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Mutagenic structure/function analysis of the cytoplasmic cysteines of the insulin receptor.

ABSTRACT: Native human insulin receptor (hIR) has been reported to contain only one free thiol group proposed to lie near the ATP-binding. domain of its beta-subunit [Finn, Ridge and Hofmann (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 419-423]. The present study investigated the role of the six cytoplasmic cysteines of the beta-subunit of the hIR using a mutagenic approach in which insulin receptors, mutated at each cytoplasmic cysteine (to alanine) in turn, were transfected into Chinese hamster ovary (CHO) cells. Cell lines expressing hIR mutation at high level were obtained which, by both flow-cytometric analysis towards an hIR-specific monoclonal antibody (83-7) and insulin-binding analysis, were similar to the well-characterized CHOT cell line which overexpresses native hIR. The ED50 and Kd values of the mutant receptors were the same as those of the wild-type hIR. Each of the mutant receptors signalled insulin action to stimulate receptor autophosphorylation and kinase activity as well as glucose utilization to levels appropriate for the receptor level expressed. In contrast, insulin-stimulated thymidine uptake and glucose-transport responses of two of the six mutant cell lines, those expressing Cys981Ala and Cys1245Ala, were impaired compared with that of the native hIR-expressing cell line, CHOT. The beta-subunits of each of the hIR cytoplasmic cysteine mutant cell lines could be alkylated specifically with N-[3H]ethylmaleimide. The kinase activity of each receptor was inhibited by N-ethylmaleimide and stimulated by iodoacetamide, indicating that none of the cytoplasmic cysteines alone contributes the single free thiol group to the hIR structure. We conclude that the cytoplasmic cysteines of the hIR have a predominantly passive role in hIR activity although Cys-981 and Cys-1245 do affect mitogenic and glucose-transport responses of the receptor. Our findings indicate that the stoicheiometry of a single free thiol group/mol of insulin-binding activity noted in previous studies is either spread fractionally over a number of the cytoplasmic cysteines or is one of the four cysteines in the ectodomain of the hIR beta-subunit. Alternatively, the mutagenesis performed in the present study may enable differential exposure of a second titratable cysteine in wild-type and mutant receptors.

SUBMITTER: Macaulay SL

PROVIDER: S-EPMC1136593 | biostudies-other | 1995 Mar

REPOSITORIES: biostudies-other

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Project description:Cytoplasmic I?B proteins are primary regulators that interact with NF-?B subunits in the cytoplasm of unstimulated cells. Upon stimulation, these I?B proteins are rapidly degraded, thus allowing NF-?B to translocate into the nucleus and activate the transcription of genes encoding various immune mediators. Subsequent to translocation, nuclear I?B proteins play an important role in the regulation of NF-?B transcriptional activity by acting either as activators or inhibitors. To date, molecular basis for the binding of I?B?, I?B? and I?B? along with their partners is known; however, the activation and inhibition mechanism of the remaining I?B (I?BNS, I?B? and Bcl-3) proteins remains elusive. Moreover, even though I?B proteins are structurally similar, it is difficult to determine the exact specificities of I?B proteins towards their respective binding partners. The three-dimensional structures of I?BNS, I?B? and I?B? were modeled. Subsequently, we used an explicit solvent method to perform detailed molecular dynamic simulations of these proteins along with their known crystal structures (I?B?, I?B? and Bcl-3) in order to investigate the flexibility of the ankyrin repeat domains (ARDs). Furthermore, the refined models of I?BNS, I?B? and Bcl-3 were used for multiple protein-protein docking studies for the identification of I?BNS-p50/p50, I?B?-p50/p65 and Bcl-3-p50/p50 complexes in order to study the structural basis of their activation and inhibition. The docking experiments revealed that I?B? masked the nuclear localization signal (NLS) of the p50/p65 subunits, thereby preventing its translocation into the nucleus. For the Bcl-3- and I?BNS-p50/p50 complexes, the results show that Bcl-3 mediated transcription through its transactivation domain (TAD) while I?BNS inhibited transcription due to its lack of a TAD, which is consistent with biochemical studies. Additionally, the numbers of identified flexible residues were equal in number among all I?B proteins, although they were not conserved. This could be the primary reason for their binding partner specificities.

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Mutagenic structure/function analysis of the cytoplasmic cysteines of the insulin receptor.

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