An intact N terminus of the gamma subunit is required for the Gbetagamma stimulation of rhodopsin phosphorylation by human beta-adrenergic receptor kinase-1 but not for kinase binding.
Cleavage after lysine 32 in the Ggamma2 subtype and after lysine 36 in the Ggamma3 subtype of purified mixed brain Gbetagamma by endoproteinase Lys-C blocks Gbetagamma-mediated stimulation of phosphorylation of rhodopsin in urea-extracted rod outer segments by recombinant human beta-adrenergic receptor kinase (hbetaARK1) holoenzyme while hbetaARK1 binding to rod outer segments is partially affected. This treatment does not attenuate the binding of the treated Gbetagamma to C-terminal fragments of hbetaARK1 containing the pleckstrin homology domain. Lys-C proteolysis also does not alter the association of the Gbetagamma with phospholipids, its ability to support pertussis toxin-catalyzed Galphao/Galphai ADP-ribosylation, or its ability to inhibit forskolin-stimulated platelet adenylate cyclase. The Gbeta subunit remains noncovalently associated with the cleaved Ggamma fragments. Thus, in addition to recruiting hbetaARK1 to its receptor substrate, Ggamma contributes secondary and/or tertiary structural features to activate the kinase.
Haske
T N
TN
Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, Michigan 48105, USA.
DeBlasi
A
A
LeVine
H
H
D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000097:blood; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001792:Blood Platelets / Q000201:enzymology; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008239:Lysine; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D010743:Phospholipids / Q000378:metabolism; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000378:metabolism; D012243:Rhodopsin / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism; D051552:beta-Adrenergic Receptor Kinases
D000067956:Adenylyl Cyclase Inhibitors; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010743:Phospholipids; D011994:Recombinant Proteins; D012243:Rhodopsin; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D008239:Lysine