Cargando…

Constitutive Activation and Inactivation of Mutations Inducing Cell Surface Loss of Receptor and Impairing of Signal Transduction of Agonist-Stimulated Eel Follicle-Stimulating Hormone Receptor

In the present study, we investigated the signal transduction of mutants of the eel follicle-stimulating hormone receptor (eelFSHR). Specifically, we examined the constitutively activating mutant D540G in the third intracellular loop, and four inactivating mutants (A193V, N195I, R546C, and A548V). T...

Descripción completa

Detalles Bibliográficos
Autores principales: Byambaragchaa, Munkhzaya, Kim, Jeong-Soo, Park, Hong-Kyu, Kim, Dae-Jung, Hong, Sun-Mee, Kang, Myung-Hwa, Min, Kwan-Sik
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7583038/
https://www.ncbi.nlm.nih.gov/pubmed/32992880
http://dx.doi.org/10.3390/ijms21197075
_version_ 1783599329601126400
author Byambaragchaa, Munkhzaya
Kim, Jeong-Soo
Park, Hong-Kyu
Kim, Dae-Jung
Hong, Sun-Mee
Kang, Myung-Hwa
Min, Kwan-Sik
author_facet Byambaragchaa, Munkhzaya
Kim, Jeong-Soo
Park, Hong-Kyu
Kim, Dae-Jung
Hong, Sun-Mee
Kang, Myung-Hwa
Min, Kwan-Sik
author_sort Byambaragchaa, Munkhzaya
collection PubMed
description In the present study, we investigated the signal transduction of mutants of the eel follicle-stimulating hormone receptor (eelFSHR). Specifically, we examined the constitutively activating mutant D540G in the third intracellular loop, and four inactivating mutants (A193V, N195I, R546C, and A548V). To directly assess functional effects, we conducted site-directed mutagenesis to generate mutant receptors. We measured cyclic adenosine monophosphate (cAMP) accumulation via homogeneous time-resolved fluorescence assays in Chinese hamster ovary (CHO-K1) cells and investigated cell surface receptor loss using an enzyme-linked immunosorbent assay in human embryonic kidney (HEK) 293 cells. The cells expressing eelFSHR-D540G exhibited a 23-fold increase in the basal cAMP response without agonist treatment. The cells expressing A193V, N195I, and A548V mutants had completely impaired signal transduction, whereas those expressing the R546C mutant exhibited little increase in cAMP responsiveness and a small increase in signal transduction. Cell surface receptor loss in the cells expressing inactivating mutants A193V, R546C, and A548V was clearly slower than in the cell expressing the wild-type eelFSHR. However, cell surface receptor loss in the cells expressing inactivating mutant N195I decreased in a similar manner to that of the cells expressing the wild-type eelFSHR or the activating mutant D540G, despite the completely impaired cAMP response. These results provide important information regarding the structure–function relationships of G protein-coupled receptors during signal transduction.
format Online
Article
Text
id pubmed-7583038
institution National Center for Biotechnology Information
language English
publishDate 2020
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-75830382020-10-28 Constitutive Activation and Inactivation of Mutations Inducing Cell Surface Loss of Receptor and Impairing of Signal Transduction of Agonist-Stimulated Eel Follicle-Stimulating Hormone Receptor Byambaragchaa, Munkhzaya Kim, Jeong-Soo Park, Hong-Kyu Kim, Dae-Jung Hong, Sun-Mee Kang, Myung-Hwa Min, Kwan-Sik Int J Mol Sci Article In the present study, we investigated the signal transduction of mutants of the eel follicle-stimulating hormone receptor (eelFSHR). Specifically, we examined the constitutively activating mutant D540G in the third intracellular loop, and four inactivating mutants (A193V, N195I, R546C, and A548V). To directly assess functional effects, we conducted site-directed mutagenesis to generate mutant receptors. We measured cyclic adenosine monophosphate (cAMP) accumulation via homogeneous time-resolved fluorescence assays in Chinese hamster ovary (CHO-K1) cells and investigated cell surface receptor loss using an enzyme-linked immunosorbent assay in human embryonic kidney (HEK) 293 cells. The cells expressing eelFSHR-D540G exhibited a 23-fold increase in the basal cAMP response without agonist treatment. The cells expressing A193V, N195I, and A548V mutants had completely impaired signal transduction, whereas those expressing the R546C mutant exhibited little increase in cAMP responsiveness and a small increase in signal transduction. Cell surface receptor loss in the cells expressing inactivating mutants A193V, R546C, and A548V was clearly slower than in the cell expressing the wild-type eelFSHR. However, cell surface receptor loss in the cells expressing inactivating mutant N195I decreased in a similar manner to that of the cells expressing the wild-type eelFSHR or the activating mutant D540G, despite the completely impaired cAMP response. These results provide important information regarding the structure–function relationships of G protein-coupled receptors during signal transduction. MDPI 2020-09-25 /pmc/articles/PMC7583038/ /pubmed/32992880 http://dx.doi.org/10.3390/ijms21197075 Text en © 2020 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
Byambaragchaa, Munkhzaya
Kim, Jeong-Soo
Park, Hong-Kyu
Kim, Dae-Jung
Hong, Sun-Mee
Kang, Myung-Hwa
Min, Kwan-Sik
Constitutive Activation and Inactivation of Mutations Inducing Cell Surface Loss of Receptor and Impairing of Signal Transduction of Agonist-Stimulated Eel Follicle-Stimulating Hormone Receptor
title Constitutive Activation and Inactivation of Mutations Inducing Cell Surface Loss of Receptor and Impairing of Signal Transduction of Agonist-Stimulated Eel Follicle-Stimulating Hormone Receptor
title_full Constitutive Activation and Inactivation of Mutations Inducing Cell Surface Loss of Receptor and Impairing of Signal Transduction of Agonist-Stimulated Eel Follicle-Stimulating Hormone Receptor
title_fullStr Constitutive Activation and Inactivation of Mutations Inducing Cell Surface Loss of Receptor and Impairing of Signal Transduction of Agonist-Stimulated Eel Follicle-Stimulating Hormone Receptor
title_full_unstemmed Constitutive Activation and Inactivation of Mutations Inducing Cell Surface Loss of Receptor and Impairing of Signal Transduction of Agonist-Stimulated Eel Follicle-Stimulating Hormone Receptor
title_short Constitutive Activation and Inactivation of Mutations Inducing Cell Surface Loss of Receptor and Impairing of Signal Transduction of Agonist-Stimulated Eel Follicle-Stimulating Hormone Receptor
title_sort constitutive activation and inactivation of mutations inducing cell surface loss of receptor and impairing of signal transduction of agonist-stimulated eel follicle-stimulating hormone receptor
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7583038/
https://www.ncbi.nlm.nih.gov/pubmed/32992880
http://dx.doi.org/10.3390/ijms21197075
work_keys_str_mv AT byambaragchaamunkhzaya constitutiveactivationandinactivationofmutationsinducingcellsurfacelossofreceptorandimpairingofsignaltransductionofagoniststimulatedeelfolliclestimulatinghormonereceptor
AT kimjeongsoo constitutiveactivationandinactivationofmutationsinducingcellsurfacelossofreceptorandimpairingofsignaltransductionofagoniststimulatedeelfolliclestimulatinghormonereceptor
AT parkhongkyu constitutiveactivationandinactivationofmutationsinducingcellsurfacelossofreceptorandimpairingofsignaltransductionofagoniststimulatedeelfolliclestimulatinghormonereceptor
AT kimdaejung constitutiveactivationandinactivationofmutationsinducingcellsurfacelossofreceptorandimpairingofsignaltransductionofagoniststimulatedeelfolliclestimulatinghormonereceptor
AT hongsunmee constitutiveactivationandinactivationofmutationsinducingcellsurfacelossofreceptorandimpairingofsignaltransductionofagoniststimulatedeelfolliclestimulatinghormonereceptor
AT kangmyunghwa constitutiveactivationandinactivationofmutationsinducingcellsurfacelossofreceptorandimpairingofsignaltransductionofagoniststimulatedeelfolliclestimulatinghormonereceptor
AT minkwansik constitutiveactivationandinactivationofmutationsinducingcellsurfacelossofreceptorandimpairingofsignaltransductionofagoniststimulatedeelfolliclestimulatinghormonereceptor