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Sphingolipid-Cholesterol Domains (Lipid Rafts) in Normal Human and Dog Thyroid Follicular Cells Are Not Involved in Thyrotropin Receptor Signaling

Institute of Interdisciplinary Research, Free University of Brussels, School of Medicine (M.J.C., Y.S., P.M., C.M., S.C., J.E.D., J.V.S., V.V.), B-1070 Brussels B-1070, Belgium; and Faculty of Medicine, University of Porto (M.J.C.), Porto 4200-319, Portugal; and Histology Unit, Catholic University of Leuven, Medical School (M.C.M.), B-1200 Brussels, Belgium

Address all correspondence and requests for reprints to: Dr. J. Van Sande, Institute of Interdisciplinary Research, Free University of Brussels, School of Medicine, Campus Erasme, 808 Route de Lennik, Building C, B-1070 Brussels, Belgium. E-mail: jvsande{at}ulb.ac.be.

Partition of signaling molecules in sphingolipid-cholesterol-enriched membrane domains, among which are the caveolae, may contribute to signal transduction efficiency. In normal thyroid, nothing is known about a putative TSH/cAMP cascade compartmentation in caveolae or other sphingolipid-cholesterol-enriched membrane domains. In this study we show for the first time that caveolae are present in the apical membrane of dog and human thyrocytes: caveolin-1 mRNA presence is demonstrated by Northern blotting in primary cultures and that of the caveolin-1 protein by immunohistochemistry performed on human thyroid tissue. The TSH receptor located in the basal membrane can therefore not be located in caveolae. We demonstrate for the first time by biochemical methods the existence of sphingolipid-cholesterol-enriched domains in human and dog thyroid follicular cells that contain caveolin, flotillin-2, and the insulin receptor. We assessed a possible sphingolipid-cholesterol-enriched domains compartmentation of the TSH receptor and the - subunit of the heterotrimeric G_s and G_q proteins using two approaches: Western blotting on detergent-resistant membranes isolated from thyrocytes in primary cultures and the influence of 10 mM methyl-ß-cyclodextrin, a cholesterol chelator, on basal and stimulated cAMP accumulation in intact thyrocytes. The results from both types of experiments strongly suggest that the TSH/cAMP cascade in thyroid cells is not associated with sphingolipid-cholesterol-enriched membrane domains.

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