Structure and function in rhodopsin. Studies of the interaction between the rhodopsin cytoplasmic domain and transducin.
Structural requirements for the activation of transducin by rhodopsin have been studied by site-specific mutagenesis of bovine rhodopsin. A variety of single amino acid replacements and amino acid insertions and deletions of varying sizes were carried out in the two cytoplasmic loops CD (amino acids 134-151) and EF (amino acids 231-252). Except for deletion mutant delta 137-150, all the mutants bound 11-cis-retinal and displayed normal spectral characteristics. Deletion mutant delta 236-239 in loop EF caused a 50% reduction of transducin activation, whereas deletion mutant delta 244-249 and the larger deletions in loop EF abolished transducin activation. An 8-amino acid deletion in the cytoplasmic loop CD as well as a replacement of 13 amino acids with an unrelated sequence showed no transducin activation. Several single amino acid substitutions also caused significant reduction in transducin activation. The conserved charged pair Glu-134/Arg-135 in the cytoplasmic loop CD was required for transducin activation; its reversal or neutralization abolished transducin activation. Three amino acid replacements in loop EF (S240A, T243V, and K248L) resulted in significant reduction in transducin activation. We conclude that 1) both the cytoplasmic loops CD and EF are required for transducin activation, and 2) effective functional interaction between rhodopsin and transducin involves relatively large peptide sequences in the cytoplasmic loops.
Franke
R R
RR
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Sakmar
T P
TP
Graham
R M
RM
Khorana
H G
HG
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism*; D004072:Digitonin / Q000494:pharmacology; D004789:Enzyme Activation; D020558:GTP Phosphohydrolases / Q000378:metabolism; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012172:Retinaldehyde / Q000378:metabolism; D012243:Rhodopsin / Q000235:genetics* / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D015293:Transducin / Q000378:metabolism*
D012243:Rhodopsin; D020558:GTP Phosphohydrolases; D015293:Transducin; D004072:Digitonin; D012172:Retinaldehyde