title: Localization and regulation of thyrotropin receptors within lipid rafts.
abstract: The TSH receptor (TSHR) is a prototypic G protein-coupled receptor with a large extracellular domain. We have previously demonstrated homophilic interactions of TSHRs and their existence as constitutive oligomers. However, we have also shown that TSH itself promotes the formation of receptor monomers. We hypothesized, therefore, that TSHR monomers induced by TSH ligand may move into lipid rafts before effective TSH-induced signaling by bringing the cognate signaling molecules resident in such rafts together with the TSHRs. Thus, we aimed to determine whether the TSHRs would partition into these lipid rafts. The B subunit of cholera toxin (CTxB) binds to lipid raft-enriched GM1 ganglioside and has been widely exploited to visualize lipid rafts. Using such a method, we demonstrated the presence of these GM1-enriched lipid microdomains in Chinese hamster ovary cells by using CTxB labeled with a red dye (Alexa 594). To provide evidence for the presence of TSHRs in lipid rafts, we stained Chinese hamster ovary cells expressing TSHRGFP with labeled CTxB. Our results demonstrated that the TSHRGFP complexes localized to GM1-enriched lipid raft microdomains as evidenced by colocalization of the green fluorescent protein tag with the labeled CTxB. Hence, we concluded that a significant proportion of TSHRs were constitutively associated with lipid rafts. Furthermore, upon activation of these stained raft-receptor complexes with increasing concentrations of TSH, we observed that the raft-receptor complexes decreased significantly. The relevance of such receptor movement out of the rafts suggested that these may be the receptors critical in the initiation of signal transduction
authors.affiliation: Division of Endocrinolgy, Diabetes and Bone Diseases, Department of Medicine, Box 1055, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, New York 10029-6574, USA. rauf.latif@mssm.edu
authors.forename: R
authors.initials: R
authors.lastname: Latif
authors.forename: T
authors.initials: T
authors.lastname: Ando
authors.forename: S
authors.initials: S
authors.lastname: Daniel
authors.forename: T F
authors.initials: TF
authors.lastname: Davies
mesh_terms: D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents; D008024:Ligands; D050356:Lipid Metabolism; D008164:Luminescent Proteins; D011989:Receptors, Thyrotropin / Q000187:drug effects / Q000378:metabolism*; D013972:Thyrotropin / Q000494:pharmacology; D014018:Tissue Distribution
chemical_list: D007202:Indicators and Reagents; D008024:Ligands; D008164:Luminescent Proteins; D011989:Receptors, Thyrotropin; D049452:Green Fluorescent Proteins; D013972:Thyrotropin