title: ARF6 activated by the LHCG receptor through the cytohesin family of guanine nucleotide exchange factors mediates the receptor internalization and signaling.
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 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.
authors.affiliation: Institute of Life Science, College of Medicine, Swansea University, Singleton Park, Swansea SA2 8PP, United Kingdom. k.venkateswarlu@swansea.ac.uk
authors.forename: Venkateswarlu
authors.initials: V
authors.lastname: Kanamarlapudi
authors.forename: Aiysha
authors.initials: A
authors.lastname: Thompson
authors.forename: Eamonn
authors.initials: E
authors.lastname: Kelly
authors.forename: Andrés
authors.initials: A
authors.lastname: López Bernal
mesh_terms: D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism*; D006063:Chorionic Gonadotropin / Q000235:genetics / Q000378:metabolism*; D002966:Clathrin / Q000235:genetics / Q000378:metabolism; D000242:Cyclic AMP / Q000235:genetics / Q000378:metabolism; D034281:Dynamins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D018260:Gelsolin / Q000235:genetics / Q000378:metabolism; D006801:Humans; D054778:NM23 Nucleoside Diphosphate Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011974:Receptors, LH / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*
chemical_list: D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors; D006063:Chorionic Gonadotropin; D002966:Clathrin; D000242:Cyclic AMP; D034281:Dynamins; D020690:GTPase-Activating Proteins; D018260:Gelsolin; D054778:NM23 Nucleoside Diphosphate Kinases; D010446:Peptide Fragments; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D011974:Receptors, LH; C102474:cytohesin-2; C527698:gelsolin (160-169); C000715348:ARF6 protein, human