Growth Factor Receptor Regulation Insulin Receptor and Epidermal Patricia

A. Goodman,

Paolo Sbraccia,

in the Minn-1 Growth Factor

Antonio

Brunetti,

Leprechaun: Defects in Both Receptor Gene Expression

Kwok-Ying

Wong, Jacqueline D. Carter,

Stephen M. Rosenthal, and Ira D. Goldfine Leprechaunism is a disorder characterized by intrauterine growth retardation, distinctive dysmorphology, and extreme insulin resistance due to structural abnormalities of the insulin receptor (IR). In addition to the IR, it has been suggested that abnormalities of the other growth factor receptors may occur in this syndrome. Using fibroblasts from the Minn-1 leprechaun, we have now investigated the expression of three different growth factor receptor genes: the IR, the insulin-like growth factor-l receptor (IGF-IR), and the epidermal growth factor receptor (EGFR). In agreement with previous studies, we found decreased insulin binding to fibroblasts from the Minn-1 lephrechaun. In these cells, the IR transcription rate was not decreased, and sequence analysis of the IR promoter region of the patient showed no abnormalities. Both single-stranded conformational polymorphism analysis (SSCP) and DNA sequencing confirmed a previously reported nonsense mutation in one of the patient’s two IR alleles at exon 14. mRNA levels for the IR were markedly decreased, suggesting that IR mRNA turnover was enhanced. We then studied the expression of the closely related IGF-IR Ligand binding, mRNA content, and transcription rate were all normal. In contrast to the IGF-IR, when the EGFR was studied, ligand binding and mRNA content were markedly decreased. These studies therefore raise the possibility that the phenotypic expression of leprechaunism results from defects in the expression of both the IR and the EGFR. Copyright 0 1992 by W. B. Saunders Company

L

EPRECHAUNISM is a disorder characterized by unusual facies, intrauterine growth retardation, and extreme insulin resistance.‘.” Other clinical characteristics of leprechaunism include hyperkeratosis, precocious tooth eruption, and precocious mammary development.*,3 Leprechaunism is part of a wider spectrum of insulin-resistant disorders that are associated with defects in insulin receptor (IR) structure and function.4-‘0 However, not all individuals with IR defects have the morphological features of leprechaunism.” The possibility therefore arises that other receptor defects may be present in leprechaunism. Growth factor receptors such as the insulin-like growth factor-I receptor (IGF-IR) have been measured in patients with leprechaunism, but there has been no consensus regarding their expression in this syndrome.7-9 This result is not surprising, since the leprechaun syndrome is most likely heterogeneousl’ and includes a variety of disorders with defects affecting growth factor expression.‘3,‘4 Previous studies of fibroblasts from one patient with leprechaunism, the Minn-1, have established that there is decreased insulin binding as a consequence of decreased IR mRNA expression. ‘s” This decrease in IR mRNA is the result of a nonsense mutation in one allele of the IR gene.‘” In the present study, using cells from this individual, we have concomitantly investigated the gene expression of IR and two major growth factor receptors: the IGF-IR and the

From the Division of Diabetes and Endocrine Research, Mount Zion Medical Center of the University of California, San Francisco, CA; and the Depatiments of Medicine and Physiology and the Department of Pediatrics, Universityof California, San Francisco, CA. Supported by a Feasibility Grant from the American Diabetes Association (P.A.G.), and a grant from the “Dottorato di Ricerca,” Universityof Rome “La Sapienza, “Rome, Italy (P.S.). Address reprint requests to Patricia A. Goodman, PhD, Division of Endocrinology and Diabetes, Becker B-131, Cedars Sinai Hospital. Los Angeles, CA 90048. Copyright 0 I992 by W.B. Saunders Company 0026-0495192/4105-OOIr1$03.OOlO 504

epidermal growth factor receptor (EGFR). In Minn-1 fibroblasts, we now find defects in the expression of both the IR and the EGFR, but not in the expression of the IGF-IR. Decreased EGF binding to cells from this patient has also recently been reported.” These studies therefore raise the possibility that multiple growth factor receptor gene expression defects contribute to the phenotypic expression of leprechaunism. MATERIALS

AND METHODS

The following were purchased from New England NuclearDuPont, Chicago, IL: A14-‘251-insulin(2,200 Ci/mol), [a-“P]dCTP (3,000 Ciimmol), [u-‘*P]UTP (3,000 Ci/mmol), and [“S]dCTP (500 Ci/mmol). ‘*‘I-EGF (2,000 Ciimmol) and rZSI-IGF-I (2,000 Cii mmol) were obtained from Amersham (Arlington Heights, IL). Porcine insulin was obtained from Elanco Products (Indianapolis, IN), and IGF-I was a gift from Ciba-Geigy (Summit, NJ). EGFR’” and IGF-IR2’ probes were obtained from American Type Culture Collection (ATCC, Rockville, MD). The IR-*’ and IGF-IRpromoter region probes were a gift of Dr Paul Mamula. Cell Culture

Minn-1 fibroblasts (GM 5241) were obtained from NIGMS (National Institute of General Medical Sciences, Camden, NJ) Human Genetic Cell Repository. Age- and sex-matched control cells (GM 1797, GM 5241) were obtained from NIGMS Human Genetic Mutant Cell Repository. Additional control fibroblasts, HS-27 and GM 2036, were obtained from the University of California, San Francisco, Tissue Culture Facility and from NIGMS Human Genetic Mutant Cell Repository, respectively. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) and passaged once a week at a one in four dilution. Cells were used before passage 18. Radioimmunoassay

and Binding

Radioimmunoassay was performed on detergent-soluble extracts of intact cells. Assays were performed at multiple dilution on several different occasions.” All binding assays were performed in triplicate on several different occasions, using several control-cell lines. EGF binding was performed on plates at 4°C with the method of Reddy and Kahn.19 The IGF-I and insulin-binding Metabolism, Vol41, No 5 (May), 1992: pp 504-509

RECEPTOR REGULATION

505

IN MINN-1 LEPRECHAUN

studies were performed on cells in suspension. EDTA was used to remove cells for insulin binding, and a mild trypsinization (1 minute; 0.1% trypsin, 0.03% EDTA) was used for IGF-I binding to reduce interference with IGF-I-binding proteinsZ4 The cells were washed with phosphate-buffered saline (PBS) and then incubated for 6 hours at 4°C in DMEM (bicarbonate-free) supplemented with 15 mmol/L HEPES (pH 7.4) and 10% fetal bovine serum, containing radiolabeled ligand and varying concentrations of unlabeled ligand. After incubation, the cells were washed with TRISbutl’ered saline (pH 7.8) at 4°C and the cells were collected and Iysed with 0.03% sodium dodecyl sulfate (SDS). Proteins were determined by the method of Lowry et al.” Northern and Transcriptional Analysis Total RNA was prepared by a new proteinase K-sodium dodecyl sulfate (PK-SDS) method.lb Poly(A)+ RNAwas isolated by oligo-dt cellulose chromatography. This poly(A)’ RNA was immobilized to nitrocellulose using a slot blot apparatus. RNA was normalized for poly(A)’ content by hybridization with kinase-labeled oligo-dt (Collaborative Research, Bedford, MA).” Poly(A)’ mRNA, 10 to 30 kg, was denatured and subjected to electrophoresis on formaldehyde gels. with subsequent capillary transfer to nitrocellulose. Probes were labeled to a high specific activity (10’ cpmipg) with [“P]CTP (3,000 Ci/mmol) by random priming.” Nuclear run-on assays were perlonned as previously described by Brunetti and Goldfine?

4 3 1 2 1 1

01 0

Po&nerase Chain Reaction Amplification of Exon 14 Oligonucleotide primers for analysis of exon 14 were synthesized based on published sequences.‘“,3’ Amplification was performed in a reaction volume of 0.1 mL, containing 1 wg genomic DNA, 100 pmol of each primer, 10 IJ.L 10x buffer [200 mmol/L TRIS, 10 mmol/L MgCI, 250 mmol/L KCL, 0.5% Tween 201, 4 FL dNTP, and 2.5 U Taq polymerase. DNA was denatured at 96°C for 20 seconds, primers were annealed at 58°C for 30 seconds, and the extension reaction was performed at 72°C for 1 minute and 30 seconds. This was repeated for 30 cycles, with a 2-second extension of the last cycle each round. A. similar protocol was followed for asymmetric polymerase than reaction (PCR), except only one of the two primers was used in the second PCR.” The substrate for the second PCR was symmetrically amplified genomic DNA. The asymmetric product was purified by filtration by Amicon(Danvers, MA) microfiltration before sequencing. Single-Stranded Conformational Polymorphism (SSCP) Anulysis A with then and

standard PCR reaction was performed as described above, the addition of 1 I_LL[‘*P]UTP to the reaction. Samples were diluted either I/?0or 1/4t1 in 0.1% SDS and 10 mmol/L EDTA, mixed 2 pL:2 PL with sequencing 95% formamide stop

-10

-9

]N&N

10-a

I

lo-’

[Ml

Fig 1. Decreased insulin binding in Minn-1 leprechaun fibroblasts. Specific “‘l-insulin binding to Minn-1 and control fibroblasts is shown. Representative of three individual experiments are shown with control cell line 1497. Similar results were seen with control cell line HS27.

solution (USB). Samples were heated to 90°C for 3 minutes and loaded on a 4.5% 40-cm nondenaturing Tris borate EDTA (TBE) gel, and electrophoresis was performed at 4°C.“”

Cloning and Sequencing Minn-1 genomic DNA was prepared by the method of Payne et al.‘” The DNA was digested to completion with EcoRI and cloned into EMBL 4 (Promega, Madison, WI). Screening of 7 x 105clones with a human IR-promoter clone yielded three positive clones, with each one containing a 13-kb EcoRI fragment. A 9-kb EcoRIXBAI fragment from all three clones was subcloned into pUC 18. Two fragments containing the Minn-1 promoter region (BglII-SStI) and the first exon and first intron (SSfI-&I) were cloned into pBluescript (Stratagene, La Jolla, CA) and/or ml3 for sequence analysis.” Sequencing was performed with a Sequenase kit (USB, Cleveland, OH).

r

10

RESULTS IR Analysis IR content of the patient’s fibroblasts was measured by two methods. IR radioimmunoassay’ demonstrated a marked reduction in receptor content in this patient (1.3 2 0.6 ng IR/lO” cells; controls, 10.5 -t 2.9 ng IR/lOh cells [n = lo]). Ligand-binding studies in fibroblasts also demonstrated decreased IR content (Fig 1). There was over a fivefold decrease in insulin binding to these cells when compared with age- and sex-matched controls. Northern analysis showed a marked decrease in IR mRNA content in these cells (Fig 2). The extremely low abundance of IR mRNA prevented measurement of mRNA

IR Kb 11 .oa *5C Fig 2. Northern analysis of insulin receptor mRNA in Minn-1 (M) and control fibroblasts (Cj. Poly(A)’ mRNA, 30 pg. from either control or Minn-1 fibroblasts was subjected to electrophoresis on formaldehyde gels, transferred to nitrocellulose, and hybridized with the IR probe.

GOODMAN

IR

IGF-1

R

EGFR

ET AL

/Y-ACTIN

C

Fig 3. Transcriptional analysis of IR, EGFR, IGF-IR, and (3-actin in Minn-1 and control fibroblasts. Plasmids containing the IR promoter, the IGF-I promoter, the 5’ end of the EGFR cDNA,’ and 5-actin were transferred to nitrocellulose with a slot blot apparatus (Schleicher and Schull) and hybridized with radiolabeled RNAfrom in vitro transcription reactions using either Minn-1 fibroblast nuclei (M) or control fibroblast nuclei (CL

half-life. The transcription rate of the IR was measured by nuclear “run-on” analysis. No detectable difference at the level of sensitivity of this assay was observed between nuclei from the patient and the control (Fig 3). It has been reported that the IR defect in the Minn-1 leprechaun is due, in part, to a nonsense mutation in exon 14.18Accordingly, we amplified exon 14 from genomic DNA and analyzed the two alleles by SSCP analysis (Fig 4). This technique detected two separate and distinct alleles in this exon. We were also able to confirm this defect by direct sequencing of the amplified DNA. This analysis revealed the substitution of thymidine for cytosine, resulting in a stop codon at amino acid 897, which is in the extracellular portion of the p-subunit (data not shown). The genomic DNA containing the Minn-1 IR promoter region was sequenced from multiple clones. No significant changes in base sequence were noted in the IR promoter in the region 5’ of the ATG codon upstream to the beginning of the ALU sequence at -1.8 kb.31 We did, however, note the presence of a “hypervariable” region at position - 1747. This region consists of a long stretch of T’s. We noted 34 T’s in the two clones sequenced. In normal individuals, Seino et a13’reported 27 T’s in this position, whereas Tewari et a13’ reported 45 T’s

this observation, there were no discernible changes in either IGF-IR mRNA or transcription rate (Figs 3 and 6). DISCUSSION

In the present study, we have analyzed the IR in fibroblasts from the Minn-1 leprechaun. Employing two

A

B

C

D

EGFR Analysis The binding of EGF to fibroblasts was measured in both the Minn-1 and in three control lines (Fig 5). There was approximately a 50% to 60% decrease at all EGF concentrations tested, and Scatchard analysis revealed a decrease in receptor-binding capacity (data not shown). When EGFR mRNA was investigated (Fig 6) poly(A)+ RNA from both control and Minn-1 cells showed a strong band at 9.5 kb and a fainter band at 5.6 kb. There was a marked reduction in intensity of both EGFR mRNA bands in Minn-1 fibroblasts. We observed no dramatic decrease in EGFR transcription rate in Minn-1 cells when compared with the striking decrease observed in both EGF binding and EGFR mRNA abundance. IGF-IR Analysis

In contrast to the IR and the EGFR, there were no major differences in binding to fibroblasts (Fig 5). In concert with

MINN

CONTROL

Fig 4. SSCP analysis of exon 14 from genomic DNA of Minn-1 and normal control. Genomic DNA, 1 Pg. was amplified and UP-labeled with PCR, heat denatured, and subjected to nondenaturing electrophoresis.2’-z Lanes A and 6, %Q and % (respectively) dilution of amplified Minn-l DNA; lanes C and D. Yloand ‘40 (respectively) dilution of amplified control DNA. Arrow indicates mutant allele in exon 14 of the Minn-1 IR gene.

RECEPTOR REGULATION IN MINN-1 LEPRECHAUN

allele. Run-on analysis of Minn-1 nuclei revealed no decrease in IR transcription rate. These results are in agreement with our sequence data, which revealed no abnormalities of the Minn-1 IR promoter sequence. However, the nonsense allele of the Minn-1 IR gene is likely to be transcribed at a normal rate. It is difficult, therefore, to determine from these “run-on” assays whether the transcription rate of the second IR allele is also reduced. Also, since only a single control cell line was used for this study, these results are still preliminary. Thus, further analysis will be necessary to resolve this issue. Other patients have been reported with decreased IR content due to IR defects, but most do not have leprechaunism. Since leprechaunism is associated not only with insulin resistance, but also with dysmorphia, other investigators have studied related growth factors of the tyrosine kinase family that are known to play a role in normal development and differentiation.‘.’ Since it was previously suggested that IGF-IR may have been abnormal in leprechaunism,“,‘J we studied this receptor, which has a close sequence and structural homology to the IR. However, no abnormalities in the expression of the IGF-IR were observed. Reddy and Kahn”’ reported that in several leprechaun

30 1

IGF-1

A

0-B

,, , ,, 0

1 -11 10

I

-10

EGFR

Kb

I 1o-g

R

1o-a

ll.O-

EG:O [M] Fig 5. ‘zsI-IGF-l and ‘“I-EGF binding to Minn-1 and control fibroblasts. (A) Specific “51-lGF-1 binding to Minn-1 and control fibroblasts. (B) Specific WEGF binding to Minn-1 and control fibroblasts. Representative of three individual experiments are shown with control cell line. Similar results were seen with two other control cell lines.

separate types of analysis, radioimmunoassay and ligand binding, we found a severe decrease in IR content. This observation is in agreement with previous studies reporting decreased IR content in fibroblasts and other cells from this patient,‘c’7.3h41 We also found a severe decrease in IR mRNA abundance, indicating that the defect in IR was not due to decreased IR protein stability. Analysis of exon 14 of one allele of the IR gene by both SSCP analysis and direct sequencing revealed a substitution at codon 897, which caused premature termination of the IR protein. Our data thus confirm the report of Kadowaki et al,” and suggest that this premature stop codon destabilizes the corresponding IR transcript. The question arises, therefore, as to why, if there is an abnormality in only one of the two IR alleles in this patient, there is more than a 50% to 60% reduction in IR mRNA content. In this subject, the gene for the allele that had the stop codon at 897 was inherited from the father. Analysis of IR cDNA from the mother revealed that her IR allele, which was inherited by the patient, was also expressed at a very low level in the maternal cells.‘” These results are also consistent with the hypothesis that there is a mutation in the maternal IR

7.05.8-

*::v_ .

c

B

M

c

B_ACTIN

Kb 2 * lC

M

Fig 6. Northern analysis of EGFR and IGF-IR mRNA in the Minn-1 leprechaun. (A) 10 pg of poly(A)+ RNA from either control (C) or Minn-1 (M) fibroblasts was subjected to electrophoresis and transferred as described in fig 3, and subsequently hybridized with the IGF-IR cDNA (IGF-IR) or EGFR cDNA (EGFR). (6) Same filter hybridized with a @actin cDNA (actin).

GOODMAN ET AL

patients, including the Minn-1, EGFR binding and function were decreased. However, in patients with IR defects without leprechaunism, normal EGFR function was noted. Others have also reported high EGF levels in leprechaunism,2 raising the possibility that this elevated ligand level may be compensating for decreased EGFR expression. Accordingly, we studied the EGFR and found a marked decrease in EGF binding and EGFR mRNA content, but only a small change in EGFR gene transcription. These data suggest a change in EGFR mRNA stability. Thus, our studies and those of others concerning the EGFR in leprechaunism suggest that defects in EGFR expression may account for some of the dysmorphological changes observed. Leprechaunism is most likely a heterogeneous disorder, since different phenotypes have been reported for cells

from various leprechaun patients.‘-‘Y.3”-4’Although it is possible that this patient had mutations in both her EGFR and her IR gene, the chances of one individual receiving two mutant IR alleles and a mutant EGFR allele(s) are slim. More recent studies suggest that the IR, itself, may regulate EGFR expression.42 It is known that insulin, via its receptor, has multiple effects on the expression of a variety of genes.43-45Thus, decreased IR function in cells from patients with leprechaunism may be responsible for the EGFR defects.

ACKNOWLEDGMENT

We thank Klaus Hartmann for assistance with mRNA preparation, Paul Mamula for technical advice, and Robbie Butler and Betty Maddux for assistance in the preparation of this manuscript.

REFERENCES

1. Donohue WL, Uchida I: Leprechaunism euphemism for a rare familial disorder. J Pediatr 45505519,1954 2. Frindik JP, Kemp SF, Fiser RH, et al: Phenotypic expression in Donohue syndrome (leprechaunism): A role for epidermal growth factor. J Pediatr 107:428-430,1985 3. Roth SI, Schedewie HK, Herzberg VK, et al: Cutaneous manifestations of leprechaunism. Arch Dermatol 117:531-535, 1981 4. Taylor Sl, Kadowaki T, Kadowaki H, et al: Mutations in insulin-receptor gene in insulin-resistant patients. Diabetes Care 131257-279,1990 5. Klinkhamer M, Groen NA, Van der Zon GCM, et al: A leucine-to-proline mutation in the insulin receptor in a family with insulin resistance. EMBO J 8:2503-2507,1989 6. Longo N, Griffen LD, Shuster RC, et al: Increased glucose transport by human fibroblasts with a heritable defect in insulin binding. Metabolism 38:690-697,1989 7. Rosenberg AM, Haworth JC, Degroot GW, et al: A case of leprechaunism with severe hyperinsulinemia. Am J Dis Child 134170.175,198O 8. D’Ercole J, Underwood LE, Groelke J, et al: Leprechaunism: Studies of the relationship among hyperinsulinism, insulin resistance and growth retardation. J Clin Endocrinol Metab 48:495-502,1979 9. Grigorescu F, Herzberg V, King G, et al: Defects in insulin binding and autophosphorylation of erythrocyte insulin receptors in patients with syndromes of severe insulin resistance and their parents. J Clin Endocrinol Metab 64:549-656, 1987 10. Maasen JA, Klinkhamer MP, Van der Zon GCM, et al: Fibroblasts from a leprechaun patient have defects in insulin binding and insulin receptor autophosphorylation. Diabetologia 31:612-617, 1988 11. Kahn CR, Flier JS, Bar RS, et al: The syndromes of insulin resistance and acanthosis nigricans: Insulin-receptor disorders in man. N Engl J Med 14:739-745,1976 12. Geffner ME, Kaplan SA, Bersch N: Lcprechaunism: In vitro insulin action despite genetic resistance. Pediatr Res 22:286-291 f3. Kaplowitz PB, D’Ercole J: Fibroblasts from a patient with leprechaunism are resistant to insulin, epidermal growth factor and somatomedin C. J Clin Endocrinol Metab 55:741-748,1982 14. Craig JW, Larner J, Locker EF, et ah Mechanisms of insulin resistance in cultured fibroblasts from a patient with leprechaunism: Impaired post-binding actions of insulin and multiplication-stimulating activity. Metabolism 33:1084-1096,1984 15. Reddy SS-K, Lauris V, Kahn CR: Insulin receptor function in fibroblasts from patients with leprechaunism: Differential alter-

ations in binding, autophosphorylation, kinase activity, and receptormediated internalization. J Clin Invest 82:1359-1365, 1988 16. Podskalny JM, Kahn CR: Cell culture studies on patients with extreme insulin resistance. I. Receptor defects on cultured fibroblasts. J Clin Endocrinol Metab 54:261-268, 1982 17. Podskalny JM, Kahn CR: Cell culture studies on patients with extreme insulin resistance. II. Abnormal biological responses in cultured fibroblasts. J Clin Endocrinol Metab 54:269-275,1982 18. Kadowaki T, Kadowaki H, Taylor SI: A nonsense mutation causing decreased levels of insulin receptor mRNA: Detection by a simplified technique for direct sequencing of genomic DNA amplified by the polymerase chain reaction. Proc Nat] Acad Sci USA 87:658-662, 1990 19. Reddy SS-K, Kahn CR: Epidermal growth factor receptor defects in leprechaunism: A multiple growth factor-resistant syndrome. J Clin Invest 84:1569-1576,1989 20. Ullrich A, Coussens L, Hayllick JS, et al: Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. Nature 309:418-425, 1984 21. Ullrich A, Gray A, Tam AW, et al: Insulin-like growth factor I receptor primary structure: Comparison with insulin receptor suggests structural determinants that define functional specificity. EMBO J 5:2503-2512,1986 22. Mamula PW, Wong KY, Maddux BA, et al: Sequence and analysis of promoter region of human insulin-receptor gene. Diabetes 37:1241-1246, 1988 23. Pezzino V, Papa V, Trischitta V, et al: Human insulin receptor radioimmunoassay: Applicability to insulin-resistant states. Am J Physiol257:E451-E457,1989 24. Clemmons DR, Elgin RG, Han VKM, et al: Cultured fibroblast monolayers secrete a protein that alters the cellular binding of somatomedin-C/insulin like growth factor I. J Clin Invest 77:1548-1556, 1986 25. Lowry OH, Rosebrough NJ, Farr AL, et al: Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275,195l 26. Hartmann K, Papa V, Brown EJ, et al: A rapid and simple one step method for isolation of poly(A)’ RNA from cells in monolayer. Endocrinology 127:2038-2040.1990 27. Harley CB: Hybridization of oligo(dT) to RNA on nitrocellulose. Gene Anal Techn 4:17-22, 1987 28. Feinberg AP, Vogelstein B: A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 137:266-267,1984 29. Brunetti A, Goldfine ID: Role of myogenin in myoblast

RECEPTOR REGULATION

differentiation

IN MINN-1

and its regulation

LEPRECHAUN

by fibroblast

growth

factor.

J Biol

Chem11:5960-5963,1990 30. Payne GS. Courtneidge SA, Crittenden LB, et al: Analysis of avian leukosis virus DNA and RNA in bursal tumors; viral gene expression is not required for maintenance of the tumor state. Cell 23:311-322, 1981 31. Seino S, Seino M, Nishi S, et al: Structure of the human insulin receptor gene and characterization of its promoter. Proc Nat1 Acad Sci USA 86:114-l 18, 1989 32. Seino S, Seino M, Bell GI: Human insulin receptor gene: Partial sequence and amplification using the polymerase chain reacl ion. Diabetes 39:123-128. 1990 33. Cawthon RM, Weiss R, Gangfeng X, et al: A major segment of the neurofibromatosis type 1 gene: cDNA sequence, genomic structure, and point mutations. Cell 62:193-201, 1990 34. Orita M. Suzuki Y, Seiya T, et al: Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5:874-879, 1989 35. Tewari DS, Cook DM, Taub R: Characterization of the promoter region and 3’ end of the human insulin receptor gene. J Biol Chem 27:1623X-16245, 1989 36. 0,jama K, Hedo JA. Roberts CT Jr, et al: Defects in human insulin receptor gene expression. Mol Endocrinol2:242-247, 1988 37. McElduff A, Hedo JA. Taylor SI, et al: Insulin receptor degradation is accelerated in cultured lymphocytes from patients with genetic syndromes of extreme insulin resistance. J Clin Invest 74: 1366-1374. 1984

38. Maron R, Taylor SI, Jackson receptors on human lymphoblastoid Diabetologia 27:118-120, 1984

R, et al: Analysis of insulin cell lines by flow cytometry.

39. Hedo JA, Moncada VY, Taylor SI: Insulin receptor biosynthesis in cultured lymphocytes from insulin-resistant patients. J Clin Invest 76:2355-2361. 1985 40. Taylor SI, Samuels B, Roth J, et al: Decreased insulin binding in cultured lymphocytes from two patients with extreme insulin resistance. J Clin Endocrinol Metab 54:919-930. 1982 41. Kadowaki T, Kadowaki H, Rechler MM. et al: Five mutant alleles of the insulin receptor gene in patients with genetic forms of insulin resistance. J Clin Invest 86:254-264, 1990 42. Watari T. Iwana N, Nomura M, et al: Decreased EGF binding in cultured fibroblasts from a patient with Type A insulin resistance. Diabetes 40:239A, 1991 (suppl 1) 43. Osborn L. Rosenberg MP, Keller SA. et al: Insulin response of a hybrid amylase/CAT gene in transgenic mice. J Biol Chem 263:16519-16522,198s 44. Nasrin N, Ercolani L, Denaro M, et al: An insulin response element in the glyceraldehyde-3-phosphate dehydrogenase gene binds a nuclear protein induced by insulin in cultured cells and by nutritional manipulation in vivo. Proc Natl Acad Scl USA 87:52735277,199O 45. deHerreros GA, Birnbaum MJ: The regulation by insulin of glucose transporter gene expression in 3T3 adipocytes. J Biol Chem 264:9885-9890.1989