Characterization of beta-adrenergic receptor sequestration by newly developed whole cell binding assays.
Upon agonist binding, beta-adrenergic receptors sequestrate from the cell surface plasma membrane to cytosol. In the present study, we examine the kinetics of sequestration of beta1-adrenergic receptor and beta3-adrenergic receptor subtypes by radioligand binding assays using whole cells ('whole cell binding assays'). We found that HEK293T cells, but not COS1 cells, were readily and uniformly detached from the culture dish upon exposure to ice-cold phosphate-buffered saline. Using this property of HEK293T cells, we conducted whole cell binding assays using a hydrophilic antagonist ([3H]CGP-12177) and HEK293T cells transiently overexpressing human beta1-adrenergic receptor or beta3-adrenergic receptor. The Bmax and Kd values were 5.96 +/- 0.97 pmol/mg protein and 1 +/- 0.23 nM for the beta1-adrenergic receptor, and were 1.84 +/- 0.13 pmol/mg protein and 44.7 +/- 2.5 nM for the beta3-adrenergic receptor, respectively. Isoproterenol treatment, but not 6-[3-(dimethylamino)propionyl]forskolin treatment, for 2 h resulted in a dose-dependent loss of the number of the cell surface beta1-adrenergic receptor. At 100 microM, 36.6 +/- 5.7% of the cell surface beta1-adrenergic receptor was lost. In contrast, the cell surface beta3-adrenergic receptor number remained unchanged with isoproterenol treatment. Thus, beta1-adrenergic receptor sequestrates upon agonist stimulation but the same agonist stimulation does not induce beta3-adrenergic receptor sequestration, as demonstrated by our whole cell binding assays.
Iwatsubo
Kousaku
K
Department of Physiology, Yokohama City University School of Medicine, Kanazawa-Ku, Yokohama, Japan.
Tao
Yang
Y
Onda
Takeshi
T
Toya
Yoshiyuki
Y
Schwencke
Carsten
C
Fujita
Takayuki
T
Ebina
Toshiaki
T
Iwamoto
Tamio
T
Hori
Hideaki
H
Minamisawa
Susumu
S
Umemura
Satoshi
S
Ishikawa
Yoshihiro
Y
D000818:Animals; D019556:COS Cells; D002452:Cell Count / Q000379:methods; D002522:Chlorocebus aethiops; D006801:Humans; D011412:Propanolamines / Q000378:metabolism; D011869:Radioligand Assay / Q000379:methods*; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*
D011412:Propanolamines; D011943:Receptors, Adrenergic, beta; C033394:CGP 12177