Analysis of the molecular interaction of the farnesyl moiety of transducin through the use of a photoreactive farnesyl analogue.
Farnesylation of the gamma-subunit of the retinal G-protein, transducin (Talpha/Tbetagamma), is indispensable for light-initiated signaling in photoreceptor cells. However, the farnesyl-mediated molecular interactions important for signaling are not well understood. To explore this issue, we created a functional Tbetagamma analogue in which the farnesyl group was replaced with a (3-azidophenoxy)geranyl (POG) group, a novel farnesyl analogue with a distal photoreactive azido group. In the presence of lipid membranes and/or Talpha-GDP, UV irradiation of POG-modified Tbetagamma (POG-Tbetagamma) invariably yielded a cross-linked product Tgamma-Tbeta, reflecting a constitutive interaction of the Tgamma C-terminal lipid with Tbeta. In addition to the Tgamma-Tbeta adduct, a Tgamma-Talpha cross-link was detected in the aqueous fraction. Reconstitution of POG-Tbetagamma with Talpha and light-activated rhodopsin (Rh) in photoreceptor membranes resulted in cross-linking of Tgamma with a glycerophospholipid, indicating molecular interaction of the farnesyl group with cellular membranes. The Tgamma-phospholipid cross-link was observed only in the presence of both Talpha-GDP and Rh, and was abolished by the addition of GTPgammaS or by replacing Rh with opsin. These findings suggest a transient farnesyl-membrane interaction occurs only in a signaling state formed in a transducin-Rh ternary complex. On the other hand, UV irradiation of POG-Tbetagamma in a soluble complex with phosducin, a negative regulator of G-protein, yielded a Tgamma-phosducin adduct in addition to the Tgamma-Tbeta cross-link. These results illustrate that, rather than being a static membrane anchor, the farnesyl moiety plays an active role in the dynamics of protein-protein and protein-membrane interactions at defined steps in the signal transduction process.
Hagiwara
Ken'ichi
K
Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0032, Japan.
Wada
Akimori
A
Katadae
Maiko
M
Ito
Masayoshi
M
Ohya
Yoshikazu
Y
Casey
Patrick J
PJ
Fukada
Yoshitaka
Y
D000818:Animals; D002417:Cattle; D003979:Diazonium Compounds; D005136:Eye Proteins / Q000737:chemistry / Q000378:metabolism; D020555:GTP-Binding Protein Regulators; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D019920:Photoaffinity Labels / Q000378:metabolism; D011106:Polyisoprenyl Phosphates / Q000737:chemistry / Q000378:metabolism; D017368:Protein Prenylation; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012243:Rhodopsin / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D015293:Transducin / Q000737:chemistry* / Q000378:metabolism*; D014785:Vision, Ocular
D003979:Diazonium Compounds; D005136:Eye Proteins; D020555:GTP-Binding Protein Regulators; D010750:Phosphoproteins; D019920:Photoaffinity Labels; D011106:Polyisoprenyl Phosphates; D021122:Protein Subunits; D011994:Recombinant Proteins; C065631:phosducin; C015234:geranyl pyrophosphate; C046509:2-diazo-3-trifluoropropionyloxy geranyl pyrophosphate; D012243:Rhodopsin; D020962:Heterotrimeric GTP-Binding Proteins; D015293:Transducin