©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Drosophila Insulin Receptor Contains a Novel Carboxyl-terminal Extension Likely to Play an Important Role in Signal Transduction (*)

(Received for publication, October 20, 1994; and in revised form, December 12, 1994)

Yimin Ruan Chi Chen Yixue Cao Robert S. Garofalo (§)

From the Department of Anatomy and Cell Biology, State University of New York, Health Science Center at Brooklyn, Brooklyn, New York 11203

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The nucleic acid and deduced amino acid sequence of the Drosophila insulin receptor homologue (dir) has been determined. The coding sequence of dir is contained within 10 exons spanning less than 8 kilobase pairs of genomic DNA. The deduced amino acid sequence of the dir encodes a protein of 2148 amino acids, larger than the human insulin receptor due to amino- and carboxyl-terminal extensions. The overall level of amino acid identity between the DIR and human insulin and insulin-like growth factor-I receptors is 32.5 and 33.3%, respectively. Higher levels of identity are found in exon 2 (45 and 43%, respectively) and in the beta subunit (50 and 48%, respectively), and the positions of most cysteine residues in the alpha subunit cysteine-rich domain are conserved. A novel, 400-amino acid, carboxyl-terminal extension contains 9 tyrosine residues, four of which are present in YXXM or YXXL motifs, suggesting that they function as binding sites for SH2 domain-containing signaling proteins. The presence of multiple putative SH2 domain binding sites in the DIR represents a significant difference from its mammalian homologues and suggests that, unlike the human insulin and insulin-like growth factor-I receptors, the DIR forms stable complexes with signaling molecules as part of its signal transduction mechanism.


INTRODUCTION

The insulin receptor of Drosophila (DIR), like its mammalian homolog, is a tetrameric glycoprotein composed of two alpha subunits and two beta subunits joined together by disulfide bonds(1, 2) . The alpha subunits are extracellular and contain the ligand-binding domains which bind mammalian insulin with fairly high affinity (K = 15 nM; (3) ). Mammalian insulin-like growth factor I (IGF-I) (^1)fails to displace bound insulin, while IGF-II does, although at concentrations 10-20-fold higher than insulin(3, 4) . The beta subunits contain the transmembrane domain and a ligand-activated tyrosine kinase. The kinase domain exhibits a very high degree of sequence homology with other members of the insulin receptor family(1, 5) . Like the mammalian receptors, the alpha and beta subunits are produced as a single proreceptor precursor polypeptide which is proteolytically cleaved to yield the mature subunits(2, 5) .

The structure of the beta subunit kinase domain and the carboxyl terminus of the alpha subunit have been determined by molecular cloning (1, 5) . However, these data provide little insight into the basis of DIR ligand binding specificity and fail to account for a larger beta subunit form observed in autophosphorylation studies(2) . Therefore, the complete structure of the DIR protein and the organization of the dir gene have been determined as a step toward understanding the function of this receptor and its relationship to mammalian homologues. The results further reinforce the evolutionary relatedness of the Drosophila and mammalian proteins, although the DIR exhibits large extensions at both the amino and carboxyl termini. The carboxyl-terminal extension is particularly intriguing because it includes motifs known to be involved in binding of SH2 domain-containing proteins(6) . This suggests that, unlike mammalian insulin and IGF-I receptors(7) , signal transduction from the DIR involves stable interactions with other proteins to form multimeric signaling complexes.


MATERIALS AND METHODS

Isolation and Characterization of dir cDNA Clone pY19

A cDNA library prepared from 12 to 24-h embryos was generously provided by the laboratory of F. C. Kafatos (Harvard University) and screened according to the methods of Brown and Kafatos (8) using a 0.9-kb BamHI-XhoI subfragment of cDNA clone IIalpha (Fig. 1) as a probe. Filters were prehybridized in hybridization buffer (6 times SSC, 5 times Denhardt's solution, 0.5% SDS, 50% formamide, and 100 µg/ml denatured salmon sperm DNA) at 42 °C for 1 h prior to the addition of probe. Hybridization was performed at 42 °C for 24 h followed by washing twice at 22 °C with 2 times SSC, 0.1% SDS for 5 min each, and once at 50 °C with 0.1 times SSC, 0.5% SDS for 45 min. Positive hybridization was detected by autoradiography. Clone pY19 was characterized by restriction mapping according to standard protocols and DNA sequencing as described below.


Figure 1: Map of the dir gene. A, the location of restriction sites in the dir genomic region as determined by Southern analysis. (B, BamHI; E, EcoRI; H, HindIII; K, KpnI; N, NotI; P, PstI; S, SmaI; X, XhoI.) The stippled area indicates the region of genomic DNA that has been sequenced. Above the restriction map, the extent of genomic clones 1C, 19-12, and S5-1 is indicated. The SmaI site at the extreme left is >23 kb from the SmaI site in exon 2. B, the intron-exon structure of the dir gene. C, cDNA clones of dir. IIIalpha, IIalpha, Ibeta, and IVbeta are fragments generated by PCR. D, schematic representation of the deduced DIR protein and comparison with the HIR. The cysteine-rich regions (stippled box), transmembrane domains (hatched box), and binding sites for AbP2 (circle) and AbP5 (stripedbox) are indicated.



Isolation and Characterization of dir Genomic Clone 1C

Drosophila genomic libraries were constructed in the Fix II vector (Stratagene Cloning Systems, La Jolla, CA) and generously provided by the laboratory of Dennis Ballinger (Memorial Sloan-Kettering Cancer Center, New York). Library screening was carried out using standard procedures (9) with the 5`-most 1.7-kb subfragment of the IIIalpha cDNA clone as a probe. Filters were prehybridized at 42 °C for 2 h in 6 times SSPE, 0.05 times Blotto (1 times Blotto is 5% nonfat dry milk, 1% Nonidet P-40), and 50% formamide, prior to the addition of probe labeled by random priming (Random Primed DNA Labeling Kit, U. S. Biochemical Corp., Cleveland, OH). Hybridization was carried out for 18 h at 42 °C. Filters were washed twice at 22 °C in 2 times SSC, 0.1% SDS for 5 min each, followed by several washes at 68 °C in 1 times SSC, 0.1% SDS for 30-60 min each. Positive clones were characterized by restriction mapping and Southern hybridization with other cDNA and genomic clones.

Cloning of dir cDNA Fragments by Polymerase Chain Reaction (PCR) Amplification

Poly(A) RNA prepared from 0 to 20-h embryos was subjected to reverse transcription and PCR amplification using the GeneAmp RNA PCR kit (Perkin-Elmer Corp.) according to the manufacturer's instructions. PCR amplification consisted of 40 cycles of denaturation at 95 °C for 40 s, annealing at 60 °C for 1 min, and polym-erization at 72 °C for 2 min. The forward/reverse primers were 5`-GTAACGGCGAACGAGTGCATC-3`(2786)/5`-AATTCCTTGGCAAACA CCTCA-3` (4750) and 5`-GAAGGAGAGACGTTGCGCTCG-3` (4567)/5`-GCAAGATCACGATGGACGAAC-3`(6010) for the IIalpha and Ibeta fragments, respectively. Numbers indicate the position of the initial nucleotide of the primer in the dir gene, with the 5` EcoRI site in genomic clone 1C as nucleotide number 1. The 12-24 h cDNA library described above was used as a template for amplification of the IIIalpha cDNA. Reactions were carried out as above and utilized a set of nested primers. Forward/reverse primers were 5`-CAGCAGCAACAACAGCAA-3` ((45) ) (859)/5`-GGAGCACTCCTTGTCACACT-3`(2956) and 5`-CTGCACGCTTGGACACAT-3` ((45) ) (887)/5`-CGCTTGTTCTCTGACTTCTG-3`(2910), for the primary and secondary amplifications, respectively. 1% of the products of the primary PCR amplification was used as a template for secondary amplification with internal primers. Amplified cDNA fragments were ligated into the pCR-1000 vector using the TA Cloning Kit (Invitrogen, San Diego, CA) according to the manufacturer's instructions.

Southern Analysis

Genomic DNA from adult Oregon R flies was prepared according to standard methods(10) , digested with restriction enzymes (New England Biolabs), subjected to electrophoresis on 0.7% agarose gels, and transferred to nylon membranes (GeneScreen Plus, DuPont NEN) according to the procedures described(9) . Gel-purified restriction fragments of genomic clones 1C, 19-12, and S5-1 and cDNA 3alpha were P-labeled by random primed labeling according to the manufacturer's instructions (U. S. Biochemical Corp.) and hybridized to blots for 18 h at 42 °C in a solution of 50% formamide, 1 M NaCl, 1% SDS, 10% dextran sulfate, 10 times Denhardt's solution, 0.1% tetrasodium pyrophosphate, and 0.5 mg/ml of denatured salmon sperm DNA. Filters were then washed twice for 5 min each in 2 times SSC at room temperature, twice for 30 min each in 2 times SSC, 0.1% SDS at 68 °C, and twice for 30 min each in 0.1 times SSC at room temperature, followed by autoradiography at -70 °C for 1-2 days using preflashed x-ray films.

DNA Sequencing

The Sequenase Version 2.0 kit (U. S. Biochemical) was utilized for DNA sequencing according to the manufacturer's instructions. Sequencing reactions were carried out on genomic clones p19-12, the 8-kb EcoRI subfragment of 1C, and cDNA clones pY19, IIalpha, and IIIalpha. For IIIalpha, several independent clones were obtained, subfragments of these cloned into pUC19, and sequencing carried out using the M13 forward and reverse sequencing primers. dir-specific sequencing primers were utilized for determination of the complete genomic sequence. In some cases, PCR products were sequenced directly utilizing the Taq Dye Deoxy Terminator Cycle Sequencing Kit (Applied Biosystems) and an Applied Biosystems 383A Automated DNA sequencer. Any differences with published dir sequence information (1, 5, 45) were confirmed by sequencing at least two independent clones.


RESULTS AND DISCUSSION

cDNA and genomic clones encompassing the entire coding sequence of the dir were isolated through a combination of library screening and PCR amplification. A 5.6-kb cDNA clone (Fig. 1C, pY19) isolated from a cDNA library prepared from 12 to 24-h Drosophila embryos (generously provided by the laboratory of F. C. Kafatos; (8) ) encodes 277 amino acids of the alpha subunit, all of the beta subunit, and a longer 3`-untranslated region than that found in the beta 2.9 cDNA (Fig. 1C; (11) ). This and other cDNA clones were used to isolate and characterize a 15-kb genomic clone which spans the dir coding region and extends approximately 4 kb further in both the 5` and 3` directions (Fig. 1A, clone 1C). The IIIalpha and IIalpha cDNA fragments (Fig. 1C) encompass the alpha subunit and were obtained by PCR amplification of the above cDNA library and reverse transcriptase-PCR of embryo RNA, respectively. Southern analysis with the 19-12 genomic clone and subfragments thereof, the IIIalpha and Ibeta cDNAs and S5-1 (gift of Manfred Frasch, Mount Sinai School of Medicine) was used to generate a restriction map of the dir locus (Fig. 1A). S5-1 extends into the beginning of the S59 gene (12) which is located proximally to dir on the third chromosome. The sequence of 8 kb of genomic DNA has been determined and the intron-exon structure of the dir gene deduced from comparison of genomic and cDNA sequences (Fig. 1B, Table 1). The introns are small in size, ranging from 56 to 102 nucleotides, making the dir gene relatively compact as compared to the human insulin or IGF-I receptor genes(13, 14) although similar in size to the insulin receptor-related receptor gene (HIRR, (15) ). The dir coding region is contained within 10 exons (Fig. 1B). However, the transcription start site and extent of 5`-untranslated sequence has not yet been determined, and another exon containing only 5`-untranslated sequence or an alternative first exon may be found upstream.



Although the intron-exon structure of the dir gene differs from that of the mammalian members of the insulin receptor gene family (10 versus 21 or 22 exons, respectively; (13, 14, 15) ), an evolutionary relationship is suggested by the conservation of many intron-exon boundaries (Table 2). Exon 2 is similar in size in all insulin receptor family members. Exons 3 and 4 differ in size between the dir and mammalian receptors, although the size of both exons together is nearly identical. Similarly, exons 5 and 6 of the mammalian receptors are equivalent to exon 5 of dir; exons 7, 8, and 9 of the mammalian receptors are equivalent to exons 6 and 7 of dir; exons 10, 11, and 12 of the mammalian receptors correspond to exon 8 of dir; exons 13 and 14 of the mammalian receptors are equivalent to exon 9 of dir. A major difference between dir and the mammalian receptor genes is that dir exon 10 encodes the entire beta subunit beginning 7 residues before the transmembrane domain, whereas in the human insulin receptor, the beta subunit extending from 9 residues before the transmembrane domain to the carboxyl terminus is encoded by 8 exons (exons 15-22). Thus, the organization of the dir gene is clearly related to, but less complex than that of the mammalian genes. Comparison of their structures suggests the existence of an even simpler predecessor gene. dir exons 3 and 4, and likewise 6 and 7, may have comprised single exons in an earlier insulin receptor gene, that were later divided differently into multiple exons during divergent evolution.



The longest open reading frame begins with a translational start sequence that matches the Drosophila consensus (16) in six of seven positions and encodes a protein of 2148 amino acids (Fig. 2), although 5 nearby downstream in-frame methionine codons with similar or slightly weaker translational start consensus sites are found. Assuming that dir resembles most other eukaryotic mRNAs in that the 5` proximal ATG is utilized(17) , the deduced coding region would comprise 6,444 base pairs of the mature mRNAs which are 8.6 and 11 kb in length(11) . Data suggest that the two mature mRNA species differ only in their untranslated sequence, because probes encoding alpha subunit, beta subunit kinase domain, or beta subunit carboxyl-terminal extension hybridize with both mRNAs(11) . Therefore, the 8.6- and 11.0-kb mature mRNAs are predicted to contain approximately 2.1 and 4.5 kb of untranslated sequence, respectively. cDNA clone pY19 extends to an apparent polyadenylation site and contains approximately 1.6 kb of 3`-untranslated sequence. If this cDNA is derived from the 8.6-kb mRNA, it would follow that the 5`-untranslated region is approximately 0.5 kb in length. This is in the same size range as the 5`-untranslated sequence(s) of the human insulin receptor (18) although smaller than that of the human IGF-I receptor(19, 20) .


Figure 2: Nucleotide and deduced amino acid sequence of the longest open reading frame of dir. Exon boundaries, determined from comparison of genomic and cDNA sequences, are indicated above the nucleotide sequence. The putative signal or membrane anchoring sequence (amino acids 270-297), and the transmembrane domain in the beta subunit (amino acids 1315-1338) are underlined. The endopeptidase cleavage site is indicated by bold lettering, and potential sites of N-linked glycosylation are italicized. The beginning of the carboxyl-terminal extension is indicated by the arrow at position 1750.



The predicted DIR protein is larger than the HIR (2148 versus 1355 amino acids) due to extensions at both the amino- and carboxyl-terminal ends (Fig. 1D). Interestingly, the first complete codon in exon 2 in both receptors is a conserved cysteine residue ( Fig. 2and Fig. 3). Likewise, other receptors in this family, the IGF-I and HIRR, also begin exon 2 at a homologous cysteine (Fig. 3). Most of the size difference in the alpha subunits is accounted for by the first exon (341 versus 33 amino acids in DIR and HIR, respectively), which has no significant homology with the first exon of either the insulin or IGF-I receptors. Hydropathy analysis does not reveal an amino-terminal signal sequence (Fig. 4A), but a stretch of 28 hydrophobic amino acids located at positions 270-297 (Fig. 4A, open arrow) suggests an internal signal or membrane anchoring sequence (Fig. 2, underlined). A similar internal hydrophobic sequence is found in the Sevenless receptor(21) . The carboxyl-terminal end of this hydrophobic stretch conforms reasonably well with the(-3, -1) rule for identification of signal sequence cleavage sites(22) , suggesting that the mature alpha subunit may begin at His-298. The size of dir alpha subunits determined by gel electrophoresis after cross-linking to iodinated insulin (2) is similar to that of the human insulin receptor (110-120 versus 135 kDa, respectively), suggesting that most of the leader sequence encoded by exon 1 is removed.


Figure 3: Comparison of amino acid sequences of the DIR and other insulin receptor family receptor tyrosine kinases. The single letter amino acid code is used. The amino acid sequences are aligned from the beginning of exon 2 and the carboxyl-terminal extension of the DIR is excluded. Amino acid identities with the DIR are indicated by capitalized letters, and positions of identical amino acids in all four receptors are indicated by stippled boxes underlying the sequences. The positions of DIR cysteine residues which are conserved in at least three of the four receptors are indicated by asterisks. The endopeptidase processing sites and transmembrane domains are underlined. Receptor identity and amino acid numbers are indicated on the left. The sequences were aligned with the ALIGN program.




Figure 4: Hydrophilicity analysis of the predicted DIR amino acid sequence. Positive values indicate more hydrophobic regions. A, the alpha subunit. The putative internal signal sequence or membrane anchoring domain (open arrow) and two smaller more amino-terminal hydrophobic domains (arrowheads) are indicated. B, the beta subunit. The transmembrane domain is indicated (open arrow).



The DIR amino-terminal region encoded by exon 1 contains an unusual arrangement of sequence motifs. Two hydrophobic sequences of 17 amino acids each are found amino-terminal to the putative signal sequence, at positions 156-173 and 206-223 ( Fig. 2and Fig. 4A, arrowheads). Immediately following the most amino-terminal 17-amino acid hydrophobic stretch is the sequence KRRRR, which may represent a proteolytic processing site similar to that which is used to separate the alpha and beta subunits (residues 1086-1089). A Gln- and His-rich sequence follows this stretch of 5 basic residues, consisting of 26 residues of which 23 are either Gln or His. This Gln/His-rich domain terminates at residue 205, just prior to the second 17-amino acid hydrophobic domain. A search of the protein data bases for proteins exhibiting homology to this region of the DIR primarily yields a variety of transcription factors including homeobox proteins, many of which contain domains rich in glutamine or histidine residues. The functional significance of this homology is currently unknown.

The overall level of amino acid identity between the DIR and HIR is 32.5% (excluding exon 1 and the carboxyl-terminal extension), although the level of homology is higher if conservative substitutions are considered. The level of identity with the IGF-I receptor is similar (33.3%) and slightly lower with the IRR (30.7%). A higher level of identity is found in the insulin receptor beta subunits (48%) than in the alpha subunits (31%) because of the highly conserved tyrosine kinase domain. However, within the alpha subunit, the identity in exon 2 rises to 45%. The homology is highest in the amino-terminal portion of exon 2 (Fig. 3), which has been shown to contain determinants responsible for high affinity insulin binding in the human insulin receptor (23, 24, 25, 26) . dir exons 3-5 also exhibit greater than 30% amino acid identity with all of the insulin receptor family members (Table 2). Similar to the mammalian receptors, the cysteine-rich domain begins in exon 2 and extends through exons 3 and 4 (Fig. 3, asterisks). The DIR has 25 cysteine residues in this region, of which 18 occupy conserved positions in all four of the insulin receptor family members compared (Fig. 3). The positions of 24 cysteines are conserved in the human insulin receptor, and 22 in the IGF-I receptor (Fig. 3). Both the cysteine-rich domain (27, 28) and regions located carboxyl-terminal to the cysteine-rich domain(29) , also appear to contribute to insulin binding specificity. Thus, the ability of the DIR to bind mammalian insulin with reasonably high affinity is consistent with the high degree of sequence conservation in these regions of the alpha subunit. Conversely, the carboxyl terminus of the DIR alpha subunit encoded by dir exons 6-8 exhibits much lower levels of homology ( Table 2and Fig. 3), suggesting that the carboxyl-terminal region of the alpha subunit contributes very little to the formation of the ligand binding pocket.

The cytoplasmic portion of the DIR beta subunit contains the kinase domain which is the region of highest homology common to all the insulin receptor family members. The portion of the DIR beta subunit which can be aligned with the HIR (Fig. 3) contains 6 cysteine residues, three of which occupy conserved positions in other insulin receptor family members (Fig. 3, asterisks). One DIR cysteine, Cys, is displaced 10 residues toward the carboxyl terminus relative to the position of a conserved cysteine in the mammalian receptors (Cys in the HIR, Fig. 3). Interestingly, the c-ros protooncogene product resembles the DIR in that it contains a cysteine residue in the identical position (30) . There are 18 tyrosine residues in the portion of the DIR beta subunit which is colinear with the HIR. The positions of 9 tyrosines are conserved in the HIR and HIRR, and 10 in the IGF-I receptor (Fig. 3). The conserved tyrosines include DIR Tyr which, like its counterpart Tyr in the juxtamembrane domain of the HIR, is found in an NPXY motif (see below). This residue in the HIR is a major site of autophosphorylation (31) and appears to play a critical role in the interaction with and/or phosphorylation of substrates necessary for signal transduction(32, 33) . Notably, the equivalent tyrosine residue in all three mammalian receptors is preceded by an acidic amino acid (Glu), a feature common to many tyrosine phosphorylation sites(34) , whereas in the DIR, this tyrosine is preceded by a hydrophobic residue (Phe).

The DIR beta subunit contains a carboxyl-terminal extension of approximately 400 amino acids (Fig. 2, arrow). It was previously reported that a termination codon (TGA) followed Pro (numbering according to Fig. 2), yielding a DIR beta subunit comparable in size to that of the human insulin receptor(5) . However, that sequence included an additional cytosine residue (3287, numbering according to (5) ) which was not found in the cDNA and genomic clones described here. The absence of this residue leads to a frameshift which extends the open reading frame for an additional 392 amino acids. The extension has no significant homology with the HIR although its structure predicts an important role in DIR signal transduction. The carboxyl-terminal extension contains 9 tyrosine residues, some of which represent potential phosphorylation sites. Residues Tyr, Tyr, Tyr, Tyr, and Tyr have nearby acidic residues. Three tyrosines, Tyr, Tyr, and Tyr, are found in YXXM motifs which serve as excellent substrates for the insulin receptor kinase (35) and are involved in the binding and activation of phosphatidylinositol 3`-kinase by IRS-1(36) . However, none of the DIR YXXM motifs exhibit amino-terminal acidic residues which have been shown to significantly enhance the efficiency of tyrosine phosphorylation(35) . Tyr is part of the sequence YRLL, which resembles the YXXL motif found in the cytoplasmic domains of Fc, B cell, and T cell receptor subunits (37) which are involved in binding tyrosine kinases of the src and syk family (38, 39, 40) . Interestingly, these 4 tyrosines are located downstream from another apparent motif, SXNPN, beginning at the position -5 relative to the tyrosine residue. This suggests the consensus sequence SXNPNYXXM/L as a functional domain in this part of the DIR. The only other DIR beta subunit tyrosine found in a similar context is Tyr in the juxtamembrane domain, which is found in the sequence VNPFYASM. The presence of asparagine and proline at the -3 and -2 positions, respectively, relative to a putative tyrosine phosphorylation site is not a feature common to many receptor tyrosine kinases or known substrates(6) . Although the HIR, IGF-I receptor, and IRR all contain an NPXY sequence in their juxtamembane domains, this motif is not found elsewhere in the receptors, nor in their major substrate, insulin receptor substrate-1 (IRS-1; 41). It is found in the Trk and epidermal growth factor receptor tyrosine kinases(42, 43) . In the Trk receptor, it precedes Tyr, phosphorylation of which is necessary for the association of the activated receptor with SHC proteins(44) . Thus, this motif may represent a site of interaction with a subset of signaling proteins, and its prevalence in the carboxyl terminus of the DIR suggests that this domain is likely to play an important role in signaling via direct associations with particular SH2-domain proteins. This would represent a departure from mammalian insulin and IGF-I receptors, which seem not to form direct, stable interactions with signaling molecules(7) . Instead, the mammalian receptors primarily employ the substrate IRS-1 to recruit SH2 domain proteins into signaling complexes(7) . Although direct comparison of the amino acid sequences of IRS-1 (41) and the DIR carboxyl-terminal extension reveals only a very weak homology over a short region (18% in 132 amino acid overlap based primarily on the alignment of two YXXM motifs), the presence of these motifs in both molecules suggests a functional, and possibly an evolutionary, relationship between them.

The presence of a carboxyl-terminal extension as determined by DNA sequencing suggests that a larger DIR beta subunit protein will be found. This is consistent with previous observations of a 170-kDa putative receptor subunit which is directly recognized by an antibody against the HIR tyrosine kinase domain(2) . Two additional antibodies raised against peptides derived from the DIR beta subunit also recognize the larger subunit, as well as smaller 93-102 kDa subunits. (^2)In addition, expression of a dir cDNA in human cells results in the appearance of a beta subunit protein of approximately 180 kDa.^2 Thus, consistent with the sequence analysis, the DIR beta subunit is substantially larger than its mammalian counterparts. The relationship between this larger form and the smaller 93-102 kDa beta subunits which are also recognized by the same three receptor-specific antibodies remains to be determined. It seems likely that post-translational processing, e.g. proteolysis, and not alternative splicing will account for the different receptor forms because the transmembrane and cytoplasmic domains of the beta subunit including the carboxyl-terminal extension are encoded by a single exon (Fig. 1).

In summary, the determination of the complete cDNA sequence and genomic organization of the dir gene reveals that it is highly related to its mammalian counterparts, and provides some insight into the basis for ligand binding specificity. While the overall structure of the DIR protein is conserved, the presence of large amino- and carboxyl-terminal extensions represents a departure from other members of the insulin receptor family. In particular, the carboxyl-terminal extension contains structural features suggesting an important role in signal transduction, possibly in the interaction with other signaling proteins. Such direct interactions would resemble the signal transduction paradigm utilized by other receptor tyrosine kinases, such as the receptors for platelet-derived growth factor and epidermal growth factor. Therefore, this paradigm, while retained by some classes of receptor tyrosine kinases, may be the evolutionary predecessor of that utilized by mammalian insulin and IGF-I receptors in which the functions of phosphorylation and signal complex formation appear to have been largely dissociated.


FOOTNOTES

*
This work was supported by Grant IBN 92-13992 from the National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom reprint requests and correspondence should be addressed. Tel.: 718-270-1060; Fax: 718-270-3732.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U18351[GenBank].

(^1)
The abbreviations used are: IGF, insulin-like growth factor; kb, kilobase pair(s); PCR, polymerase chain reaction; IRS-1, insulin receptor substrate 1.

(^2)
M. Marin-Hincapie and R. S. Garofalo, unpublished observations.


ACKNOWLEDGEMENTS

We thank F. C. Kafatos and D. Ballinger for providing cDNA and genomic libraries, respectively; M. Frasch for providing the S5-1 genomic clone; and J. Lewis and M. A. Q. Siddiqui for critical reading of the manuscript.


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