The expanding roles of Gßγ subunits in G protein-coupled receptor signaling and drug action.

Gßγ subunits from heterotrimeric G proteins perform a vast array of functions in cells with respect to signaling, often independently as well as in concert with Gα subunits. However, the eponymous term "Gßγ" does not do justice to the fact that 5 Gß and 12 Gγ isoforms have evolved in mammals to serve much broader roles beyond their canonical roles in cellular signaling. We explore the phylogenetic diversity of Gßγ subunits with a view toward understanding these expanded roles in different cellular organelles. We suggest that the particular content of distinct Gßγ subunits regulates cellular activity, and that the granularity of individual Gß and Gγ action is only beginning to be understood. Given the therapeutic potential of targeting Gßγ action, this larger view serves as a prelude to more specific development of drugs aimed at individual isoforms.

A novel, radiolabel-free pulse chase strategy to study Kir3 channel ontogeny.

Kir3 channels are essential regulators of cellular excitability, maintaining cells at resting membrane potentials. While much research has been dedicated to elucidating the mechanisms regulating Kir3 channel gating, little is known regarding the channel's early associations with signaling partners, its stability at the plasma membrane or mechanisms regulating its internalization and degradation. To address these issues we have established an inducible Kir3.1 cell line that allows monitoring of a discrete "pulse" of channel as it progresses along the biosynthetic pathway. Using this system, we have been able to track Kir3 maturation and the influence of partner subunits on Kir3 lifetime and stability. Of note, we show that Kir3.1, in the absence of trafficking partner subunits, can exit the endoplasmic reticulum (ER) and reach the Golgi (though not the plasma membrane), and that expression of Kir3.3 subunits drastically reduced levels of Kir3.1 in the cell. We also show that interfering with trafficking from the ER to Golgi has a pronounced inhibitory effect on Kir3.1-Kir3.2 interactions, suggesting that this complex is stabilized either en route to the Golgi or in the Golgi itself. Finally, we showed that the Kir3 channel can reach the cell surface as early as 6 h post-induction and that removal of cell surface-localized channel occurs within 48 h. This system can be adapted to study the life cycle of any cellular protein without the confounds associated with radioactive labeling or the complications noted with expressing supraphysiological levels of proteins.

A division of labor: asymmetric roles for GPCR subunits in receptor dimers.

The functional architecture of dimeric or oligomeric GPCR signaling remains incompletely understood. Using a clever combination of receptor-G protein fusions and various receptor mutations, new research provides a glimpse into how oligomers might be arranged with respect to the G proteins they interact with.

Combining protein complementation assays with resonance energy transfer to detect multipartner protein complexes in living cells.

A variety of fluorescent proteins with different spectral properties have been created by mutating green fluorescent protein. When these proteins are split in two, neither fragment is fluorescent per se, nor can a fluorescent protein be reconstituted by co-expressing the complementary N- and C-terminal fragments. However, when these fragments are genetically fused to proteins that associate with each other in cellulo, the N- and C-terminal fragments of the fluorescent protein are brought together and can reconstitute a fluorescent protein. A similar protein complementation assay (PCA) can be performed with two complementary fragments of various luciferase isoforms. This makes these assays useful tools for detecting the association of two proteins in living cells. Bioluminescence resonance energy transfer (BRET) or fluorescence resonance energy transfer (FRET) occurs when energy from, respectively, a luminescent or fluorescent donor protein is non-radiatively transferred to a fluorescent acceptor protein. This transfer of energy can only occur if the proteins are within 100A of each other. Thus, BRET and FRET are also useful tools for detecting the association of two proteins in living cells. By combining different protein fragment complementation assays (PCA) with BRET or FRET it is possible to demonstrate that three or more proteins are simultaneous parts of the same protein complex in living cells. As an example of the utility of this approach, we show that as many as four different proteins are simultaneously associated as part of a G protein-coupled receptor signalling complex.

Kir3 channel ontogeny - the role of Gßγ subunits in channel assembly and trafficking.

The role of Gßγ subunits in Kir3 channel gating is well characterized. Here, we have studied the role of Gßγ dimers during their initial contact with Kir3 channels, prior to their insertion into the plasma membrane. We show that distinct Gßγ subunits play an important role in orchestrating and fine-tuning parts of the Kir3 channel life cycle. Gß1γ2, apart from its role in channel opening that it shares with other Gßγ subunit combinations, may play a unique role in protecting maturing channels from degradation as they transit to the cell surface. Taken together, our data suggest that Gß1γ2 prolongs the lifetime of the Kir3.1/Kir3.2 heterotetramer, although further studies would be required to shed more light on these early Gßγ effects on Kir3 maturation and trafficking.

The role of G proteins in assembly and function of Kir3 inwardly rectifying potassium channels.

Kir3 channels (also known as GIRK channels) are important regulators of electrical excitability in both cardiomyocytes and neurons. Much is known regarding the assembly and function of these channels and the roles that their interacting proteins play in controlling these events. Further, they are one of the best studied effectors of heterotrimeric G proteins in general and Gßγ subunits in particular. However, our understanding of the roles of multiple Gßγ binding sites on Kir3 channels is still rudimentary. We discuss potential roles for Gßγ in channel assembly and trafficking in addition to their known role in cellular signaling.

The NECAP PHear domain increases clathrin accessory protein binding potential.

AP-2 is a key regulator of the endocytic protein machinery driving clathrin-coated vesicle (CCV) formation. One critical function, mediated primarily by the AP-2 alpha-ear, is the recruitment of accessory proteins. NECAPs are alpha-ear-binding proteins that enrich on CCVs. Here, we have solved the structure of the conserved N-terminal region of NECAP 1, revealing a unique module in the pleckstrin homology (PH) domain superfamily, which we named the PHear domain. The PHear domain binds accessory proteins bearing FxDxF motifs, which were previously thought to bind exclusively to the AP-2 alpha-ear. Structural analysis of the PHear domain reveals the molecular surface for FxDxF motif binding, which was confirmed by site-directed mutagenesis. The reciprocal analysis of the FxDxF motif in amphiphysin I identified distinct binding requirements for binding to the alpha-ear and PHear domain. We show that NECAP knockdown compromises transferrin uptake and establish a functional role for NECAPs in clathrin-mediated endocytosis. Our data uncover a striking convergence of two evolutionarily and structurally distinct modules to recognize a common peptide motif and promote efficient endocytosis.