Prolonged agonist stimulation does not alter the protein composition of membrane domains in spite of dramatic changes induced in a specific signaling cascade.
Protein composition of membrane domains prepared by three different procedures (mechanical homogenization, alkaline treatment with 1 M Na2CO3 [pH 11.0], or extraction with nonionic detergent Triton X-100), and isolated from the bulk of plasma membranes by flotation on equilibrium sucrose density gradients, was analyzed by two-dimensional (2D) electrophoresis and compared in preparations from control (quiescent) and agonist-stimulated human embryonic kidney cells (HEK)293 or S49 cells. HEK293 cells (clone e2m11) stably expressing high levels of thyrotropin-releasing hormone receptor and G11alpha protein were stimulated by thyrotropin-releasing hormone and S49 lymphoma cells by the beta-adrenergic receptor agonist isoprenaline. Whereas sustained exposure (16 h) of both cell lines to the appropriate hormones led to substantial cellular redistribution and downregulation of the cognate G proteins (G(q)alpha/G11alpha and G(s)alpha, respectively), the distribution and levels of nonstimulated G(i) proteins remained unchanged. The 2D electrophoretic analysis of membrane domains distinguished approx 150-170 major proteins in these structures and none of these proteins was significantly altered by prolonged agonist stimulation. Furthermore, specific immunochemical determination of a number of plasma membrane markers, including transmembrane and glycosyl-phosphatidylinositol-anchored peripheral proteins, confirmed that their detergent-extractability/solubility was not influenced by hormone treatment. Collectively, our present data indicate that sustained hormone stimulation of target cells does not alter the basic protein composition of membrane domain/raft compartments of the plasma membrane in spite of marked changes proceeding in a given signaling cascade.
Matousek
Petr
P
Institute of Physiology, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20, Czech Republic.
Novotny
Jirí
J
Rudajev
Vladimír
V
Svoboda
Petr
P
D000318:Adrenergic beta-Agonists / Q000494:pharmacology*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D015180:Electrophoresis, Gel, Two-Dimensional; D044385:GTP-Binding Protein alpha Subunits / Q000378:metabolism*; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology*; D007668:Kidney / Q000166:cytology; D008223:Lymphoma / Q000473:pathology; D008565:Membrane Proteins / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000187:drug effects; D015398:Signal Transduction; D013973:Thyrotropin-Releasing Hormone / Q000378:metabolism*
D000318:Adrenergic beta-Agonists; D044385:GTP-Binding Protein alpha Subunits; D008565:Membrane Proteins; D013973:Thyrotropin-Releasing Hormone; D007545:Isoproterenol