Purification and functional characterization of β‐adrenergic receptor kinase expressed in insect cells

Abstract

The B-adrenerglc receptor kinase mediates agomst-dependent phosphorylatlon of /&adrenerglc receptors, which 1s thought to represent the first step of homologous desensitization.We have expressed bovine and human BARK1 in St9 cells and purified them to apparent homogeneity m milligram quantities. The &-values of the enzyme were 3.8 PM for rhodopsin and 22 PM for ATP: the r',,,,, -value was 9.9 mol phosphate/m01 BARK/mm.These data indicate that the two recombinant kmases were at least as active as preparations previously obtained from bovme brain There were no differences in the functional activity of human and bovine BARK.

Mutation of Asn293 to Asp in Transmembrane Helix VI Abolishes Agonist-Induced but Not Constitutive Activity of the β2-Adrenergic Receptor

“…These membrane preparations contained ␤ 2 AR levels of up to 150 pmol/mg protein. Recombinant bovine GRK2 was expressed in Sf9 insect cells and purified to Ͼ95% homogeneity as described earlier (Söhlemann et al, 1993).…”

Specific enhancement of beta-adrenergic receptor kinase activity by defined G-protein beta and gamma subunits.

“…Urea-treated rod outer segments and purified (32-adrenergic receptors reconstituted into phospholipid vesicles were phosphorylated by purified recombinant ,BARK essentially as described (37 Incubations were carried out at 30°C for 25 min in the presence of 10 ,uM (-)-isoproterenol. The samples were analyzed as stated above.…”

Selectivity of β‐adrenergic receptor kinase 2 for G protein βγ subunits

“…The expression and purification procedure for ßARK2 was adapted from that for ßARKl [19] and resulted in preparations of > 90% purity (not shown). Due to its higher isoelectric point, ßARK2 eluted at 140 mM NaCl from the CMFractogel matrix, whereas ßARKl already eluted at 100 mM NaCl [19].…”

“…Due to its higher isoelectric point, ßARK2 eluted at 140 mM NaCl from the CMFractogel matrix, whereas ßARKl already eluted at 100 mM NaCl [19]. The concentrations of ßARKl and 2 were determined from Coomassie blue R250-stained polyacrylamide gels in order to apply equal amounts in the functional assays.…”

“…Recombinant G protein ßy subunits, bovine ßARKl and ßARK2 were expressed using the baculovirus expression system [15,19]. The cDNAs for the y5 [20] and ^ [21] subunits were isolated from a bovine brain cDNA.…”