title: Selectivity of commonly used inhibitors of clathrin-mediated and caveolae-dependent endocytosis of G protein-coupled receptors.
abstract: Among the multiple G protein-coupled receptor (GPCR) endocytic pathways, clathrin-mediated endocytosis (CME) and caveolar endocytosis are more extensively characterized than other endocytic pathways. A number of endocytic inhibitors have been used to block CME; however, systemic studies to determine the selectivity of these inhibitors are needed. Clathrin heavy chain or caveolin1-knockdown cells have been employed to determine the specificity of various chemical and molecular biological tools for CME and caveolar endocytosis. Sucrose, concanavalin A, and dominant negative mutants of dynamin blocked other endocytic pathways, in addition to CME. In particular, concanavalin A nonspecifically interfered with the signaling of several GPCRs tested in the study. Decreased pH, monodansylcadaverine, and dominant negative mutants of epsin were more specific for CME than other treatments were. A recently introduced CME inhibitor, Pitstop2™, showed only marginal selectivity for CME and interfered with receptor expression on the cell surface. Blockade of receptor endocytosis by epsin mutants and knockdown of the clathrin heavy chain enhanced the β2AR-mediated ERK activation. Overall, our studies show that previous experimental results should be interpreted with discretion if they included the use of endocytic inhibitors that were previously thought to be CME-selective. In addition, our study shows that endocytosis of β2 adrenoceptor through clathrin-mediated pathway has negative effects on ERK activation.
authors.affiliation: Department of Pharmacology, College of Pharmacy, Chonnam National University, Gwang-Ju 500-757, Republic of Korea.
authors.forename: Shuohan
authors.initials: S
authors.lastname: Guo
authors.affiliation: Department of Pharmacology, College of Pharmacy, Chonnam National University, Gwang-Ju 500-757, Republic of Korea.
authors.forename: Xiaohan
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Pharmacology, College of Pharmacy, Chonnam National University, Gwang-Ju 500-757, Republic of Korea.
authors.forename: Mei
authors.initials: M
authors.lastname: Zheng
authors.affiliation: Department of Pharmacology, College of Pharmacy, Chonnam National University, Gwang-Ju 500-757, Republic of Korea.
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Pharmacology, College of Pharmacy, Chonnam National University, Gwang-Ju 500-757, Republic of Korea.
authors.forename: Chengchun
authors.initials: C
authors.lastname: Min
authors.affiliation: Department of Medicinal Chemistry, College of Pharmacy, Chonnam National University, Gwang-Ju 500-757, Republic of Korea.
authors.forename: Zengtao
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Department of Medicinal Chemistry, College of Pharmacy, Chonnam National University, Gwang-Ju 500-757, Republic of Korea.
authors.forename: Seung Hoon
authors.initials: SH
authors.lastname: Cheon
authors.affiliation: College of Pharmacy, Mokpo National University, Muan-gun, Jeollanamdo 534-729, Republic of Korea.
authors.forename: Min-Ho
authors.initials: MH
authors.lastname: Oak
authors.affiliation: Department of Physiology, College of Veterinary Medicine and Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, Republic of Korea.
authors.forename: Seung-Yeol
authors.initials: SY
authors.lastname: Nah
authors.affiliation: Department of Pharmacology, College of Pharmacy, Chonnam National University, Gwang-Ju 500-757, Republic of Korea. Electronic address: kmkim@jnu.ac.kr.
authors.forename: Kyeong-Man
authors.initials: KM
authors.lastname: Kim
mesh_terms: D000818:Animals; D019556:COS Cells; D021941:Caveolae / Q000378:metabolism*; D002522:Chlorocebus aethiops; D002966:Clathrin / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D057809:HEK293 Cells; D006801:Humans; D043562:Receptors, G-Protein-Coupled / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013449:Sulfonamides / Q000008:administration & dosage*; D053778:Thiazolidines / Q000008:administration & dosage*
chemical_list: D002966:Clathrin; D043562:Receptors, G-Protein-Coupled; D013449:Sulfonamides; D053778:Thiazolidines; C578512:pitstop 2
keywords: Caveolae; Clathrin; Endocytosis; GPCR; RNA interference