Quantitation of the class I disulfides of the insulin receptor

Chiacchia, Kenneth B.

doi:10.1016/0006-291x(91)90409-z

Cited by 24 publications

(30 citation statements)

References 18 publications

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“…The results of experiments in which Cys 647 was mutated to serine suggested that the IR has only one ␣-␤ disulfide bond (19). Current models of the IR thus suggest ␣-chains linked to each other to form the dimer with ␤-chains linked only to ␣-chains within each monomer (13,14).In this report the disulfide bond arrangement in the Cterminal half of the IR ectodomain is established. It is shown that the ␣-␤ disulfide link is between Cys 647 in the first Fn III repeat and Cys 872 in the second Fn III repeat, which is consistent with the mutagenesis data (19); also identified is an ␣-␣ dimer linkage, in addition to that involving Cys 524 (16), involving one or more of the triplet of cysteine residues at positions 682, 683, and 685 in the insert domain.…”

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confidence: 70%

“…Digestion of the ␤Ј subunit derived from NEM-labeled ectodomain lead to the isolation of a labeled peptide that confirmed that the thiol was Cys 884 . A number of studies have indicated that the ␣␤ monomer of the IR has a single thiol group that is located on the ␤-chain and is readily alkylated by NEM in the absence of disaggregating agents (13,14). Alkylation of the thiol of the native receptor with NEM has been shown to inhibit the tyrosine kinase activity of the receptor (35); inhibition by NEM is more effective in the presence than in the absence of insulin (36) and the IR is protected from alkylation with NEM by ATP or a nonhydrolyzable analogue (14).…”

Section: Discussionmentioning

confidence: 99%

“…Alkylation of the IR suggests that it has one free thiol group per ␣␤ monomer and that this is on the ␤-chain (12)(13)(14). Mild reduction of the insulin receptor cleaves a small number of disulfide bonds (the class I bonds) giving rise to disulfide-linked ␣␤ monomers that can still bind ligand; more vigorous reduction cleaves the ␣-␤ bond(s) (the class II bonds) leading to free ␣-and ␤-chains and to denaturation of the two protein chains (15).…”

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confidence: 99%

“…However, mutation of Cys 524 to Ala (17) or Ser (18) resulted in an IR that was still dimeric, thereby indicating that more than one disulfide bond is involved in dimer formation. Labeling of monomeric IR showed that there are at least two cysteines per monomer involved in class I disulfide bonds (13,14). The results of experiments in which Cys 647 was mutated to serine suggested that the IR has only one ␣-␤ disulfide bond (19).…”

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confidence: 99%

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confidence: 99%

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The Disulfide Bonds in the C-terminal Domains of the Human Insulin Receptor Ectodomain

Sparrow

McKern

Gorman

et al. 1997

Journal of Biological Chemistry

116

101

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The human insulin receptor is a homodimer consisting of two monomers linked by disulfide bonds. Each monomer comprises an ␣-chain that is entirely extracellular and a ␤-chain that spans the cell membrane. The ␣-chain has a total of 37 cysteine residues, most of which form intrachain disulfide bonds, whereas the ␤-chain contains 10 cysteine residues, four of which are in the extracellular region. There are two classes of disulfide bonds in the insulin receptor, those that can be reduced under mild reducing conditions to give ␣-␤ monomers (class I) and those that require stronger reducing conditions (class II). The number of class I disulfides is small and includes the ␣-␣ dimer bond Cys 524 . In this report we describe the use of cyanogen bromide and protease digestion of the exon 11 plus form of the receptor ectodomain to identify disulfide linkages between the ␤-chain residues Cys 798 and Cys 807 and between the ␣-chain Cys 647 and the ␤-chain Cys 872 . The latter bond is the sole ␣-␤ link in the molecule and implies a side-byside alignment of the two fibronectin III domains of the receptor. Also presented is evidence for additional ␣-␣ dimer bond(s) involving at least one of the cysteine residues of the triplet at positions 682, 683, and 685. Evidence is also presented to show that Cys 884 exists as a buried thiol in the soluble ectodomain. The insulin receptor (IR)1 is a homodimer, (␣␤) 2 , held together by disulfide bonds. Each ␣␤ monomer comprises an ␣-chain that is entirely extracellular and a ␤-chain that spans the cell membrane via a single transmembrane link to an intracellular segment that includes the protein-tyrosine kinase domain and the domains involved in binding signal transduction proteins (1). The ␣␤ monomer of the IR is coded for by 22 exons (2), is synthesized with a 27-residue signal sequence, and is glycosylated, S-S linked, and proteolytically processed in that order during transport to the cell surface (3, 4). The extracellular region of the mature receptor (the ectodomain) contains the ␣-chain (1-735) and 194 residues of the ␤-chain (the ␤Ј-chain, residues 736 -929). Two isoforms of the IR, which arise by alternative splicing of the mRNA, are expressed in a tissue-specific fashion; they differ by the presence (plus exon 11) or the absence (minus exon 11) of a 12-residue segment close to the C terminus of the ␣-chain (5).The ectodomain of the IR has been shown (6, 7) to contain two homologous domains (L1 and L2) separated by a single cysteine-rich region (residues 159 -310). The L2 domain is joined by a connecting domain (residues 471-593) to the Cterminal portion (from residue 594) that is comprised of two fibronectin (Fn) III repeats, the first of which contains an insert domain that includes both the ␣-␤ cleavage site and the alternately spliced exon 11 (8, 9). A schematic diagram showing the relative arrangement of the domains is shown in Fig. 7.The ␣-chain has a total of 37 cysteine residues, whereas the ␤-chain has 4 extracellular and 6 intracellular cysteine residues. Homologous re...

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mentioning

confidence: 70%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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The Disulfide Bonds in the C-terminal Domains of the Human Insulin Receptor Ectodomain

Sparrow

McKern

Gorman

et al. 1997

Journal of Biological Chemistry

116

101

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The insulin‐binding domain of insulin receptor is encoded by exon 2 and exon 3

Yip

1992

J of Cellular Biochemistry

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Insulin receptors are disulfide-linked oligotetramers composed of two heterodimers each containing a 130-kDa alpha subunit and a 90-kDa beta subunit. Insulin binds to the extracellular alpha subunit, and in the process stimulates the autophosphorylation of the beta subunit and the expression of tyrosine kinase activity. Studies combining the use of photoaffinity labeling and immunoprecipitation with anti-peptide antibody have directly demonstrated that the cysteine-rich domain, encoded by exon 3, in the alpha subunit is part of the insulin-binding site of the receptor. Experiments with chimeric insulin receptors and chimeric insulin-like growth factor I receptors have confirmed that the cysteine-rich domain constitutes a part of the insulin-binding site. In addition, results from these experiments suggest that the N-terminal sequence, encoded by exon 2, in the alpha subunit also participates in insulin binding. In this review it is proposed that, assuming two insulin-binding sites per each holoreceptor oligotetramer, each insulin-binding domain may contain respectively two sub-domains for hydrophobic and charge contact with insulin, and that high-affinity binding would require the interaction of both subunits with the possibility of each subunit reciprocally contributing one of the sub-domains.

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Insulin and epidermal growth factor receptors contain the cysteine repeat motif found in the tumor necrosis factor receptor

1995

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The insulin receptor (INSR) and epidermal growth factor receptor (EGFR) are representatives of two structurally related subfamilies of tyrosine kinase receptors. Using the Wisconsin GCG sequence analysis programs, we have demonstrated that the cysteine-rich regions of INSR and EGFR conform to the structural motif found in the tumor necrosis factor receptor (TNFR) family. The study also revealed that these regions were not composed of simple repeats of eight cysteine residues as previously proposed and that the second Cys-rich region of EGFR contained one fewer TNFR repeat than the first. The sequence alignments identified two cysteine residues in INSR that could be responsible for the additional disulfide bonds known to be involved in dimer formation. The published data on the alignments for the fibronectin type III repeat region of the INSR together with previous cysteine mutagenesis studies indicated that there were two disulfide bonds linking the alpha and beta chains of the INSR, but only one alpha-beta linkage in the insulin-like growth factor 1 receptor (IG1R). Database searches and sequence alignments showed that the TNFR motif is also found in the cysteine-rich repeats of laminins and the noncatalytic domains of furin-like proteases. If the starting position of the repeat is altered the characteristic laminin repeat of eight cysteine residues can be shown to consist of a TNFR-like motif fused to the last half of an EGF-like repeat. The overlapping regions of these two motifs are known to have identical disulfide bonding patterns and similar protein folds.

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Quantitation of the class I disulfides of the insulin receptor

Cited by 24 publications

References 18 publications

The Disulfide Bonds in the C-terminal Domains of the Human Insulin Receptor Ectodomain

The Disulfide Bonds in the C-terminal Domains of the Human Insulin Receptor Ectodomain

The insulin‐binding domain of insulin receptor is encoded by exon 2 and exon 3

Insulin and epidermal growth factor receptors contain the cysteine repeat motif found in the tumor necrosis factor receptor

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