Regulatory mechanism for the stability of the meta II intermediate of pinopsin.
Pinopsin is a chicken pineal photoreceptive molecule with a possible role in photoentrainment of the circadian clock. Sequence comparison among members of the rhodopsin family has suggested that pinopsin might have properties more similar to cone visual pigments than to rhodopsin, but the lifetime of the physiologically active intermediate (meta II) of pinopsin is rather similar to that of metarhodopsin II, which is far more stable than meta II intermediates of cone visual pigments [Nakamura, A. et al., (1999) Biochemistry 38, 14738-14745]. In the present study, we investigated the amino acid residue(s) contributing to this unique property of pinopsin by using site-directed mutagenesis to pinopsin-specific structural features, (i) Ser171, (ii) Asn184, and (iii) the second extracellular loop two-amino acids shorter than that of cone visual pigments. The meta II stability of the 171/184 double mutant of pinopsin (S171R/N184D) is almost the same as that of wild-type pinopsin. In contrast, the meta II lifetime is markedly shortened (one third) by introduction of the third mutation (replacement of a six-amino acid stretch, 188-193, by the corresponding eight residues of chicken green-sensitive cone pigment) to the 171/184 double mutant of pinopsin. Consistently, meta II of the green-sensitive pigment mutant, in which the eight-amino acid stretch is inversely replaced by the corresponding six residues of pinopsin, is more stable than meta II of the wild-type pigment. These results strongly suggest that the specific sequence and/or the number of residues at amino acids 188-193 in pinopsin play an important role in the stabilization of the meta II intermediate.
Nakamura
A
A
Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Hongo, Bunkyo-Ku, Tokyo 113-0033, Japan.
Kojima
D
D
Okano
T
T
Imai
H
H
Terakita
A
A
Shichida
Y
Y
Fukada
Y
Y
D019943:Amino Acid Substitution; D000818:Animals; D030161:Avian Proteins; D002645:Chickens; D002940:Circadian Rhythm / Q000502:physiology*; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics; D010786:Photoreceptor Cells / Q000737:chemistry / Q000502:physiology; D010870:Pineal Gland / Q000166:cytology; D017510:Protein Folding; D012168:Retinal Pigments / Q000737:chemistry* / Q000502:physiology; D012243:Rhodopsin / Q000031:analogs & derivatives* / Q000502:physiology; D017299:Rod Opsins / Q000737:chemistry* / Q000235:genetics
D030161:Avian Proteins; D009419:Nerve Tissue Proteins; D012168:Retinal Pigments; D012243:Rhodopsin; D017299:Rod Opsins; C090139:OPNP protein, chicken