According to the prevailing paradigm, G-proteins are composed of three subunits, an ␣ subunit with GTPase activity and a tightly associated ␤␥ subunit complex. In the yeast Saccharomyces cerevisiae there are two known G␣ proteins (Gpa1 and Gpa2) but only one G␤␥, which binds only to Gpa1. Here we show that the yeast ortholog of RACK1 (receptor for activated protein kinase C1) Asc1 functions as the G␤ for Gpa2. As with other known G␤ proteins, Asc1 has a 7-WD domain structure, interacts directly with the G␣ in a guanine nucleotide-dependent manner, and inhibits G␣ guanine nucleotide exchange activity. In addition, Asc1 binds to the effector enzyme adenylyl cyclase (Cyr1), and diminishes the production of cAMP in response to glucose stimulation. Thus, whereas Gpa2 promotes glucose signaling through elevated production of cAMP, Asc1 has opposing effects on these same processes. Our findings reveal the existence of an unusual G␤ subunit, one having multiple functions within the cell in addition to serving as a signal transducer for cell surface receptors and intracellular effectors.

G proteins are key intermediates in cellular signaling and act in response to a variety of extracellular stimuli. The prevailing paradigm is that G protein subunits form a heterotrimeric complex and function principally at the plasma membrane. However, there is growing evidence for localization at, and signaling by, G proteins at intracellular compartments. Moreover, different cellular pools of G proteins may be composed of distinct subunit subtypes, including some binding partners that function in the place of G protein ␥ subunits. An article in this issue of Molecular Pharmacology (Yost et al., p. 812) describes the use of an innovative fluorescent cell imaging technique to study interactions of the G protein ␤ 5 subunit with a panel of G␥ subunits as well as regulator of G protein signaling (RGS) proteins that contain a G␥-like subdomain. The approach used here provides a new strategy to elucidate the spatial and temporal properties of G proteins, including a growing number of atypical G␤␥ pairings.Heterotrimeric G proteins normally consist of ␣, ␤, and ␥ subunits and are coupled to seven transmembrane receptors at the plasma membrane. Agonist binding to the receptor induces a conformational change of the G␣ subunit promoting the release of GDP and binding to GTP. This exchange triggers G␤␥ disassociation from the G␣, freeing both components to modulate downstream signals. Hydrolysis of GTP to GDP by the G␣ results in reassociation of the heterotrimer and termination of the signal (Sprang, 1997).So far, 23 G␣, 5 G␤, and 12 G␥ subunits have been identified in the mammalian genome. Of the G␤ isoforms, types 1 to 4 are highly conserved, sharing 80% sequence identity, but G␤ 5 is divergent, sharing only 50% identity. Like other ␤ isoforms, G␤ 5 interacts with G␥ subunits; unlike the others, G␤ 5 can also interact with RGS proteins from the R7 family (RGS6, RGS7, RGS9, and RGS11) (Witherow and Slepak, 2003). Most RGS proteins regulate signaling by acting as GTPase-accelerating proteins, increasing the rate of GTP hydrolysis, causing a more rapid termination of the signal. Members of the R7 family of RGS proteins are defined as having a C-terminal RGS domain, a central G␥-like domain, and an N-terminal DEP (Dishevelled, Egl-10, Pleckstrin) domain. It is not clear why R7 RGS and G␤ 5 proteins interact; however, it has been shown that the interaction stabilizes the heterodimer against proteolysis (McCudden et al., 2005).The RGS/G␤ 5 complex could be thought of as a highly atypical G␤␥ pair. Others are likely to exist (see below). With the identification of such atypical subunit complexes, new techniques are needed to ascertain their function within the cell. Bimolecular fluorescence complementation (BiFC) is one promising technique (Kerppola, 2006a). BiFC uses fragments of green fluorescent protein derivatives (YFP or CFP) each fused to interacting proteins. When not assembled, the individual fusion proteins do not fluoresce, but when associated, they produce a fluorescent signal. This technique allows for...

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