High level functional expression of human beta 1-adrenergic receptor in baculovirus-infected cells screened by a rapid in situ procedure.
A novel screening assay for the identification of baculovirus infected cells expressing membrane receptors was developed by using a replica transfer technique. Sf9 cells were cotransfected with wild type baculoviral DNA and the transfer vector pVL941-beta 1 containing the coding region of the human beta 1-adrenergic receptor gene. Infected cells embedded in agarose were incubated with [125I]-iodocyanopindolol and transferred onto filters that were subsequently autoradiographed. This procedure resulted in the isolation of recombinant baculoviruses that expressed beta 1-adrenergic receptors. Binding assays carried out with [125I]-ICYP indicated that more than 600,000 receptors were expressed per cell, the highest level noted so far for this receptor in genetically engineered cells. Sf9 cells expressing the beta 1-AR were analysed by ligand binding, competition experiments, adenylyl cyclase stimulation and photoaffinity labeling. These cells express a homogenous population of receptors and display the known pharmacological properties of beta 1-AR in human tissues.
Ravet
V
V
Laboratoire d'Immuno-Pharmacologie Moléculaire, Centre National de la Recherche Scientifique, Paris, France.
Blin
N
N
Guillaume
J L
JL
Petitjean
F
F
Cabanié
L
L
Strosberg
A D
AD
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000502:physiology; D005822:Genetic Vectors; D006801:Humans; D009036:Moths / Q000235:genetics / Q000378:metabolism; D011943:Receptors, Adrenergic, beta / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D011994:Recombinant Proteins / Q000096:biosynthesis; D013997:Time Factors
D011943:Receptors, Adrenergic, beta; D011994:Recombinant Proteins; D000262:Adenylyl Cyclases