Probing functional interfaces of rod PDE gamma-subunit using scanning fluorescent labeling.
In the dark, the activity of the rod cGMP phosphodiesterase (PDE) catalytic alpha- and beta-subunits (P alpha beta) is inhibited by two gamma-subunits (P gamma). On light stimulation of the photoreceptor cells, the GTP-bound alpha-subunit of visual G-protein transducin (GtaGTP) displaces the P gamma-subunits from their inhibitory sites on P alpha beta, leading to the effect or enzyme activation. We designed a number of P gamma mutants, each with a single cysteine residue evenly distributed at a different position along the P gamma polypeptide chain. These cysteine residues served as sites for the introduction of the environmentally sensitive fluorescent probe, 3-(bromoacetyl)-7-diethyl aminocoumarin (BC). Analysis of the interactions of P alpha beta and Gta with the fluorescently labeled P gamma mutants suggests two distinct functional interfaces of P gamma. The P alpha beta/P gamma interface is formed essentially by the C-terminus of P gamma and by the N-terminal portion of the P gamma polycationic region, P gamma-24-45, whereas the P gamma/Gta interface includes the C-terminal portion of P gamma-24-45 and the region surrounding P gamma Cys68. Such functional organization of P gamma may represent an important element for the PDE activation mechanism during transduction of visual signals.
Granovsky
A E
AE
Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City 52242.
McEntaffer
R
R
Artemyev
N O
NO
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D003067:Coenzymes / Q000378:metabolism; D003374:Coumarins; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D003545:Cysteine; D004789:Enzyme Activation; D005456:Fluorescent Dyes; D009154:Mutation; D010446:Peptide Fragments / Q000378:metabolism; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011485:Protein Binding; D012374:Rod Cell Outer Segment / Q000201:enzymology*; D013050:Spectrometry, Fluorescence / Q000379:methods; D015293:Transducin / Q000378:metabolism; D014357:Trypsin
C105553:3-bromoacetyl-7-(diethylamino)coumarin; D003067:Coenzymes; D003374:Coumarins; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D010726:Phosphodiesterase Inhibitors; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D014357:Trypsin; D015293:Transducin; D003545:Cysteine