A yeast two-hybrid approach was used to discern possible new effectors for the ␤␥ subunit of heterotrimeric G proteins. Three of the clones isolated are structurally similar to G␤, each exhibiting the WD40 repeat motif. Two of these proteins, the receptor for activated C kinase 1 (RACK1) and the dynein intermediate chain, coimmunoprecipitate with G␤␥ using an anti-G␤ antibody. The third protein, AAH20044, has no known function; however, sequence analysis indicates that it is a WD40 repeat protein. Further investigation with RACK1 shows that it not only interacts with G␤ 1 ␥ 1 but also unexpectedly with the transducin heterotrimer G␣ t ␤ 1 ␥ 1 . G␣ t alone does not interact, but it must contribute to the interaction because the apparent EC 50 value of RACK1 for G␣ t ␤ 1 ␥ 1 is 3-fold greater than that for G␤ 1 ␥ 1 (0.1 versus 0.3 M). RACK1 is a scaffold that interacts with several proteins, among which are activated ␤IIPKC and dynamin-1 (1). ␤IIPKC and dynamin-1 compete with G␤ 1 ␥ 1 and G␣ t ␤ 1 ␥ 1 for interaction with RACK1. These findings have several implications: 1) that WD40 repeat proteins may interact with each other; 2) that G␤␥ interacts differently with RACK1 than with its other known effectors; and/or 3) that the G protein-RACK1 complex may constitute a signaling scaffold important for intracellular responses.Heterotrimeric G proteins are a family of proteins that transduce an extracellular signal to an intracellular response via a seven helical transmembrane G protein-coupled receptor (GPCR).1 Upon activation, the receptor facilitates the exchange of GDP for GTP in the G␣ subunit. G␣ is then thought to dissociate from the G␤␥ heterodimer allowing both complexes to individually activate a number of effectors (2, 3). Free G␤␥ interacts with a large assortment of effector proteins, including phospholipases (4), adenylyl cyclases (5), ion channels (6), and G protein-coupled receptor kinases (7). There are, however, G protein-coupled receptor responses, such as MAP kinase activation (8 -10), receptor internalization (11, 12), and organelle transport (13-15) that are mediated through the G␤␥ subunit but that have not been definitively linked to known G␤␥ effectors.G␤ is the prototypical member of a family of proteins known as WD40 repeat proteins, which seem to function as adaptors and enzyme regulators (16,17). G␤ is the only WD40 repeat protein whose three-dimensional structure is known, and it exhibits a toroidal bladed ␤-propeller structure, with each blade consisting of 4 anti-parallel ␤-strands (18). Because the WD repeat motif is a structural element of the ␤-propeller, all of these proteins are thought to be ␤-propeller proteins with a variable number of blades. Furthermore, G␤ subunits are known to interact with G␥ subunits, proteins containing a G␥-like domain (19), a pleckstrin homology domain (20), a QXXER domain (found in adenylyl cyclases) (21), and a domain contained within phosducin and its relatives (22). In this work we propose that G␤␥ also interacts with certain other WD40 repeat prote...

We showed previously that G␤␥ interacts with Receptor for Activated C Kinase 1 (RACK1), a protein that not only binds activated protein kinase C (PKC) but also serves as an adaptor/scaffold for many signaling pathways. Here we report that RACK1 does not interact with G␣ subunits or heterotrimeric G proteins but binds free G␤␥ subunits released from activated heterotrimeric G proteins following the activation of their cognate receptors in vivo. The association with G␤␥ promotes the translocation of RACK1 from the cytosol to the membrane. Moreover, binding of RACK1 to G␤␥ results in inhibition of G␤␥-mediated activation of phospholipase C ␤2 and adenylyl cyclase II. However, RACK1 has no effect on other functions of G␤␥, such as activation of the mitogen-activated protein kinase signaling pathway or chemotaxis of HEK293 cells via the chemokine receptor CXCR2. Similarly, RACK1 does not affect signal transduction through the G␣ subunits of G i , G s , or G q . Collectively, these findings suggest a role of RACK1 in regulating specific functions of G␤␥.Heterotrimeric G proteins transduce extracellular signals from a large family of G protein-coupled receptors and mediate intracellular responses critical for many cellular processes, such as vision, taste, metabolism, and neuronal and cardiovascular functions (1). G proteins consist of three subunits, ␣, ␤, and ␥. The signaling function of G proteins was once attributed totally to the G␣ subunit. The G␤␥ subunit was considered merely a membrane anchor and a negative regulator of the G␣. However, it is now clear that it itself plays a prominent role in signal transduction. G␤␥ has a long list of effector and interacting proteins and has been shown to play a dominant role in certain cellular functions. For example, in yeast, G␤␥ is the principal transducer of the mating signal for cell cycle arrest and differentiation (2). Chemotactic responses of leukocytes (3, 4) and Dictyostelium discoideum amoeba (5, 6) are mediated through G␤␥. Recently, G␤␥ has also been implicated in smooth muscle cell proliferation and arterial restenosis (7).The diversity of G␤␥ target proteins raises the question of how the specificity and efficiency of G␤␥ signaling are regulated. Several G␤␥-interacting proteins have been shown to regulate its function. For example, phosducin and phosducinlike proteins (PhLPs) 1 bind G␤␥ with high affinities and are regarded as scavengers of G␤␥ (8). Binding of these proteins to G␤␥ limits the amount of G␤␥ available to interact with G␣ and to form functional G protein heterotrimers, resulting in inhibition of signal transmission from receptors to G proteins (9, 10). Moreover, they block the activation of effectors by G␤␥, because their binding sites on G␤␥ overlap with the contact residues for G␤␥ effectors (11,12). Some G␤␥ effectors, such as G proteincoupled receptor kinases (GRK) 2 and 3, could also exert regulatory roles in G␤␥ signaling, because binding of these proteins impede the access of other effectors to G␤␥ (13). Notably, these G␤␥-interacting pr...

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hen the Nobel Prize for Chemistry was awarded to Osamu Shimomura, Martin Chalfie, and Roger Y. Tsien for the discovery and development of the green fluorescent protein (GFP) in 2008, researchers around the world were already using GFP (and the many other variants) in a wide range of cell-based assays. W One of the most important uses for GFP is monitoring a variety of cellular processes in real-time. The many applications that use fluorescent proteins include: