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Cyclic AMP-dependent phosphoprotein components I and II interact with beta gamma subunits of transducin in frog rod
1Department of Physiology and Biophysics, University of Illinois at Chicago College of Medicine, 60612-7342, USA.
Abstract:
Components I and II (CI&II) in frog rod outer segments (ROS) are prominent cAMP-dependent protein kinase (PK-A) substrates. Their phosphorylation level is high in the dark, and illumination causes dephosphorylation. In order to understand their physiological role in phototransduction, biochemical characterization of CI&II phosphorylation was performed. Fractionation of phosphorylated ROS proteins showed that CI&II in the soluble fraction were highly phosphorylated by endogenous PK-A, whereas those in the membrane-associated protein fractions were not. The latter proteins could be phosphorylated by purified catalytic subunit of PK-A (PK-Acat) while the former proteins were not, suggesting that membrane-bound CI&II are normally much less phosphorylated. Treatments that dissociate the alpha subunit (alpha t) of transducin (Gt) from beta gamma subunits (beta gamma t) and thus produce excess free subunits of Gt in the soluble fraction caused inhibition of CI&II phosphorylation in the soluble fraction and enhancement of CI&II phosphorylation in the peripheral membrane fractions containing less Gt. Unphosphorylated CI&II tightly associated with the washed ROS membranes could be extracted after phosphorylation by PK-Acat. Phosphorylation also caused elution of beta gamma t from the membrane under the same conditions. Cross-linking by the maleimidobenzoyl-N-hydroxysuccinimide ester of the peripheral membrane fraction produced a distinct phosphorylated 50 kDa product with concurrent disappearance of the beta subunit of transducin (beta t) and phosphorylated CI&II. This phosphorylated cross-linked product was not recognized by a monoclonal anti-alpha t antibody but was recognized by antiserum against beta t, suggesting that the 50 kDa protein is a complex of beta gamma t and CI&II. Amino terminal sequencing of components I and II suggests that they are identical proteins with a unique sequence unrelated to other proteins in protein data bases. Phosphopeptide mapping of phosphorylated CI&II in the soluble fraction yielded two trypsinized phosphopeptides, while that in the peripheral membrane fractions showed only one phosphopeptide. These data suggest that multiple phosphorylation of CI&II alters their cellular localization. We conclude that phosphorylation of CI&II controls their localization in frog ROS and an interaction of CI&II with subunits of Gt regulates their phosphorylation.
Insights
Phosphorylation of Components I and II (CI&II) controls their location in frog rod outer segments (ROS). This process is regulated by interactions with transducin (Gt) subunits, impacting phototransduction.
Area of Science:
- Molecular Biology
- Cellular Biology
- Biochemistry
- Phototransduction
Background:
- Components I and II (CI&II) are key cAMP-dependent protein kinase (PK-A) substrates in frog rod outer segments (ROS).
- CI&II phosphorylation is high in the dark and decreases upon illumination, suggesting a role in phototransduction.
- Understanding the regulation and function of CI&II phosphorylation is crucial for elucidating phototransduction mechanisms.
Purpose of the Study:
- To biochemically characterize the phosphorylation of Components I and II (CI&II) in frog rod outer segments (ROS).
- To investigate the physiological role of CI&II phosphorylation in phototransduction.
- To determine how CI&II phosphorylation affects its localization and interaction with other proteins.
Main Methods:
- Fractionation of ROS proteins to separate soluble and membrane-associated components.
- In vitro phosphorylation assays using purified PK-A and ROS protein fractions.
- Treatment with subunits of transducin (Gt) to study effects on CI&II phosphorylation.
- Protein cross-linking and phosphopeptide mapping to identify protein complexes and phosphorylation sites.
Main Results:
- Soluble CI&II are highly phosphorylated by endogenous PK-A, while membrane-associated CI&II are less phosphorylated.
- Dissociation of transducin (Gt) subunits alters CI&II phosphorylation, inhibiting it in soluble fractions and enhancing it in membrane fractions.
- Phosphorylation of CI&II promotes its extraction from membranes and causes the elution of beta gamma t subunits.
- A 50 kDa cross-linked product suggests a complex between phosphorylated CI&II and beta gamma t subunits.
Conclusions:
- Phosphorylation of CI&II is a key regulator of its cellular localization within frog ROS.
- The interaction between CI&II and transducin (Gt) subunits modulates CI&II phosphorylation.
- These findings provide insights into the molecular mechanisms of phototransduction and the role of CI&II.