ^ Val Trp 1200 -» Ser
17 21
Japanese American
15 14
>10-kb deletion
17-22
Japanese
Decreased tyrosine kinase activity Decreased receptor affinity and autophosphorylation Decreased tyrosine kinase activity
Subject
Ref.
a-Subunit 1
2 3 Proreceptor processing site 4 (3-Subunit 5 6 7
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.
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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 INSRs 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 hINSR 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 hINSR gene that are associated with insulin resistance, population-association studies suggest that there is also genetic variation in the region of the hINSR 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 hINSR haplotypes might be due to genetic variation that increases expression of the hINSR 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 hINSR gene that alter the sequence of the gene and mRNA but not the protein have been described (8,10,19f). Several restriction-fragment-length polymorphisms have also been reported at the hINSR 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 NIDDM in a small but measurable subpopulation of patients. The isolation and characterization of the hINSR gene and recent technical developments provide a strategy for examining the contribution of this gene to the development of NIDDM by amplifying individual exons of the hINSR gene with the polymerase chain reaction (PCR) and then sequencing the amplified DNA. Where should we look for hINSR mutations? Because mutations in the tyrosine kinase domain of the hINSR are associated with severe insulin resistance in the heterozygous state, whereas heterozygosity for mutations in the a-subunit appears to be associated with lesssevere insulin resistance, the NIDDM 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 hINSR, 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 hINSR gene in 50-100 well-characterized NIDDM 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 NIDDM are unclear. Although impaired p-cell function and insulin resistance are both features of NIDDM, controversy exists as to which defect is primary. Studies of patients expressing abnormal insulin (43-45) or INSR proteins (10-16) have indicated that mutations in either of these genes can be associated with NIDDM. In fact, insulin gene mutations represent a paradigm for p-cell defects that can cause diabetes. Similarly, INSR mutations are an example of a defect in a responsive cell that can contribute to the development of this disorder. In contrast to insulin gene mutations, which are extremely rare, mutations in the hINSR gene may be more common. Moreover, because heterozygous individuals who express both normal and mutant receptors may exhibit mild insulin resistance, e.g., parents of subjects 1 and 3 (Table 1; 10,12), it seems reasonable to consider that codominant expression of normal and abnormal hINSR proteins may contribute to the development of NIDDM. Although linkage studies of 20 American Black families in which at least two siblings had
•[Comparison of hINSR cDNA and genomic sequences (19) suggested that residue 421 of the a-subunit could be either He or Thr and that residue 465 could be Gin or Lys; i.e., these sites were polymorphic. We have reexamined the sequence of the kidney hINSR cDNA described in ref. 22 by hybridization with allele-specific oligonucleotides, and it appears to encode lie 421 and Gin 465 rather than Thr 421 and Lys 465 as suggested in Table 2 of ref. 12. We have also examined the hINSR alleles of 3 unrelated individuals by sequence or hybridization with allele-specific oligonucleotides, and all encode proteins having lie 421 and Gin 465. The polymorphism of amino acids 421 and 465 still requires confirmation.
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REFERENCES 1. Kahn CR: Current concepts of the molecular mechanism of insulin action. Annu Rev Med 36:429-51, 1985 2. Czech MP: The nature and regulation of the insulin receptor. Annu Rev Physiol 47:357-81, 1985 3. Rosen OM: After insulin binds. Science 237:1452-58, 1987 4. Goldfine ID: The insulin receptor: molecular biology and transmembrane signalling. Endocr Rev 8:235-55, 1987 5. Czech MP, Klarlund JK, Yagaloff KA, Bradford AP, Lewis RE: Insulin receptor signalling: activation of multiple serine kinases. J Biol Chem 263:11017-20, 1988 6. Kahn CR, White MF: The insulin receptor and the molecular mechanism of insulin action. J Clin Invest 82:1151-56, 1988 7. Ullrich A, Bell JR, Chen EY, Herrera R, Petruzzelli LM, Dull TJ, Gray A, Coussens L, Liao Y-C, Tsubokawa M, Mason A, Seeburg PH, Grunfeld C, Rosen OM, Ramachandran J: Human insulin receptor and its relationship to the tyrosine kinase family of oncogenes. Nature (Lond) 313:75661, 1985 8. Ebina Y, Ellis L, Jarnagin K, Edery M, Graf L, Clauser E, Ou J-H, Masiarz F, Kan YW, Goldfine ID, Roth RA, Rutter WJ: The human insulin receptor cDNA: the structural basis for hormone activated transmembrane signalling. Cell 40:747-58, 1985 9. Ellis L, Morgan DO, Clauser E, RothRA, Rutter WJ: A membrane-anchored cytoplasmic domain of the human insulin receptor mediates a constitutively elevated insulin-independent uptake of 2-deoxyglucose. Mol Endocrinol 1:15-24, 1987 10. Kadowaki T, Bevins CL, Cama A, Ojamaa K, Marcus-Samuels B, Kadowaki H, Beitz L, McKeon C, Taylor SI: Two mutant alleles of the insulin receptor gene in a patient with extreme insulin resistance. Science 240:787-90, 1988 11. Accili D, Frapier C, Mosthaf L, McKeon C, Elbein S, Permutt MA, Ramos E, Lander E, Ullrich A, Taylor SI: A mutation in the insulin receptor gene that impairs transport of the receptor to the plasma membrane and causes insulin resistant diabetes. EMBO J 8:2509-17, 1989 12. Klinkhamer MP, Groen NA, van der Zon GCM, Lindhout D, Sandkuyl LA, Krans HMJ, Moller W, Maassen JA: A leucine-to-proline mutation in the insulin receptor in a family with insulin resistance. EMBO J 8:2503-507, 1989
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S. SEINO, M. SEINO, AND G.I. BELL 13. Yoshimasa Y, Seino S, Whittaker J, Kakehi A, Kuzuya H, Imura H, Bell Gl, Steiner DF: Insulin resistant diabetes due to a point mutation that prevents insulin receptor processing. Science 240:784-87, 1988 14. Moller DE, Flier JS: Detection of an alteration in the insulin receptor gene in a patient with insulin resistance, acanthosis nigricans, and the polycystic ovary syndrome (type A insulin resistance). N Engl J Med 319:1526-29, 1988 15. Odawara M, Kadowaki T, Yamamoto R, Shibasaki Y, Tobe K, Accili D, Bevins C, Mikami Y, Matsuura N, Akanuma Y, Takaku F, Taylor SI, Kasuga M: Human diabetes associated with a mutation in the tyrosine kinase domain of the insulin receptor. Science 245:66-68, 1989 16. Taira M, Taira M, Hashimoto N, Shimada F, Suzuki Y, Kanatsuka A, Nakamura F, Ebina Y, Tatibana M, Makino H, Yoshida S: Human diabetes associated with a deletion of the tyrosine kinase domain of the insulin receptor. Science 245:63-66, 1989 17. Yang-Feng TL, Franke U, Ullrich A: Gene for human insulin receptor: localization to a site on chromosome 19 involved in pre-B-cell leukemia. Science 228:728-31, 1985 18. Shaw DJ, Meredith AL, Brook JD, Sarfarazi M, Harley HG, Huson SM, Bell Gl, Harper PS: Linkage relationships of the insulin receptor gene with the complement component 3, LDL receptor, apolipoprotein C2 and myotonic dystrophy loci on chromosome 19. Hum Genet 74:267-69, 1986 19. Seino S, Seino M, Nishi S, Bell Gl: Structure of the human insulin receptor gene and characterization of its promoter. Proc Natl Acad Sci USA 86:114-18, 1989 20. Muller-Wieland D, Taub R, Tewari DS, Kriauciunas KM, Sethu S, Reddy K, Kahn CR: Insulin-receptor gene and its expression in patients with insulin resistance. Diabetes 38:31-38, 1989 21. DeMeyts P, Gu J-L, Shymko RM, Bell G, Whittaker J: Identification of an insulin receptor binding domain complementary to the cooperative site of insulin (Abstract). Diabetologia 31:484A, 1988 22. Yip CC, Hsu H, Patel RG, Hawley DM, Maddux BA, Goldine ID: Localization of the insulin binding site to cysteine rich domain of the a subunit. Biochem Biophys Res Commun 157:321-29, 1988 23. Kadowaki H, Kadowaki T, Marcus-Samuels B, Cama A, Rovira A, Taylor SI: Site-directed mutagenesis of position 460 in the alpha-subunit of the insulin receptor alters cooperative site-site interactions in insulin binding (Abstract). Diabetes 38 (Suppl. 2):2A, 1989 24. Seino S, Bell Gl: Alternative splicing of human insulin receptor messenger RNA. Biochem Biophys Res Commun 159:312-16, 1989 25. Brown MS, Goldstein JL: A receptor-mediated pathway for cholesterol homeostasis. Science 232:34-47, 1986 26. Russell DW, Esser V, Hobbs HH: Molecular basis of familial hypercholesterolemia. Arteriosclerosis 9 (Suppl. 1): 18-13, 1989 27. Ebina Y, Edery M, Ellis L, Standring D, Beaudoin J, Roth RA, Rutter WJ: Expression of a functional human insulin receptor from a cloned cDNA in Chinese hamster ovary cells. Proc Natl Acad Sci USA 82:8014-18, 1985 28. Chou CK, Dull TJ, Russell DS, Gherzi R, Lebwohl D, Ullrich A, Rosen OM: Human insulin receptors mutated at the ATP-binding site lack protein tyrosine kinase activity and fail to mediate postreceptor effects of insulin. J Biol Chem 262:1842-47, 1987
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29. Whittaker J, Okamoto AK, Thys R, Bell Gl, Steiner DF, Hofmann CA: Highlevel expression of human insulin receptor cDNA in mouse NIH3T3 cells. Proc Natl Acad Sci USA 84:5239-41, 1987 30. McClain D, Mosthaf L, Ullrich A: Properties of the two naturally occurring alternative forms of the insulin receptor (Abstract). Diabetes 38 (Suppl. 2):1A, 1989 31. Taylor SI: Receptor defects in patients with extreme insulin resistance. Diabetes Metab Rev 1:171-202, 1985 32. McClain DA, Henry RR, Ullrich A, Olefsky JM: Restriction-fragment-length polymorphism in insulin-receptor gene and insulin resistance in NIDDM. Diabetes 37:1071-75, 1988 33. Xiang K-S, Cox NJ, Sanz N, Huang P, Karam JH, Bell Gl: Insulin-receptor and apolipoprotein genes contribute to development of NIDDM in Chinese Americans. Diabetes 38:17-23, 1989 34. Shaw DJ, Bell Gl: Rsa1 polymorphism at the insulin receptor locus (INSR) on chromosome 19. Nucleic Acids Res 13:8661, 1985 35. Elbein SC, Corsetti L, Ullrich A, Permutt MA: Multiple restriction fragment length polymorphisms at the insulin receptor locus: a highly informative marker for linkage analysis. Proc Natl Acad Sci USA 83:5223-27, 1986 36. Sanna MA, Bell Gl, Cao A, Pirastu M: Three RFLPs for the insulin receptor gene INSR: EcoRI, Pst I, Hin6\\\. Nucleic Acids Res 14:6776, 1986 37. TakedaJ, Seino Y, FukumotoH, KohG, Imura H, Bell GhPvull polymorphic sites in the human insulin receptor gene INSR. Nucleic Acids Res 14:6777, 1986 38. Patel P, O'Rahilly S, Ullrich A, Turner RC, Wainscoat JS: A new Sst 1 RFLP associated with human insulin receptor locus. Nucleic Acids Res 16:5700, 1988 39. Cox NJ, Spielman RS, Kahn CR, Muller-Wieland D, Kriauciunas KM, Taub R: Four RFLPs of the human insulin receptor gene: Pst\, Kpn\, Rsa\ (2 RFLPs). Nucleic Acids Res 16:8204, 1988 40. Accili D, Elbein S, McKeon C, Taylor SI: A new fcoRI polymorphism for the insulin receptor gene. Nucleic Acids Res 17:821, 1989 41. Sten-Linder M, Stern I, Bell Gl, Luthman H: Human insulin receptor RFLPs detected by H/ndlll and Dra I. Nucleic Acids Res 17:1277, 1989 42. Elbein SC: Molecular and clinical characterization of an insertional polymorphism of the insulin-receptor gene. Diabetes 38:737-43, 1989 43. Haneda M, Polonsky KS, Bergenstal RM, Jaspan JB, Shoelson SE, Blix PM, Chan SJ, Kwok SCM, Wishner WB, Zeidler A, Olefsky JM, Freidenberg G, Tager HS, Steiner DF, Rubenstein AH: Familial hyperinsulinemia due to a structurally abnormal insulin: definition of an emerging new clinical syndrome. N Engl J Med 310:1288-94, 1984 44. Nanjo K, Sanke T, Miyano M, Okai K, Sowa R, Kondo M, Nishimura S, Iwo K, Miyamura K, Given BD, Chan SJ, Tager HS, Steiner DF, Rubenstein AH: Diabetes due to secretion of a structurally abnormal insulin (insulin Wakayama): clinical and functional characteristics of [LeuA3]insulin. J Clin Invest 77:514-19, 1986 45. Nanjo K, Miyano M, Kondo M, Sanke T, Nishimura S, Miyamura K, Inouye K, Given BD, Chan SJ, Polonsky KS, Tager HS, Steiner DF, Rubenstein AH: Insulin Wakayama: familial mutant insulin syndrome in Japan. Diabetologia 30:87-92, 1987 46. Cox NJ, Epstein PA, Spielman RS: Linkage studies of NIDDM and the insulin and insulin-receptor genes. Diabetes 38:653-58, 1989
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The human insulin-receptor (hINSR) 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 hINSR gene into segments that encode structural and/or functional elements of the hINSR protein. The exonintron organization of the hINSR 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 hINSR 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 hINSR gene in the development of diabetes mellitus. Diabetes 39:129-33, 1990
I
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 INSR (hINSR) is an integral membrane protein present on the surface of all cells. The surface concentration
From the Howard Hughes Medical Institute and the Departments of Medicine, Biochemistry, and Molecular Biology, The University of Chicago, Chicago, Illinois. Address correspondence and reprint requests to Susumu Seino, MD, DMS, Howard Hughes Medical Institute, The University of Chicago, 5841 South Maryland Avenue, Box 391, Chicago, IL 60637. Received for publication 30 May 1989 and accepted in revised form 9 August 1989.
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of receptors varies widely from as few as 40 on circulating erythrocytes to >200,000 on adipocytes and hepatocytes. The mature hINSR is a heterotetramer of two a-subunits of 719 or 731 amino acids* and two 620-amino acid (3-subunits (7,8). The insulin-binding a-subunit and the membranespanning protein tyrosine kinase (3-subunit are generated by proteolytic processing of a common single-chain precursor. Cleavage of the proreceptor occurs during intracellular 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 serine/threonine residues (O linked). Thus, the aand (3-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 Mr of 82,000 or 84,000 and 70,000, respectively. In the absence of the a-subunit, the endogenous tyrosine kinase of the (3-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 hINSR that are associated with extreme insulin resistance and glucose intolerance has provided new insight into the possible role of the hINSR in the development of non-insulin-dependent diabetes mellitus (NIDDM) (10-16). It is appropriate to consider the contribution of genetic variation in the hINSR gene in relation to the development of the common forms of NIDDM. Is there a subpopulation of diabetic patients who have mutations in the hINSR 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 residues of the hINSR is for a-subunit and precursor of 731 and 1355 amino 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.
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and contribute to the cooperative site-site binding interactions of the hINSR (23). Exon 11 is the smallest exon, only 36 bp in size, and alternative splicing of this exon results in the synthesis of hINSR proteins having a-subunits with different COOH-temninal 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 NH2-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 NH2-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 hINSR, 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 kinase on 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/structural domains of the hINSR protein and that the hINSR gene, like the LDL-receptor gene (25,26), is a mosaic constructed from exons recruited from other sources.
EXON-INTRON ORGANIZATION OF hINSR GENE
The hINSR gene is located on the distal short arm of human chromosome 19 in the region of bands p13.3-> 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 - 1 0 - 1 5 x 106 base pairs (bp) from the hINSR gene (18). Most of the hINSR gene has been isolated as a series of overlapping DNA fragments in the bacteriophage-\ (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 bpof thehlNSR, 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 hINSR mRNA), whereas the 21 introns that separate the exons vary in size from - 5 0 0 to >25,000 bp (19). All of the introns interrupt protein-coding regions of the gene. The actual size of the hINSR 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 hINSR 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 1-11, 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 hINSR gene has also been characterized, and its features are reviewed in Seino et al. (19). The exon organization of the hINSR 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 hINSR
ALTERNATIVE SPLICING GENERATES AMINO ACID-SEQUENCE ISOFORMS OF hINSR
The a- and p-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 >10 kb
Mutations
4
Exon Protein domains
OO
D O
5 6 789
ion
• ••••• Signal peptide
Cysteine rich
Insulin binding
II
O
O
•• •••••••••
12 13
14 I5I6 I7I8I92O2I 22
Tyrosine a-subunit Alternatively Proreceptor Kinase association spliced exon processing site Transmembrane Receptor recycling (?)
a-and /3-subunit association
EGF receptor homology a-subunit _J L 10
20
30
40
50
J 60
70
80
90
100
/3-subunit I 110
L 120
130 kb
FIG. 1. Map of human insulin-receptor (hINSR) gene. Relative locations and sizes of exons ( • ) and introns are indicated. Oblique lines, regions of introns 2, 3, 9, and 11 that have not been cloned. Location of mutations in hINSR gene are indicated: 0 , missense; Q, nonsense; Pro Phe 382 -> Val Lys 460 -» Glu Gin 672 -» AM
3 5 6 10
Dutch Venezuelan American
Delayed transport to cell surface Delayed transport to cell surface Qualitative abnormalities in insulin binding Truncated a-subunit
12 11 10
Arg. 735 -> Ser
12
Japanese
Impaired proreceptor processing
13
Gly 1 0 0 8 ^ Val Trp 1200 -» Ser
17 21
Japanese American
15 14
>10-kb deletion
17-22
Japanese
Decreased tyrosine kinase activity Decreased receptor affinity and autophosphorylation Decreased tyrosine kinase activity
Subject
Ref.
a-Subunit 1
2 3 Proreceptor processing site 4 (3-Subunit 5 6 7
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, VOL. 39, FEBRUARY 1990
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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 INSRs 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 hINSR 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 hINSR gene that are associated with insulin resistance, population-association studies suggest that there is also genetic variation in the region of the hINSR 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 hINSR haplotypes might be due to genetic variation that increases expression of the hINSR 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 hINSR gene that alter the sequence of the gene and mRNA but not the protein have been described (8,10,19f). Several restriction-fragment-length polymorphisms have also been reported at the hINSR 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 NIDDM in a small but measurable subpopulation of patients. The isolation and characterization of the hINSR gene and recent technical developments provide a strategy for examining the contribution of this gene to the development of NIDDM by amplifying individual exons of the hINSR gene with the polymerase chain reaction (PCR) and then sequencing the amplified DNA. Where should we look for hINSR mutations? Because mutations in the tyrosine kinase domain of the hINSR are associated with severe insulin resistance in the heterozygous state, whereas heterozygosity for mutations in the a-subunit appears to be associated with lesssevere insulin resistance, the NIDDM 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 hINSR, 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 hINSR gene in 50-100 well-characterized NIDDM 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 NIDDM are unclear. Although impaired p-cell function and insulin resistance are both features of NIDDM, controversy exists as to which defect is primary. Studies of patients expressing abnormal insulin (43-45) or INSR proteins (10-16) have indicated that mutations in either of these genes can be associated with NIDDM. In fact, insulin gene mutations represent a paradigm for p-cell defects that can cause diabetes. Similarly, INSR mutations are an example of a defect in a responsive cell that can contribute to the development of this disorder. In contrast to insulin gene mutations, which are extremely rare, mutations in the hINSR gene may be more common. Moreover, because heterozygous individuals who express both normal and mutant receptors may exhibit mild insulin resistance, e.g., parents of subjects 1 and 3 (Table 1; 10,12), it seems reasonable to consider that codominant expression of normal and abnormal hINSR proteins may contribute to the development of NIDDM. Although linkage studies of 20 American Black families in which at least two siblings had
•[Comparison of hINSR cDNA and genomic sequences (19) suggested that residue 421 of the a-subunit could be either He or Thr and that residue 465 could be Gin or Lys; i.e., these sites were polymorphic. We have reexamined the sequence of the kidney hINSR cDNA described in ref. 22 by hybridization with allele-specific oligonucleotides, and it appears to encode lie 421 and Gin 465 rather than Thr 421 and Lys 465 as suggested in Table 2 of ref. 12. We have also examined the hINSR alleles of 3 unrelated individuals by sequence or hybridization with allele-specific oligonucleotides, and all encode proteins having lie 421 and Gin 465. The polymorphism of amino acids 421 and 465 still requires confirmation.
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