Functional interaction between bovine rhodopsin and G protein transducin.
To elucidate the mechanisms of specific coupling of bovine rhodopsin with the G protein transducin (G(t)), we have constructed the bovine rhodopsin mutants whose second or third cytoplasmic loop (loop 2 or 3) was replaced with the corresponding loop of the G(o)-coupled scallop rhodopsin and investigated the difference in the activation abilities for G(t), G(o), and G(i) among these mutants and wild type. We have also prepared the Galpha(i) mutants whose C-terminal 11 or 5 amino acids were replaced with those of Galpha(t), Galpha(o), and Galpha(q) to evaluate the role of the C-terminal tail of the alpha-subunit on the specific coupling of bovine rhodopsin with G(t). Replacement of loop 2 of bovine rhodopsin with that of the scallop rhodopsin caused about a 40% loss of G(t) and G(o) activation, whereas that of loop 3 enhanced the G(o) activation four times with a 60% decrease in the G(t) activation. These results indicated that loop 3 of bovine rhodopsin is one of the regions responsible for the specific coupling with G(t). Loop 3 of bovine rhodopsin discriminates the difference of the 6-amino acid sequence (region A) at a position adjacent to the C-terminal 5 amino acids of the G protein, resulting in the different activation efficiency between G(t) and G(o). In addition, the binding of region A to loop 3 of bovine rhodopsin is essential for activation of G(t) but not G(i), even though the sequence of the region A is almost identical between Galpha(t) and Galpha(i). These results suggest that the binding of loop 3 of bovine rhodopsin to region A in Galpha(t) is one of the mechanisms of specific G(t) activation by bovine rhodopsin.
Terakita
Akihisa
A
Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.
Yamashita
Takahiro
T
Nimbari
Nozomu
N
Kojima
Daisuke
D
Shichida
Yoshinori
Y
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000502:physiology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000502:physiology; D008969:Molecular Sequence Data; D012243:Rhodopsin / Q000737:chemistry / Q000502:physiology*; D013329:Structure-Activity Relationship; D015293:Transducin / Q000502:physiology*
D016244:Guanosine 5'-O-(3-Thiotriphosphate); D012243:Rhodopsin; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020962:Heterotrimeric GTP-Binding Proteins; D015293:Transducin