beta(2)-Adrenoceptor, Gs and adenylate cyclase coupling in purified detergent-resistant, low density membrane fractions

Oner S. S., Kaya A. I., ONARAN H. O., Ozcan G., Ugur O.

EUROPEAN JOURNAL OF PHARMACOLOGY, cilt.630, sa.1-3, ss.42-52, 2010 (SCI-Expanded) identifier identifier identifier

  • Yayın Türü: Makale / Tam Makale
  • Cilt numarası: 630 Sayı: 1-3
  • Basım Tarihi: 2010
  • Doi Numarası: 10.1016/j.ejphar.2009.12.035
  • Dergi Adı: EUROPEAN JOURNAL OF PHARMACOLOGY
  • Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus
  • Sayfa Sayıları: ss.42-52
  • Anahtar Kelimeler: Lipid rafts, Beta-adrenoceptor, Adenylate cyclase, Cyclodextrin, Lateral diffusion, Caveola, HETEROTRIMERIC G-PROTEINS, LIPID RAFTS, PLASMA-MEMBRANE, SIGNAL-TRANSDUCTION, CAVEOLAE MEMBRANE, SMOOTH-MUSCLE, CELL-SURFACE, RECEPTORS, CHOLESTEROL, LOCALIZATION
  • Ankara Üniversitesi Adresli: Evet

Özet

Membrane rafts and caveolae are specialized microdomains of the cell membrane that form physical platforms for compartmentalization of signalling molecules Here, we intended to gain insight into the consequences of caveolar localization in G protein-coupled receptor function We analysed beta(2)-adrenoceptor signalling in purified CRLDF (caveolin-rich low density fractions) of beta(2)-adrenoceptor-overexpressing HEK-293 cells beta(2)-adrenoceptor and Gs immunoreactivities and forskolin-stimulated adenylate cyclase activity were all detected in CRLDF obtained by the conventional raft purification method that uses Triton X-100 solubilization. However, Triton X-100 caused a complete loss of the functional Coupling between beta(2)-adrenoceptor, Gs and adenylate cyclase Therefore, we developed an optimized purification method based on n-octyl-beta-D-glucopyranoside solubilization. where the functional properties of beta(2)-adrenoceptor, Gs and adenylate cyclase were preserved in the CRLDF Using this method, we showed that isoproterenol-stimulated adenylate cyclase activity was similar in CRLDF and bulk membrane preparations of HEK-293 cells that overexpress beta(2)-adrenoceptor or beta(2)-adrenoceptor-Gs fusion Accordingly, treatment of cells with methyl-beta-cyclodextrin, a caveola-disrupting agent, did not affect beta(2)-adrenoceptor-induced cAMP response. Likewise, these responses were insensitive to caveolin 1 and 2 overexpression On the other hand, methyl-beta-cyclodextrin treatment did decrease beta(2)-adrenoceptor-induced ERK phosphorylation However. the latter effect of methyl-beta-cyclodextrin could be attributed to a non-specific effect rather than its ability to disrupt membrane microdomains We showed that localization in the raft microdomains did not affect the signalling efficiency of beta(2)-adrenoceptor-Gs-adenylate cyclase pathway, and that methyl-beta-cyclodextrin may inhibit signalling by directly affecting the signalling system independently of its caveola-disrupting property (C) 2009 Elsevier B V All rights reserved