Cargando…

Site-Directed Mutagenesis of the Fibronectin Domains in Insulin Receptor-Related Receptor

The orphan insulin receptor-related receptor (IRR), in contrast to its close homologs, the insulin receptor (IR) and insulin-like growth factor receptor (IGF-IR) can be activated by mildly alkaline extracellular medium. We have previously demonstrated that IRR activation is defined by its extracellu...

Descripción completa

Detalles Bibliográficos
Autores principales: Deyev, Igor E., Chachina, Natalia A., Zhevlenev, Egor S., Petrenko, Alexander G.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5713427/
https://www.ncbi.nlm.nih.gov/pubmed/29156593
http://dx.doi.org/10.3390/ijms18112461
_version_ 1783283423523110912
author Deyev, Igor E.
Chachina, Natalia A.
Zhevlenev, Egor S.
Petrenko, Alexander G.
author_facet Deyev, Igor E.
Chachina, Natalia A.
Zhevlenev, Egor S.
Petrenko, Alexander G.
author_sort Deyev, Igor E.
collection PubMed
description The orphan insulin receptor-related receptor (IRR), in contrast to its close homologs, the insulin receptor (IR) and insulin-like growth factor receptor (IGF-IR) can be activated by mildly alkaline extracellular medium. We have previously demonstrated that IRR activation is defined by its extracellular region, involves multiple domains, and shows positive cooperativity with two synergistic sites. By the analyses of point mutants and chimeras of IRR with IR in, we now address the role of the fibronectin type III (FnIII) repeats in the IRR pH-sensing. The first activation site includes the intrinsically disordered subdomain ID (646–716) within the FnIII-2 domain at the C-terminus of IRR alpha subunit together with closely located residues L135, G188, R244, H318, and K319 of L1 and C domains of the second subunit. The second site involves residue T582 of FnIII-1 domain at the top of IRR lambda-shape pyramid together with M406, V407, and D408 from L2 domain within the second subunit. A possible importance of the IRR carbohydrate moiety for its activation was also assessed. IRR is normally less glycosylated than IR and IGF-IR. Swapping both FnIII-2 and FnIII-3 IRR domains with those of IR shifted beta-subunit mass from 68 kDa for IRR to about 100 kDa due to increased glycosylation and abolished the IRR pH response. However, mutations of four asparagine residues, potential glycosylation sites in chimera IRR with swapped FnIII-2/3 domains of IR, decreased the chimera glycosylation and resulted in a partial restoration of IRR pH-sensing activity, suggesting that the extensive glycosylation of FnIII-2/3 provides steric hindrance for the alkali-induced rearrangement of the IRR ectodomain.
format Online
Article
Text
id pubmed-5713427
institution National Center for Biotechnology Information
language English
publishDate 2017
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-57134272017-12-07 Site-Directed Mutagenesis of the Fibronectin Domains in Insulin Receptor-Related Receptor Deyev, Igor E. Chachina, Natalia A. Zhevlenev, Egor S. Petrenko, Alexander G. Int J Mol Sci Article The orphan insulin receptor-related receptor (IRR), in contrast to its close homologs, the insulin receptor (IR) and insulin-like growth factor receptor (IGF-IR) can be activated by mildly alkaline extracellular medium. We have previously demonstrated that IRR activation is defined by its extracellular region, involves multiple domains, and shows positive cooperativity with two synergistic sites. By the analyses of point mutants and chimeras of IRR with IR in, we now address the role of the fibronectin type III (FnIII) repeats in the IRR pH-sensing. The first activation site includes the intrinsically disordered subdomain ID (646–716) within the FnIII-2 domain at the C-terminus of IRR alpha subunit together with closely located residues L135, G188, R244, H318, and K319 of L1 and C domains of the second subunit. The second site involves residue T582 of FnIII-1 domain at the top of IRR lambda-shape pyramid together with M406, V407, and D408 from L2 domain within the second subunit. A possible importance of the IRR carbohydrate moiety for its activation was also assessed. IRR is normally less glycosylated than IR and IGF-IR. Swapping both FnIII-2 and FnIII-3 IRR domains with those of IR shifted beta-subunit mass from 68 kDa for IRR to about 100 kDa due to increased glycosylation and abolished the IRR pH response. However, mutations of four asparagine residues, potential glycosylation sites in chimera IRR with swapped FnIII-2/3 domains of IR, decreased the chimera glycosylation and resulted in a partial restoration of IRR pH-sensing activity, suggesting that the extensive glycosylation of FnIII-2/3 provides steric hindrance for the alkali-induced rearrangement of the IRR ectodomain. MDPI 2017-11-19 /pmc/articles/PMC5713427/ /pubmed/29156593 http://dx.doi.org/10.3390/ijms18112461 Text en © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Deyev, Igor E.
Chachina, Natalia A.
Zhevlenev, Egor S.
Petrenko, Alexander G.
Site-Directed Mutagenesis of the Fibronectin Domains in Insulin Receptor-Related Receptor
title Site-Directed Mutagenesis of the Fibronectin Domains in Insulin Receptor-Related Receptor
title_full Site-Directed Mutagenesis of the Fibronectin Domains in Insulin Receptor-Related Receptor
title_fullStr Site-Directed Mutagenesis of the Fibronectin Domains in Insulin Receptor-Related Receptor
title_full_unstemmed Site-Directed Mutagenesis of the Fibronectin Domains in Insulin Receptor-Related Receptor
title_short Site-Directed Mutagenesis of the Fibronectin Domains in Insulin Receptor-Related Receptor
title_sort site-directed mutagenesis of the fibronectin domains in insulin receptor-related receptor
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5713427/
https://www.ncbi.nlm.nih.gov/pubmed/29156593
http://dx.doi.org/10.3390/ijms18112461
work_keys_str_mv AT deyevigore sitedirectedmutagenesisofthefibronectindomainsininsulinreceptorrelatedreceptor
AT chachinanataliaa sitedirectedmutagenesisofthefibronectindomainsininsulinreceptorrelatedreceptor
AT zhevlenevegors sitedirectedmutagenesisofthefibronectindomainsininsulinreceptorrelatedreceptor
AT petrenkoalexanderg sitedirectedmutagenesisofthefibronectindomainsininsulinreceptorrelatedreceptor