Proc. Nati. Acad. Sci. USA Vol. 86, pp. 114-118, January 1989 Biochemistry Structure of the human insulin receptor gene and characterization of its promoter (alternative splicing/tyrosine kinase/Alu sequence) SUSUMU SEINO*t, MITSUKO SEINOt, SHIGEO NISHI*t, AND GRAEME I. BELL*t# *Howard Hughes Medical Institute and Departments of tBiochemistry and Molecular Biology and of tMedicine, The University of Chicago, Chicago, IL 60637 Communicated by Rachmiel Levine, October 6, 1988 (receivedfor review July 11, 1988) ABSTRACT The human insulin receptor gene, INSR, and Isolation ofINSR. Segments of the gene were isolated from its promoter region have been isolated and characterized. The the partial Hae III/Alu I fetal liver library in phage ACh4A of gene spans >120 kilobase pairs (kbp) and has 22 exons. All Lawn et al. (8) by hybridization with fragments of the human introns interrupt protein coding regions of the gene. The 11 insulin receptor cDNA (9). DNA fragments containing exons exons encoding the a subunit ofthe receptor are dispersed over 3, 10, and 11 could not be isolated from this library. Genomic >90 kbp, whereas the 11 exons encoding the (3 subunit are DNA blotting studies indicated that exon 3 was contained located together in a region of =30 kbp. Three transcriptional within a 7-kilobase pair (kbp) BamHI fragment. BamHI initiation sites have been identified and are located 276, 282, fragments of this size were isolated after electrophoresis in a and 283 bp upstream of the translation initiation site. In 1% low-melting-point agarose gel and cloned in AEMBL3 and addition, a 247-bp fragment from the promoter region pos- phage containing exon 3 identified by hybridization. Blotting sessing 62.6% of the maximal promoter activity has been studies indicated that exon 10 was contained within a 6.3-kbp identified. This promoter-active fragment lacks a TATA-like EcoRI fragment. Despite repeated attempts, this EcoRI frag- sequence but has two possible binding regions for the tran- ment could not be cloned. A 2.9-kbp Xho IlEcoRI subfrag- scriptional factor Spl. Comparison of the exon structure of the ment was readily cloned and contained exon 11 and all but 13 tyrosine kinase domain of the INSR with the corresponding bp of exon 10. The fortuitous identification, during a popula- regions of the human SRC, ROS, and ERBB2 (NGL) protoon- tion study (K. Xiang, N. J. Cox, N. Sanz, P. Huang, J. H. cogenes indicates that the exonintron organization of this Karam, and G.I.B., unpublished data), of a subject with a rare region has not been well conserved. EcoRI restriction fragment length polymorphism facilitated the isolation ofthe remainder of exon 10. Restriction mapping Insulin initiates a variety of metabolic effects upon binding to suggested that there was a deletion of 1.2 kbp in the region of a specific receptor on the cell surface (1). The isolation and exon 10 in one of the two insulin receptor alleles of this characterization ofcDNA clones encoding the human insulin individual. EcoRl fragments of 5-7 kbp were isolated by receptor indicate that the a and f3 subunits of the insulin and receptor are derived by proteolytic processing of a common electrophoresis in a 1% low-melting-point agarose gel 1382 amino acid preproreceptor (2, 3). The 731-amino acid a cloned in AgtlO. Four phage having EcoRI inserts of 5.1 kbp subunit (Mr, 135,000) is external to the plasma membrane and and containing exons 10 and 11 were isolated; the 1.2-kbp contains the insulin-binding region. It is linked by interchain deletion in this fragment is upstream of exon 10. As no phage disulfide bonds to the 620-amino acid ,B subunit (Mr, 95,000), containing a 6.3-kbp EcoRI fragment from the other allele was which includes a 194-amino acid extracellular domain, a isolated, we believe that there is a DNA sequence upstream of 23-amino acid membrane-spanning segment and a 403-amino exon 10 that prevents the cloning of this region from normal acid cytoplasmic segment that has intrinsic tyrosine kinase chromosomes. The sequences of the exons and adjacent activity. Both the a subunit and the extracellular region ofthe introns were determined. The positions of the exon-intron 13 subunit are glycosylated. junctions were assigned by using the "GT/AG" rule (10). Recent studies have indicated that the synthesis of an Primer Extension. The start of transcription was deter- abnormal insulin receptor can contribute to the development mined by primer extension using a modification of the of non-insulin-dependent diabetes mellitus (4, 5). As a first procedure described by Gil et al. (11). About 10 ,ug of total step in characterizing potentially abnormal genes relevant to RNA from term placenta, adult liver, and IM-9 and Hep G2 diabetes, we have determined the exon-intron organization cells and 1 pmol of the 32P-labeled oligonucleotide 3'- of the human insulin receptor gene, INSR, and characterized GAGCCTCGTACTGGGGGCGCCCG-5' complementary to the 5' flanking promoter sequences that regulate its expres- nucleotides -47 to -69 of the mRNA were hybridized and sion. extended with reverse transcriptase. The primer-extended products were separated on a 5% polyacrylamide/8 M urea gel. MATERIALS AND METHODS Identification of the Promoter Region Necessary for Tran- General Methods. Standard procedures were carried out as scription ofINSR. Various fragments ofthe putative promoter described by Maniatis et al. (6). Probes were labeled by region were isolated and inserted into the Bgl II site of the nick-translation. DNA sequencing was done by the dideoxy- bacterial chloramphenicol acetyltransferase (CAT) reporter nucleotide chain-termination procedure (7) after subcloning gene plasmid pCAT3M (12). Hep G2 cells, a human hepato- appropriate DNA fragments into M13mpl8 or M13mpl9. The blastoma-derived cell line (13), were transfected with 25 ug universal primer as well as sequence-specific oligonucleo- of plasmid DNA per 100-mm dish by using the calcium- tides were used as primers. Both strands were sequenced. phosphate procedure as described by Gorman (14). After 48 hr, cells were harvested, and CAT activity in cell extracts measured (14). The publication costs of this article were defrayed in part by page charge was payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: CAT, chloramphenicol acetyltransferase. 114 Downloaded by guest on September 25, 2021 Biochemistry: Seino et al. Proc. Natl. Acad. Sci. USA 86 (1989) 115 Exon I 2 3 4 54 67 8 9 1011 12 13 14\15\ " 16171819202122/ .1 N -- -Hi--/~ ,i*. ml i s! . IZInn . a Subunit O Subunit 5.6 1 13 XhINSR-I 15 | 5.6 XhINSR-II I I ~~~~~~1.8 III .0 2.61 11 1-XhINSR-2 XhINSR-12 0.8116.2 I 1 7° S 0 N6 H-18_--4 KXhINSR-3 7.6 1 i 5 6 XhlNSR-13 6.2, XhINSR-4 2405 XhINSR-5 1.2 5.hlN R1 XhINSR-6 I ~~~~2.4 | I XhINSR-7 II 2.4 4A.813.1f246 hINSR-8 229 XhINSR-9 Wp1 XhINSR-IC II I l I 9 I I -12.5 0 10 20 30 40 50 60 70 80 90 100 110 1202 kbp FIG. 1. Map of the human insulin receptor gene, INSR. The positions of the exons are indicated by the filled boxes. The transcriptional initiation site is the 0 coordinate. The name as well as the sizes and order of the EcoRl fragments in each clone are noted. The vertical lines represent natural EcoRI sites. The EcoRI insert in clone AhINSR-10 has a 1.2-kbp deletion upstream of exon 10. RESULTS Alternative Splicing Generates Different Insulin Receptors. The insulin receptor cDNA sequences first reported by Exon-Intron Organization of Human INSR. INSR was Ullrich et al. (2) and Ebina et al. (3) predicted the sequences isolated as a series of overlapping DNA fragments that span of insulin receptor precursors of 1370 and 1382 amino acids, >130 kbp (Fig. 1). As parts of five introns were not com- respectively. This difference in size is due to the absence/ pletely cloned, the gene must be larger than indicated in Fig. presence of a 36-bp segment in the cDNA sequence following 1. The coding region of the gene comprises 22 exons (Fig. 1 the codon for amino acid 717. The gene sequence indicates and Table 1). All of the introns interrupt protein coding that this size difference results from alternative splicing ofthe regions of the gene. The overall distribution of the exons is region encoded by exon 11. Interestingly, both forms of the rather striking. Exons 1-3 are distributed over a region of>50 insulin receptor have been expressed in vivo (9, 15, 16) and kbp. By contrast, exons 4-9 and 12-22 are located together are functional; however, the properties of the two types of in regions of 15 and 20 kbp, respectively, separated by a proteins have not been compared rigorously. region of >25 kbp containing exon 10 and the 36-bp miniexon Resolution of Nucleotide and Amino Acid Sequence Differ- 11. As a consequence of this pattern of exon-intron distri- ences in the Receptor. Comparison of the INSR nucleotide bution, the 11 exons encoding the a subunit of the receptor sequence and predicted amino acid sequence of its product are dispersed over >90 kbp, whereas the 11 exons encoding with reported cDNA sequences (and deduced amino acids) the subunit are located together in a region of -30 kbp. resolves several of the reported differences (Table 2). The Table 1. Exon-intron organization of INSR Exon Exon size, Sequence at exon-intron junction Intron size, Amino acid no.