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ARF6 Activated by the LHCG Receptor through the Cytohesin Family of Guanine Nucleotide Exchange Factors Mediates the Receptor Internalization and Signaling

Venkat Kanamarlapudi Orcid Logo, Aiysha Thompson, Eamonn Kelly, Andres Lopez Bernal

Journal of Biological Chemistry, Volume: 287, Issue: 24, Start page: 20443

Swansea University Author: Venkat Kanamarlapudi Orcid Logo

DOI (Published version): 10.1074/jbc.M112.362087

Abstract

The luteinizing hormone chorionic gonadotropin receptor (LHCGR) is a G(s)-coupled GPCR that is essential for the maturation and function of the ovary and testis. LHCGR is internalized following its activation, which regulates the biological responsiveness of the receptor. Previous studies indicated...

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Published in: Journal of Biological Chemistry
Published: 2012
URI: https://cronfa.swan.ac.uk/Record/cronfa11277
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spelling 2019-07-16T13:54:32.7179999 v2 11277 2012-06-12 ARF6 Activated by the LHCG Receptor through the Cytohesin Family of Guanine Nucleotide Exchange Factors Mediates the Receptor Internalization and Signaling 63741801137148abfa4c00cd547dcdfa 0000-0002-8739-1483 Venkat Kanamarlapudi Venkat Kanamarlapudi true false 2012-06-12 BMS The luteinizing hormone chorionic gonadotropin receptor (LHCGR) is a G(s)-coupled GPCR that is essential for the maturation and function of the ovary and testis. LHCGR is internalized following its activation, which regulates the biological responsiveness of the receptor. Previous studies indicated that ADP-ribosylation factor (ARF)6 and its GTP-exchange factor (GEF) cytohesin 2 regulate LHCGR internalization in follicular membranes. However, the mechanisms by which ARF6 and cytohesin 2 regulate LHCGR internalization remain incompletely understood. Here we investigated the role of the ARF6 signaling pathway in the internalization of heterologously expressed human LHCGR (HLHCGR) in intact cells using a combination of pharmacological inhibitors, siRNA and the expression of mutant proteins. We found that human CG (HCG)-induced HLHCGR internalization, cAMP accumulation and ARF6 activation were inhibited by Gallein (βγ inhibitor), Wortmannin (PI 3-kinase inhibitor), SecinH3 (cytohesin ARF GEF inhibitor), QS11 (an ARF GAP inhibitor), an ARF6 inhibitory peptide and ARF6 siRNA. However, Dynasore (dynamin inhibitor), the dominant negative mutants of NM23-H1 (dynamin activator) and clathrin, and PBP10 (PtdIns 4,5-P2-binding peptide) inhibited agonist-induced HLHCGR and cAMP accumulation but not ARF6 activation. These results indicate that heterotrimeric G-protein, phosphatidylinositol (PI) 3-kinase (PI3K), cytohesin ARF GEF and ARF GAP function upstream of ARF6 whereas dynamin and clathrin act downstream of ARF6 in the regulation of HCG-induced HLHCGR internalization and signaling. In conclusion, we have identified the components and molecular details of the ARF6 signaling pathway required for agonist-induced HLHCGR internalization. Journal Article Journal of Biological Chemistry 287 24 20443 20 4 2012 2012-04-20 10.1074/jbc.M112.362087 COLLEGE NANME Biomedical Sciences COLLEGE CODE BMS Swansea University 2019-07-16T13:54:32.7179999 2012-06-12T19:23:59.1793501 Faculty of Medicine, Health and Life Sciences Swansea University Medical School - Medicine Venkat Kanamarlapudi 0000-0002-8739-1483 1 Aiysha Thompson 2 Eamonn Kelly 3 Andres Lopez Bernal 4 0011277-16072019135423.pdf ARF6LHRJBC12v2.pdf 2019-07-16T13:54:23.2330000 Output 4779397 application/pdf Not Applicable (or Unknown) true 2019-06-16T00:00:00.0000000 false eng
title ARF6 Activated by the LHCG Receptor through the Cytohesin Family of Guanine Nucleotide Exchange Factors Mediates the Receptor Internalization and Signaling
spellingShingle ARF6 Activated by the LHCG Receptor through the Cytohesin Family of Guanine Nucleotide Exchange Factors Mediates the Receptor Internalization and Signaling
Venkat Kanamarlapudi
title_short ARF6 Activated by the LHCG Receptor through the Cytohesin Family of Guanine Nucleotide Exchange Factors Mediates the Receptor Internalization and Signaling
title_full ARF6 Activated by the LHCG Receptor through the Cytohesin Family of Guanine Nucleotide Exchange Factors Mediates the Receptor Internalization and Signaling
title_fullStr ARF6 Activated by the LHCG Receptor through the Cytohesin Family of Guanine Nucleotide Exchange Factors Mediates the Receptor Internalization and Signaling
title_full_unstemmed ARF6 Activated by the LHCG Receptor through the Cytohesin Family of Guanine Nucleotide Exchange Factors Mediates the Receptor Internalization and Signaling
title_sort ARF6 Activated by the LHCG Receptor through the Cytohesin Family of Guanine Nucleotide Exchange Factors Mediates the Receptor Internalization and Signaling
author_id_str_mv 63741801137148abfa4c00cd547dcdfa
author_id_fullname_str_mv 63741801137148abfa4c00cd547dcdfa_***_Venkat Kanamarlapudi
author Venkat Kanamarlapudi
author2 Venkat Kanamarlapudi
Aiysha Thompson
Eamonn Kelly
Andres Lopez Bernal
format Journal article
container_title Journal of Biological Chemistry
container_volume 287
container_issue 24
container_start_page 20443
publishDate 2012
institution Swansea University
doi_str_mv 10.1074/jbc.M112.362087
college_str Faculty of Medicine, Health and Life Sciences
hierarchytype
hierarchy_top_id facultyofmedicinehealthandlifesciences
hierarchy_top_title Faculty of Medicine, Health and Life Sciences
hierarchy_parent_id facultyofmedicinehealthandlifesciences
hierarchy_parent_title Faculty of Medicine, Health and Life Sciences
department_str Swansea University Medical School - Medicine{{{_:::_}}}Faculty of Medicine, Health and Life Sciences{{{_:::_}}}Swansea University Medical School - Medicine
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description The luteinizing hormone chorionic gonadotropin receptor (LHCGR) is a G(s)-coupled GPCR that is essential for the maturation and function of the ovary and testis. LHCGR is internalized following its activation, which regulates the biological responsiveness of the receptor. Previous studies indicated that ADP-ribosylation factor (ARF)6 and its GTP-exchange factor (GEF) cytohesin 2 regulate LHCGR internalization in follicular membranes. However, the mechanisms by which ARF6 and cytohesin 2 regulate LHCGR internalization remain incompletely understood. Here we investigated the role of the ARF6 signaling pathway in the internalization of heterologously expressed human LHCGR (HLHCGR) in intact cells using a combination of pharmacological inhibitors, siRNA and the expression of mutant proteins. We found that human CG (HCG)-induced HLHCGR internalization, cAMP accumulation and ARF6 activation were inhibited by Gallein (βγ inhibitor), Wortmannin (PI 3-kinase inhibitor), SecinH3 (cytohesin ARF GEF inhibitor), QS11 (an ARF GAP inhibitor), an ARF6 inhibitory peptide and ARF6 siRNA. However, Dynasore (dynamin inhibitor), the dominant negative mutants of NM23-H1 (dynamin activator) and clathrin, and PBP10 (PtdIns 4,5-P2-binding peptide) inhibited agonist-induced HLHCGR and cAMP accumulation but not ARF6 activation. These results indicate that heterotrimeric G-protein, phosphatidylinositol (PI) 3-kinase (PI3K), cytohesin ARF GEF and ARF GAP function upstream of ARF6 whereas dynamin and clathrin act downstream of ARF6 in the regulation of HCG-induced HLHCGR internalization and signaling. In conclusion, we have identified the components and molecular details of the ARF6 signaling pathway required for agonist-induced HLHCGR internalization.
published_date 2012-04-20T03:12:58Z
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