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Localization and Regulation of Thyrotropin Receptors within Lipid Rafts

Division of Endocrinology, Diabetes and Bone Diseases, Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029

Address all correspondence and requests for reprints to: Dr. R. Latif, Department of Medicine, Box 1055, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, New York 10029-6574. E-mail: rauf.latif{at}mssm.edu.

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 G_M1 ganglioside and has been widely exploited to visualize lipid rafts. Using such a method, we demonstrated the presence of these G_M1-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 TSHR_GFP with labeled CTxB. Our results demonstrated that the TSHR_GFP complexes localized to G_M1-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