High level functional expression of human beta 1-adrenergic receptor in baculovirus-infected cells screened by a rapid in situ procedure

J Recept Res. 1993;13(1-4):541-58. doi: 10.3109/10799899309073678.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenylyl Cyclases / metabolism
  • Animals
  • Baculoviridae / genetics*
  • Cells, Cultured
  • Enzyme Activation / physiology
  • Genetic Vectors
  • Humans
  • Moths / genetics
  • Moths / metabolism
  • Receptors, Adrenergic, beta / biosynthesis*
  • Receptors, Adrenergic, beta / genetics
  • Receptors, Adrenergic, beta / physiology
  • Recombinant Proteins / biosynthesis
  • Time Factors

Substances

  • Receptors, Adrenergic, beta
  • Recombinant Proteins
  • Adenylyl Cyclases