γ ₂ subunit of G protein heterotrimer is an N-end rule ubiquitylation substrate

Abstract

Heterotrimeric G proteins transduce signals from activated transmembrane G protein-coupled receptors to appropriate downstream effectors within cells. Signaling specificity is achieved in part by the specific ␣, ␤, and ␥ subunits that compose a given heterotrimer. Additional structural and functional diversity in these subunits is generated at the level of posttranslational modification, offering alternate regulatory mechanisms for G protein signaling. Presented here is the identification of a variant of the ␥ 2 subunit of G protein heterotrimer purified from bovine brain and the demonstration that this RDTASIA ␥2 variant, containing unique amino acid sequence at its N terminus, is a substrate for ubiquitylation and degradation via the N-end rule pathway. Although N-end-dependent degradation has been shown to have important functions in peptide import, chromosome segregation, angiogenesis, and cardiovascular development, the identification of cellular substrates in mammalian systems has remained elusive. The isolation of RDTASIA ␥2 from a native tissue represents identification of a mammalian N-end rule substrate from a physiological source, and elucidates a mechanism for the targeting of G protein ␥ subunits for ubiquitylation and degradation.T he G protein heterotrimer, composed of an ␣, ␤, and ␥ subunit, is complexed with the nucleotide GDP in its inactive state. Upon activation, GDP is exchanged for GTP, resulting in subunit dissociation, and the GTP-bound ␣ or the ␤␥ dimer can both subsequently modulate the activity of a variety of downstream effectors (1, 2). The ␥ subunit, although generally associated in the cell with a ␤ subunit, is the smallest and most variable heterotrimeric G protein subunit (3, 4) and has been the least well understood with regard to its specific biological function. Several well established lipid modifications of ␥ subunits include farnesylation or geranylgeranylation of the C terminus (5, 6), and these modifications play an important role in membrane association, receptor coupling, and effector regulation (7-9). Recent work suggests a role for the ␥ subunit of transducin in ubiquitylation and destabilization of G t ␤␥ dimers (10). Although it is unclear whether such observations can be extended to other distinct ␥ isoforms, the crucial question of how a ␥ subunit could be targeted to the ubiquitylation machinery in a regulated fashion also remains unanswered. The amino terminal region of ␥ subunits is the region most divergent between the 12 known ␥ isoforms (11), an observation which suggests that the first 20 amino acids may be crucial for a specific function or subcellular localization. Indeed, presented here is the identification of an N-terminal variant of the G␥ 2 subunit isolated from bovine brain. Our discovery that this RDTASIA ␥ 2 variant undergoes N-end rule degradation highlights regulated degradation as an important function for the ␥ subunit of the G protein heterotrimer, offering a mechanism by which G protein subunits can be targeted to enzymatic components of the...

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