Dataset Information

S-acylation of the Insulin-Responsive Aminopeptidase (IRAP): Quantitative analysis and Identification of Modified Cysteines.

ABSTRACT: The insulin-responsive aminopeptidase (IRAP) was recently identified as an S-acylated protein in adipocytes and other tissues. However, there is currently no information on the extent of S-acylation of this protein, the residues that are modified, or the effects of S-acylation on IRAP localisation. In this study, we employ a semi-quantitative acyl-RAC technique to show that approximately 60% of IRAP is S-acylated in 3T3-L1 adipocytes. In contrast, S-acylation of GLUT4, a glucose transporter that extensively co-localises with IRAP, was approximately five-fold lower. Site-directed mutagenesis was employed to map the sites of S-acylation on IRAP to two cysteine residues, one of which is predicted to lie in the cytoplasmic side of the single transmembrane domain and the other which is just upstream of this transmembrane domain; our results suggest that these cysteines may be modified in a mutually-exclusive manner. Although S-acylation regulates the intracellular trafficking of several transmembrane proteins, we did not detect any effects of mutating the modified cysteines on the plasma membrane localisation of IRAP in HEK293T cells, suggesting that S-acylation is not essential for the movement of IRAP through the secretory pathway.

SUBMITTER: Werno MW

PROVIDER: S-EPMC4510526 | biostudies-other | 2015 Jul

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description: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.

Dataset Information

S-acylation of the Insulin-Responsive Aminopeptidase (IRAP): Quantitative analysis and Identification of Modified Cysteines.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure