Residues within the polycationic region of cGMP phosphodiesterase gamma subunit crucial for the interaction with transducin alpha subunit. Identification by endogenous ADP-ribosylation and site-directed mutagenesis.
Interaction between the gamma subunit (Pgamma) of cGMP phosphodiesterase and the alpha subunit (Talpha) of transducin is a key step for the regulation of cGMP phosphodiesterase in retinal rod outer segments. Here we have utilized a combination of specific modification by an endogenous enzyme and site-directed mutagenesis of the Pgamma polycationic region to identify residues required for the interaction with Talpha. Pgamma, free or complexed with the alphabeta subunit (Palphabeta) of cGMP phosphodiesterase, was specifically radiolabeled by prewashed rod membranes in the presence of [adenylate-32P]NAD. Identification of ADP-ribose in the radiolabeled Pgamma and radiolabeling of arginine-replaced mutant forms of Pgamma indicate that both arginine 33 and arginine 36 are similarly ADP-ribosylated by endogenous ADP-ribosyltransferase, but only one arginine is modified at a time. Pgamma complexed with Talpha (both GTP- and GDP-bound forms) was not ADP-ribosylated; however, agmatine, which cannot interact with Talpha, was ADP-ribosylated in the presence of Talpha, suggesting that a Pgamma domain containing these arginines is masked by Talpha. A Pgamma mutant (R33,36K), as well as wild type Pgamma, inhibited both GTP hydrolysis of Talpha and GTP binding to Talpha. Moreover, GTP-bound Talpha activated Palphabeta that had been inhibited by R33,36K. However, another Pgamma mutant (R33,36L) could not inhibit these Talpha functions. In addition, GTP-bound Talpha could not activate Palphabeta inhibited by R33,36L. These results indicate that a Pgamma domain containing these arginines is required for its interaction with Talpha, but not with Palphabeta, and that positive charges in these arginines are crucial for the interaction.
Bondarenko
V A
VA
Kresge Eye Institute, Wayne State University, School of Medicine, Detroit, Michigan 48201, USA.
Desai
M
M
Dua
S
S
Yamazaki
M
M
Amin
R H
RH
Yousif
K K
KK
Kinumi
T
T
Ohashi
M
M
Komori
N
N
Matsumoto
H
H
Jackson
K W
KW
Hayashi
F
F
Usukura
J
J
Lipkin
V M
VM
Yamazaki
A
A
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000235:genetics / Q000378:metabolism*; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000378:metabolism; D001665:Binding Sites; D002417:Cattle; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D020558:GTP Phosphohydrolases / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D037342:Pertussis Toxin; D011892:Rana catesbeiana; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015293:Transducin / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000378:metabolism
D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; D009243:NAD; D006153:Guanosine Diphosphate; D000246:Adenosine Diphosphate Ribose; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D001120:Arginine; D037342:Pertussis Toxin; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D020558:GTP Phosphohydrolases; D015293:Transducin