Characterization of transducin from bovine retinal rod outer segments. Participation of the amino-terminal region of T alpha in subunit interaction.
The GTP-induced dissociation of T alpha from T beta gamma initiates the release of transducin from photolyzed rhodopsin and the subsequent activation of the cGMP phosphodiesterase. In this study, site-specific proteolysis and immunoprecipitation were used to map the domain of T alpha that interacts with T beta gamma. We found that Staphylococcus aureus V8 protease rapidly removes a small fragment from T alpha under native conditions, resulting in the formation of a single 38-kDa polypeptide (T alpha'). Under the same conditions, T beta gamma remains intact. A 4.5-fold decrease in the rate of T alpha cleavage by S. aureus protease was observed in the presence of T beta gamma, suggesting T beta gamma binding blocks the protease-sensitive site on T alpha. Amino acid sequence analysis indicated that T alpha' is derived from the cleavage of T alpha at Glu-21. The ability of T alpha' to interact with and activate the retinal phosphodiesterase is not diminished. However, T alpha' is unable to participate in T beta gamma-dependent activities such as the light-stimulated binding of guanine nucleotides, binding to photoexcited rhodopsin, and ADP-ribosylation catalyzed by pertussis toxin. Moreover, the anti-T alpha monoclonal antibody TF16 was able to precipitate T beta gamma in the presence of T alpha, but not with either T alpha' or T alpha-guanosine 5'-O-(3-thiotriphosphate). We conclude that the amino-terminal region of T alpha participates in T beta gamma interaction and discuss our results with respect to the known structure and function of transducin.
Navon
S E
SE
Jules Stein Eye Institute, UCLA School of Medicine 90024.
Fung
B K
BK
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D002417:Cattle; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000032:analysis*; D008666:Metalloendopeptidases; D037342:Pertussis Toxin; D010786:Photoreceptor Cells / Q000032:analysis*; D012374:Rod Cell Outer Segment / Q000032:analysis*; D013873:Thionucleotides / Q000494:pharmacology; D015293:Transducin; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D008565:Membrane Proteins; D013873:Thionucleotides; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D006165:Guanylyl Imidodiphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D037342:Pertussis Toxin; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus; D015293:Transducin