Characterization of post translational modification of heterotrimeric G proteins

Lupi, Rosita (2001). Characterization of post translational modification of heterotrimeric G proteins. PhD thesis The Open University.



Mono-ADP-ribosylation is a post translational modification of cellular proteins, which has been implicated in the regulation of signal transduction, muscle cell differentiation, protein trafficking and secretion. However, the physiological role of the ADP-ribosylation reaction in intact cells remain to be clarified.

We demonstrated that the β subunit of the heterotrimeric G proteins is endogenously mono-ADP-ribosylated in intact cells. This modification occurs when the βɣ dimer is in its active dimeric conformation (the addition of increasing concentrations of GDP-αi3 in the ADP-ribosylation assay mixture results in the inhibition of the β subunit labelling), while the enzyme catalyzing this reaction is a membrane-associated ADP-ribosyltransferase that modifies arginine 129. Interestingly, this amino acid is located in the N-terminal region, which is involved in the interaction with the α subunit and with βɣ effector enzymes. The experiments described in this thesis demonstrate that the βɣ dimer, once modified, loses its ability to inhibit the calmodulin-stimulated type 1 adenylyl cyclase (Lupi et al., J. Biol. Chem., 2000) and to stimulate phospholipase Cβ2 in in vitro assays. ADP-ribosylated β subunit is deribosylated by a cytosolic hydrolase completing an ADP-ribosylation/deribosylation cycle that might thus modulate the interaction of βɣ with specific effectors.

Heterotrimeric G proteins play a key role in cell regulation, with a growing body of evidence demonstrating that the βɣ complex participates in a wide range of G protein function. Our finding of an ADP-ribosylation/deribosylation cycle acting on the βɣ subunits delineates a novel molecular mechanism that could provide crucial control of G protein-mediated signalling pathways.

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