Regulation of retinal cGMP cascade by phosducin in bovine rod photoreceptor cells. Interaction of phosducin and transducin.
Photoexcitation of retinal rod photoreceptor cells involves the activation of cGMP enzyme cascade in which sequential activation of rhodopsin, transducin, and the cGMP phosphodiesterase in the rod outer segment constitutes the signal amplification mechanism. Phosducin, a 33-kDa phosphoprotein, has been shown to form a tight complex with the T beta gamma subunit of transducin. In this study, we examined the interaction of phosducin-T beta gamma and the possible regulatory role of phosducin on the cGMP cascade. Addition of phosducin to photolyzed rod outer segment (ROS) membrane reduced the GTP hydrolysis activity of transducin as well as the subsequent activation of the cGMP phosphodiesterase. Phosducin also inhibited the pertussis toxin-catalyzed ADP-ribosylation of transducin, indicating that the interaction between the T alpha and T beta gamma subunits of transducin was interrupted upon binding of phosducin. The inhibitory effects of phosducin were reversed by the addition of exogenous T beta gamma. These results suggest that phosducin is capable of regulating the amount of T beta gamma available to interact with T alpha to form the active transducin complex and thereby functions as a negative regulator of the cGMP cascade. The phosducin-induced alteration of the subunit organization of transducin was examined by chemical cross-linking method using para-phenyl dimaleimide as cross-linker. It was found that the cross-linking among T alpha and T beta gamma was blocked in the presence of phosducin. This result implies that T beta gamma may undergo a conformational change upon phosducin binding which leads to the release of T alpha. Since phosducin is a soluble protein, the interaction with transducin only occurs when transducin is dissociated from ROS disc membrane. Indeed, phosducin failed to dissociate membrane-bound transducin and did not inhibit the initial cycle of transducin activation as measured by the presteady state GTP hydrolysis. However, phosducin interacts effectively with transducin released into solution after the initial activation and blocks the re-binding of T alpha. T beta gamma to ROS membrane by forming a tight complex with T beta gamma. This interaction may play an important role in regulating the turnover of the cGMP cascade in photoreceptor cells.
Lee
R H
RH
Department of Anatomy and Cell Biology, UCLA School of Medicine.
Ting
T D
TD
Lieberman
B S
BS
Tobias
D E
DE
Lolley
R N
RN
Ho
Y K
YK
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D006152:Cyclic GMP / Q000378:metabolism*; D005136:Eye Proteins / Q000302:isolation & purification / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000378:metabolism; D020555:GTP-Binding Protein Regulators; D019204:GTP-Binding Proteins / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism*; D037342:Pertussis Toxin; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D012374:Rod Cell Outer Segment / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015293:Transducin / Q000302:isolation & purification / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D005136:Eye Proteins; D020555:GTP-Binding Protein Regulators; D046911:Macromolecular Substances; D010750:Phosphoproteins; D010566:Virulence Factors, Bordetella; C065631:phosducin; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D006160:Guanosine Triphosphate; D037342:Pertussis Toxin; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D015293:Transducin; D006152:Cyclic GMP