The role of cytosolic and membrane factors in processing of the human beta-2 adrenergic receptor following translocation and glycosylation in a cell-free system.
The beta-2 adrenergic receptor has been proposed to have seven membrane-spanning domains. Expression of functional beta-2 adrenergic receptor was achieved in a heterologous cell-free system composed of rabbit reticulocyte lysate and microsomal membranes from Xenopus laevis oocytes. The functional state of the receptor protein can be determined by ligand-binding assays and by the ability of ligands to alter the susceptibility of the receptor to proteinase K digestion. The process by which functional receptor is made was studied. The receptor protein remains nonfunctional immediately following translocation and glycosylation, and additional processing steps are needed before the receptor is able to interact with ligands. These processing steps require intact microsomal membranes as well as several cytosolic factors including ATP and one or more high molecular mass (greater than 30 kDa) factors but do not require receptor glycosylation and are not inhibited by nonhydrolyzable GTP analogues.
Kobilka
B K
BK
Department of Medicine (Cardiology), Duke University Medical Center, Durham, North Carolina 27710.
D000818:Animals; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism*; D002474:Cell-Free System; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D010957:Plasmids; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D012313:RNA / Q000235:genetics; D011817:Rabbits; D011943:Receptors, Adrenergic, beta / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism
D011943:Receptors, Adrenergic, beta; D012313:RNA; D004247:DNA