Effect of transmembrane Ca2+ gradient on the coupling of beta-adrenergic receptors and adenylyl cyclase.
In order to investigate the effect of transmembrane Ca2+ gradient on Gs mediated coupling of beta-AR and adenylyl cyclase, beta-AR from duck erythrocytes and Gs and adenylyl cyclase from bovine brain cortices were co-reconstituted into asolectin liposomes with different transmembrane Ca2+ gradient. These proteoliposomes were proven to be impermeable to water-soluble substances. The results obtained indicate that a physiological transmembrane Ca2+ gradient (1000-fold) is essential for higher stimulation of adenylyl cyclase by hormone-activated beta-AR via coupling to Gs and can be further enhanced by the decrease of such Ca2+ gradient within certain range (100 fold) following Ca2+ influx into cells during signal transduction. Fluorescence polarization of DPH revealed that transmembrane Ca2+ gradient modulates adenylyl cyclase and its stimulation by hormones through mediating a change in lipid fluidity. Correspondent conformational changes of beta-AR were also detected from the fluorescence spectra and quenching of Acrylodan-labelled beta-AR in those proteoliposomes. It is suggested that a proper transmembrane Ca2+ gradient is essential for the optimal fluidity of the phospholipid bilayer in the proteoliposomes, which favors the formation of a suitable conformation of the reconstituted beta-AR and thus promotes the stimulation of adenylyl cyclase activities by hormone-activated beta-AR via Gs.
Fan
G F
GF
National Laboratory of Biomacromolecules, Chinese Academy of Sciences, Beijing, China.
Yang
X Y
XY
Huang
Y G
YG
Yang
F Y
FY
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D004372:Ducks; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D010539:Permeability; D010713:Phosphatidylcholines; D010743:Phospholipids / Q000378:metabolism; D011510:Proteolipids / Q000378:metabolism; D011943:Receptors, Adrenergic, beta / Q000737:chemistry / Q000378:metabolism*
D008081:Liposomes; D010713:Phosphatidylcholines; D010743:Phospholipids; D011510:Proteolipids; D011943:Receptors, Adrenergic, beta; C020485:proteoliposomes; C024990:asolectin; D019205:GTP-Binding Protein alpha Subunits, Gs; D000262:Adenylyl Cyclases; D002118:Calcium