title: Role of flotillins in the endocytosis of GPCR in salivary gland epithelial cells.
abstract: Endocytosis has numerous functions in cellular homeostasis. Defects in the endocytic pathway of receptors may lead to dysfunction of salivary gland secretion. Therefore, elucidating the complex mechanisms of endocytosis may facilitate solutions for disease treatment and prevention. The muscarinic type 3 receptor (M3R), a G-protein-coupled receptor (GPCR) located in the plasma membrane, is involved in numerous physiological activities such as smooth muscle contraction and saliva secretion. M3R enters cells through clathrin-mediated endocytosis (CME), while flotillins (flot-1 and -2), highly conserved proteins residing in lipid-raft microdomains, make use of clathrin-independent endocytosis (CIE) for their internalization. Since these two proteins use two discrete pathways for endocytic entry, the association of flotillins with CME is poorly understood. We examined whether flotillins play a role in CME of M3R using immunoblotting, immunocytochemistry, confocal immunofluorescence microscopy, co-immunoprecipitation, and RNA interference techniques in secretory epithelial cells. Upon stimulation with a cholinergic agonist, M3R, flot-1, and flot-2 each internalized from the plasma membrane into intracellular sites. The addition of chlorpromazine and cytochalasin D, well-known inhibitors of CME, inhibited internalization of M3R via CME. Filipin III and methyl-β-cyclodextrin (mβCD) acting as lipid raft inhibitors disrupted internalization of flot-1/2 via CIE. Interestingly, filipin III and mβCD slightly reduced expression level of M3R whereas chlorpromazine and cytochalasin D did not affect internalization of the flotillin isoforms. M3R and flot-1/2 colocalized and interacted with each other as they entered the cytosol during limited periods of incubation. Moreover, knockdown of flot-1 or -2 by flotillin-specific siRNA prevented internalization and reduced the endocytic efficiency of M3R. Our results suggest that flot-1 and -2 are partially involved in CME of M3R by facilitating its internalization.
authors.affiliation: Department of Physiology, School of Dentistry, Seoul National University and Dental Research Institute, Seoul 110-749, Republic of Korea.
authors.forename: Moon-Yong
authors.initials: MY
authors.lastname: Park
authors.affiliation: Department of Physiology, School of Dentistry, Seoul National University and Dental Research Institute, Seoul 110-749, Republic of Korea.
authors.forename: Nahyun
authors.initials: N
authors.lastname: Kim
authors.affiliation: Department of Physiology and Pathophysiology, Peking University Health Science Center, Beijing 100191, China.
authors.forename: Li-Ling
authors.initials: LL
authors.lastname: Wu
authors.affiliation: Department of Oral and Maxillofacial Surgery, Peking University School and Hospital of Stomatology, Beijing 100081, China.
authors.forename: Guang-Yan
authors.initials: GY
authors.lastname: Yu
authors.affiliation: Department of Physiology, School of Dentistry, Seoul National University and Dental Research Institute, Seoul 110-749, Republic of Korea. Electronic address: kppark@snu.ac.kr.
authors.forename: Kyungpyo
authors.initials: K
authors.lastname: Park
mesh_terms: D002460:Cell Line; D004705:Endocytosis; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism*; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D043587:Receptor, Muscarinic M3 / Q000032:analysis / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; D034741:RNA, Small Interfering; D043587:Receptor, Muscarinic M3; D043562:Receptors, G-Protein-Coupled; C106354:flotillins
keywords: Colocalization; Endocytosis; Flotillin; GPCR; Internalization; Lipid-raft