Cone photoreceptor betagamma-transducin: posttranslational modification and interaction with phosducin.
OBJECTIVE
To characterize the structure of cone betagamma-transducin (Tbeta3gamma8) and its interaction with phosducin (pdc).


METHODS
The Tgamma8 subunit of Tbeta3gamma8 was isolated by column chromatography for peptide mapping with mass spectrometry. Tbeta3gamma8 was compared with rod betagamma-transducin (Tbeta1gamma1) in terms of the electrophoretic mobility, pdc binding affinity, and the effects of phosphorylation and methylation, and then the correlation to the crystal structures and functional domains of Tbeta1gamma1 was determined.


RESULTS
The mature Tgamma8 is a 65-amino-acid peptide encoded by the Ggamma8 gene with an acetylated and a farnesylated-methylated N- and C-terminus, respectively. Purified Tbeta3gamma8 is similar to Tbeta1gamma1 in that (1) both are heterogeneous, containing methylated and demethylated Tgamma subunits; (2) each demethylated dimer migrates faster than its methylated counterpart during native gel electrophoresis, and the methylation-associated mobility differential is masked by pdc binding; and (3) both dimers bind pdc with the same affinity, and the affinity is reduced threefold by PKA phosphorylation of pdc and twofold by demethylation at the C-terminus of Tgamma. Tbeta3gamma8 differs from Tbeta1gamma1 in exhibiting lower intrinsic electrophoretic mobility, and the difference is unaffected by either pdc binding or the status of Tgamma methylation.


CONCLUSIONS
Tbeta3gamma8 is identical with Tbeta1gamma1 in Tgamma isoprenylation, the spatial organization, and the mode of pdc binding, indicating that its interaction with pdc does not play an important role in the specialization of cones. Changes in Tbetagamma characteristics by Tgamma methylation reveal conformational changes on a surface domain that is essential for Tbetagamma functions and support a regulatory role for reversible methylation.
Chen
Fayu
F
Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, California, USA.
Ng
Pei-San
PS
Faull
Kym F
KF
Lee
Rehwa H
RH
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins / Q000378:metabolism*; D020555:GTP-Binding Protein Regulators; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000378:metabolism*; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational; D017949:Retinal Cone Photoreceptor Cells / Q000502:physiology*; D017948:Retinal Rod Photoreceptor Cells / Q000502:physiology; D021241:Spectrometry, Mass, Electrospray Ionization; D015293:Transducin / Q000378:metabolism*
D005136:Eye Proteins; D020555:GTP-Binding Protein Regulators; D010750:Phosphoproteins; C065631:phosducin; D015293:Transducin