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Journal of Biochemistry 2005 138(2):111-125; doi:10.1093/jb/mvi114
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© 2005 The Japanese Biochemical Society

Regular Paper

Dominant Portion of Thyrotropin-Releasing Hormone Receptor Is Excluded from Lipid Domains. Detergent-Resistant and Detergent-Sensitive Pools of TRH Receptor and Gq{alpha}/G11{alpha} Protein

Vladimir Rudajev1,2, Jiri Novotny1,2, Lucie Hejnova1,2, Graeme Milligan3 and Petr Svoboda1,2,3,*

1 Institute of Physiology, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic; 2 Department of Physiology, Faculty of Natural Sciences, Charles University, Vinicna 7, 120 00 Prague 2, Czech Republic; and 3 Department of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, UK

* To whom correspondence should be addressed at: Institute of Physiology, Czech Academy of Sciences, Videnska 1083, 142 20 Prague 4, Czech Republic. Phone: +420-2-41062478, Fax: +420-2-41062488, E-mail: svobodap{at}biomed.cas.cz

Some G protein–coupled receptors might be spacially targetted to discrete domains within the plasma membrane. Here we assessed the localization in membrane domains of the epitope-tagged, fluorescent version of thyrotropin-releasing hormone receptor (VSV-TRH-R-GFP) expressed in HEK293 cells. Our comparison of three different methods of cell fractionation (detergent extraction, alkaline treatment/sonication and mechanical homogenization) indicated that the dominant portion of plasma membrane pool of the receptor was totally solubilized by Triton X-100 and its distribution was similar to that of transmembrane plasma membrane proteins (glycosylated and non-glycosylated forms of CD147, MHCI, CD29, CD44, transmembrane form of CD58, Tapa1 and Na,K-ATPase). As expected, caveolin and GPI-bound proteins CD55, CD59 and GPI-bound form of CD58 were preferentially localized in detergent-resistant membrane domains (DRMs). Trimeric G proteins Gq{alpha}/G11{alpha}, Gi{alpha}1/Gi{alpha}2, Gs{alpha}L/Gs{alpha}S and Gß were distributed almost equally between detergent-resistant and detergent-solubilized pools. In contrast, VSV-TRH-R-GFP, G{alpha}, Gß and caveolin were localized massively only in low-density membrane fragments of plasma membranes, which were generated by alkaline treatment/sonication or by mechanical homogenization of cells. These data indicate that VSV-TRH-R-GFP as well as other transmembrane markers of plasma membranes are excluded from TX-100–resistant, caveolin-enriched membrane domains. Trimeric G protein Gq{alpha}/G11{alpha} occurs in both DRMs and in the bulk of plasma membranes, which is totally solubilized by TX-100.


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